Accessing new biomedical applications by combining genetic design and chemical modification of elastin-like recombinamers

PROGRAMA DE DOCTORADO EN QUÍMICA: QUÍMICA DE SÍNTESIS, CATÁLISIS Y MATERIALES AVANZADOS TESIS DOCTORAL: ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS Presentada por Leander Aaron Adrian Siddartha Poócza para optar al grado de Doctor por la Universidad de Valladolid Dirigida por: José Carlos Rodríguez Cabello Für Jalah Für Lian AGRADECIMIENTOS - ACKNOWLEDGEMENTS III Agradecimientos – Acknowledgements Agradecimientos – Acknowledgements En las siguientes páginas me gustaría conmemorar a las personas que me han acompañado y siempre me han apoyado en esta fase emocionante e intensiva de mi vida para obtener el grado de doctorado. Este capítulo de mi vida no solo me ha educado científicamente, sino que también me ha desarrollado personalmente. Si mi vida fuera una novela, esta última etapa llenaría las paginas de más de más de un capítulo, pudiendo rellenar una pagina con cada uno de vosotros. A veces unas palabras son capaces de trasmitir un mensaje más grande, como el amor, el respeto o la amistad, que me conectan con todos de usted. En primer lugar, quisiera dar las gracias a mi director Carlos. Gracias por acogerme en el grupo, por guiarme y por darme todos los medios para poder llevar a cabo este trabajo y por tu tiempo y tus consejos indispensables. Quiero mostrar igualmente mi gratitud a Matilde y Israel, que siempre han dedicado su tiempo y su predisposición cuando lo necesitaria. Gracias a los seniors de grupo, Javi, Merche, Alessandra y Luis, por vuestra amabilidad y por vuestras ganas de ayudar. Muchas gracias a mis compañeros de despacho BIOGEL, Soraya y Filippo vosotros habéis llenado el espacio con alegría, y me habéis dado ganas de volver a trabajar no solo en momentos buenos, pero también en temporadas criticas . Tanto a mis “compañeros de doctorado y sobre mesa” (Doriana, Tatjana, Sofia, Ito, Sergio, Arturo, Juan), como a las nuevas incorporaciones Miguel, Marcos, Fernando. Me habéis apoyado muchísimo para llegar a Valladolid y habéis apoyado aprender las técnicas del grupo. Muchísimos gracias. IV ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS Rocío, muchas gracias por enseñarme los secretos de la purificación y por tu paciencia. Gracias Irene y Alicia por proporcionarme cientos de veces vuestro ánimo. Todos vosotros impregnáis esta tesis. Solo espero haber sido capaz en este tiempo de devolveros, aunque solo sea una pequeña parte de todo el afecto que me habéis dado durante estos años. This work as being part of an incredible international project was further supported by a phenomenal group of people. I am very proud that I can consider you as my friends and that we have shared such an incredible time together at so many interesting and wonderful places and that you have shared your research with me. This interdisciplinary input really leveraged my knowledge. First, I have to name Laura, Professor Möller and Elisabeth, who did an incredible job in managing and realizing this wonderful project. Further, I like to thank Alan and Paul for their input on the mechanical characterization, especially during my stay in the university of Nijmegen (the Netherlands) and the support of my project colleagues Paula and Max (Kai-zengh Li-u) it was so inspiring to work on your side. My journey also brought me to Aachen were I want to thank my resident colleagues Arturo, Luis, Marcel and Sitara and their co-workers Christopher and Yashoda for their help in the lab and for their inspiring thoughts. To complete the Biogel group, a special thanks goes to those who I unfortunately could not work with for a longer period, but not least we had an incredible time in countless meetings and I think we managed to get to know each other pretty well. Thank you Melanie, Daria, Jenny, Dominik, and Nestor, and of course Delphine who was a short member, but remains part of our group V Agradecimientos – Acknowledgements Abschließend möchte ich noch meiner Familie Danken ohne deren Unterstüzung ich diese Aufgabe in der Form nicht hätte realisieren können. Als erstes Danke Ich meiner Ehefrau Jalah für Ihre Unterstüzung und Ihre Stärke in einer Phase in der Ich gerne mehr für Sie getan hätte als es mir durch die räumliche Trennung möglich war. Ich Danke Dir, vielen Dank das du so wundervoll bist und unserem Sohn eine so tolle Mutter bist, auch wenn ich nicht immer da sein konnte. Weiterhin gilt ein Dank meinem Bruder Nico und meiner Mutter Andrea und meiner Großmutter Eva, für Ihre immer währende Unterstüzung. Ein Dank auch an meinen Vater Piet und seine Frau Anke, ich bin froh das ich in dieser prägenden Phase auf euch zählen konnte. Leander RESUMEN XV Resumen/Summary Resumen/Summary Introducción La adquisición de un conocimiento profundo de biomateriales requiere comprender su papel en diversas aplicaciones, tanto en ingeniería de tejidos como en nanotecnología. Dentro de las distintas estrategias destinadas al desarrollo de aplicaciones biomédicas, cabe destacar el uso de materiales que mimetizan las propiedades y características propias que se encuentran en materiales naturales1. En primer lugar, los materiales empleados para aplicaciones médicas deben de cumplir una serie de requisitos como la biocompatibilidad, pero además deben aportar funciones específicas para su uso, como estabilidad mecánica, o la posibilidad de ajustar la degradación del propio material. Los primeros materiales empleados en biomedicina fueron distintos metales o cerámicas, ya conocido por las antiguas civilizaciones2. Tras esto, con el avance de la química macromolecular aparecieron los polímeros sintéticos como el ácido poli(láctico-co-glicólico) (PLGA) o el ácido poliláctico (PLLA). En las últimas décadas el desarrollo de la ingeniería genética y de la tecnología del ADN recombinante ha permitido generar nuevos materiales proteicos, los cuales representan un gran paso en la fabricación de materiales biomiméticos3,4. De este modo, se abría un extenso abanico de posibilidades para el uso del biomaterial más abundante, y probablemente más complejo que podemos encontrar en la naturaleza, las proteínas. En los últimos años, la ciencia ha hecho un gran avance en la elucidación estructural de proteínas, y sigue avanzando en el estudio de las relaciones entre estructura y función3,4. Este XVI ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS conocimiento permite no sólo modificar la secuencia de un material tan complejo como las proteínas, e incluso mejorarlas. Dentro de esta clase de biomateriales recombinantes, encontramos la familia de proteínas inspirada en la secuencia de aminoácidos de la tropoelastina natural, que es el monómero soluble de la elastina5–11. De forma simplificada nos referimos a esta familia de proteínas es como recombinámeros tipo elastina (“elastin-like recombinamers”, ELRs, en inglés). Las ventajas en el uso de la tropoelastina como “proteína madre”, son las funcionalidades ésta como biocompatibilidad, elasticidad y comportamiento termo-sensible que conduce a procesos de autoensamblado12. Además, la secuencia subyacente de amino ácidos a estas propiedades es bastante simple desde el punto de vista de la complejidad de las proteínas. Dicha secuencia está basada en la repetición del pentapéptido L-Valina-L-Prolina-Glicina-X-Glicina (VPGXG), en el que X (denominado aminoácido invitado) puede ser cualquier aminoácido, excepto L-Prolina7,13– 17. Dependiendo del número de repeticiones y de la composición del amino acido invitado, X, se pueden crear una gran variedad de ELRs. Debido a su carácter recombinante, es posible fusionar secuencias de ADN que codifican distintos péptidos y proteínas. Así se puede combinar bloques de pentapéptidos con distintos aminoácidos invitados, en el lugar X, en la misma molécula del ELR. Además, es posible añadir secuencias peptídicas como secuencias biodegradables18,19, o con dominio de adhesión celular20,21 para generar materiales ‘a la carta’ de la aplicación deseada. Objetivos El objetivo de esta tesis es demostrar que la versatilidad de estos recombinámeros se puede aumentar meidante modificación química y XVII Resumen/Summary genética para la configurar la degradación, el autoensamblado y la interacción con células de los ELRs. El trabajo desarrollado en esta tesis aborda todo el proceso de diseño, producción, purificación, caracterización y aplicación directa de los nuevos ELRs. Para ello, se han utilizado una amplia variedad técnicas de ingeniería genética, microbiología, física, química junto con los correspondientes cultivos celulares. A. La tecnología del ADN recombinante permite un control total sobre el diseño de ELR, y de este modo la inserción de distintas secuencias biofuncionales, como secuencias sensibles a proteasas. Mediante el control de la disposición espacial de este tipo de secuencias proteolíticas queremos demostrar la biodegradación especifica de ELRs. Además, la capacidad de biodegradación selectiva será aplicada para la biofabricación de sustratos para detección zimográfica. B. Debido a la degradabbilidad controlable de los ELRs, su uso será estudiado como sustrato selectivo para la identificación de enzimas proteolíticas. Por lo tanto, siguiendo el estudio de la aplicación de ELRs para técnicas zimográficas diseñaremos un nuevo método de detección de proteasas con potencial para sistemas de inspección avanzados de alto rendimiento. C. Se ha demostrado que los biomateriales modificados con colesterol exhiben fuertes interacciones intermoleculares. Así, aplicaremos estas interacciones en un sistema de ELRs para generar fuerzas intermoleculares que desencadenen el autoensamblado de los ELR. D. Además, gracias a la capacidad de interacción de los grupos colesterol con membranas lipídicas, estudiaremos la capacidad de ELRs ricos en colesterol para mejorar la interacción de los éstos con ciertos tipos XVIII ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS celulares implicados en la captación de lípidos, para aumentar el recubrimiento de células vivas con proteínas ELR. Metodología del Diseño de ELRs con grupos proteolíticos (A y B) Con el objetivo de utilizar ELRs como sustrato de zimografía de geles (IGZ) más específicas, se pueden fusionar secuencias de degradación enzimática, por ejemplo la secuencia que contiene el hexapéptido L-Valina-Glicina-L- Valina-L-Alanina-L-Prolina-Glicina (VGVAPG), y cuya L-Alanina con especificad a la enzima elastasa, aumentando la biodegradabilidad de los ELRs, que es de gran importancia en el remodelado del tejido22. Además concentraciones altas en proteasas también son típicamente efectos secundarios de cáncer promovando la infiltración de tejido23. Pese a que la degradación enzimática es más estudiada en el campo de la regeneración tisular, la degradación de sustratos basados en proteínas también puede ser utilizada para desarrollar técnicas de detección de proteasas, enzimas capaces de degradar proteínas. Una de las técnicas más conocido es la zimografía de gelatina24. El método funciona como una electroforesis de poliacrilamida en que la gelatina se añade al gel, si la muestra electroforética en ese gel contiene una enzima con actividad proteolítica, la gelatina será degradada en el lugar que corresponde al peso molecular de esta proteasa. En el caso de la gelatina este método está bien establecida por los metalloproteasas de la matriz MMP2 y MMP925. Sin embargo, aparte de gelatina, otras proteínas naturales pueden utilizarse como substrato de la zimografía, y de este modo aumentar la variedad de secuencias degradables presentadas a las enzimas. Por otro, lado esa variedad en su secuencia depende de muchos factores, como el organismo XIX Resumen/Summary de origen o el proceso de purificación. Así, aunque de la gelatina se conocen muchos elementos estructurales, se desconoce su secuencia completa. Por lo que, los resultados pueden ser confusos, e inclusos erróneos debido a que no se conozca la secuencia proteolítica que está siendo degradada por la enzima. En esta tesis utilizamos ELRs como substrato para zimografia que presenten únicamente un grupo proteolítico, y que tengan una secuencia conocida, para investigar la degradación con una selección de MMPs 1 a 20. Esto facilita el uso de dicho ELRs en un nuevo método de zimografia dentro de una placa (in-well zymography (IWZ). Resultados (A y B) Los resultados mostraron que las diferencias en las concentraciones de peptidasas se pueden medir con zimografía utilizando ELRs como sustrato. Además, el uso de ELRs permitió la detección de proteasas de degradación inespecífica tipo serina, las cuales son indetectables en sustratos clásicos como la gelatina. Más concretamente, los resultados revelaron que, a diferencia de métodos zimográficos clásicos, la ELR-IGZ permite la detección de las enzimas metaloproteasas MMP2 y MMP9. La sensibilidad hacia MMP9 fue casi diez veces mayor con ELREla + FN que en gelatina, mientras que el control negativo ELR sin ninguna región proteolítica no fue degradado. El cambio a IWZ reduce la complejidad en comparación con IGZ mediante la eliminación de la separación electroforética y la desnaturalización de la muestra. La sensibilidad se limitó a un 10% respecto al control no degradado. Se pudo detectar patrones distintos de degradación de cada MMP y se redujo la cantidad necesaria para la cuantificación de 50-500 ng (en IGZ) a 10 ng por muestra. XX ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS Los resultados permitieron clasificar las enzimas proteasas en cuatro grupos diferentes respecto a los sustratos empleados: 1) sin degradación (MMP 2, 3, 10, 14, 16, 19 y 20); 2) mediada por FN (MMP1, 13, 16); 3) mediada por ELA (MMP7); 4) no específica (MMP 8,9, 12, 17). Conclusiones (A y B) Se demostró que la producción de sustratos para IGZ basados en ELRs y secuencias proteolíticas. Los cuales permiten la incrustación en matrices de gel, y la detección con CBB. Empleando dos grupos de degradación diferente, se revelaron diferencias en la especifidad frente proteasas tipo serina y distintas MMPs. Cabe mencionar que el sustrato ELR-Control, al carecer de secuencias específicas de degradación, no fue digerido por ninguna de las MMPs seleccionadas, pero sí se detectó su ligera degradación en presencia de tripsina. La degradación del control pudo deberse a la presencia de lisinas que son son un punto de corte preferente para las proteasas tipo serina, además las lisinas, debido a su cadena lateral catiónica, son sensibles a la tinción con CBB. Por otro lado, presentamos un nuevo método de detección de proteasas mediante la adaptación de la IGZ en una zimografía de placa de pocillo (IWZ), lo que permite una lectura rápida y cuantitativa. Este nuevo método proporciona una técnica de detección capaz de determinar diferencias en patrones de degradación de metaloproteasas (MMP1-20) con sólo dos secuencias de degradación. El control positivo y negativo sirvió como un circuito de retroalimentación adicional en términos del patrón derivado. Complementariamente, este método de cuantificación permitiría seguir la cinética de degradación de cada una de las enzimas sobre los sustratos específicos. La especificidad de los sustratos basados en ELRs aporta varios XXI Resumen/Summary avances a las técnicas zimográficas basadas en gel, por ejemplo gracias al control de los motivos de degradación se pueden elaborar dispositivos de reconocimiento de enzimas proteolíticas basado en un sistema binario. Metodología de la Modificación de ELRs con grupos hidrofóbicos (C) El interés por los biomateriales autoensamblados ha aumentado considerablemente en las últimas décadas debido a su compleja disposición espacial y a que puedan controlar la exposición de distintas funcionalidades. Las implicaciones directas de la estructura en el autoensamblaje en polímeros de bloque anfifílicos y proteínas, junto a la posibilidad de generar interacciones hidrofóbicas26,27 e hidrofílicas28,29, ha permitido cierto grado de control sobre estructuras jerárquicas, como micelas esféricas o cilíndricas, vesículas, membranas bimoleculares e hidrogeles30–33. Además, propiedades tales como la respuesta frente a estímulos como cambios de temperatura27,34 o pH35, han dado como resultado la aplicabilidad de estos materiales en la ingeniería de tejidos36, sistemas para la administración de fármacos35–38, dispersantes de polímeros39, agentes gelificantes físicos40 o biosensores41–43. La capacidad de respuesta térmica es la característica más investigada y mejor controlada para los copolímeros de bloque27,34 y, como tal, ha ganado un mayor interés en el diseño de proteínas recombinantes de vanguardia, especialmente a la luz de los hallazgos de Urry con respecto a la LCST de péptidos miméticos de elastina44–47. Además, debido a que los ELRs permiten distintos diseños modulares combinando bloque análogos con dominios de diferente hidrofilicidad, se han estudiado como modelos para comprender el plegamiento, el autoensamblaje y la función de otras proteínas naturales más complejas48–50. INDEX 3 Table of Content Table of Content Agradecimientos – Acknowledgements ................................................................................... III Abstract ............................................................................................................................................IX Resumen/Summary ...................................................................................................................... XV Introducción ............................................................................................................................. XV Objetivos .................................................................................................................................. XVI Metodología del Diseño de ELRs con grupos proteolíticos (A y B) ....................... XVIII Resultados (A y B) ................................................................................................................. XIX Conclusiones (A y B) ............................................................................................................. XX Metodología de la Modificación de ELRs con grupos hidrofóbicos (C) ................... XXI Resultados (C)....................................................................................................................... XXII Metodologia Interacción celular de colesteryl ELRs (D) ........................................... XXIII Resultados (D) ...................................................................................................................... XXV Conclusiones (C y D) .........................................................................................................XXVI Table of Content .............................................................................................................................. 3 Introduction ........................................................................................................................................... 9 1 Introduction ................................................................................................................................ 11 1.1 Biomimetic Materials ...................................................................................................... 11 1.1.1. Natural Elastin......................................................................................................... 12 1.1.2. Elastin bio-inspired polypeptides (Elastin-like Recombinamers) ................. 14 1.2 Biosynthesis of ELRs ....................................................................................................... 22 1.2.1 Design of the codifying gene .................................................................................... 23 1.2.2 Construction of the multi block ............................................................................. 24 1.2.3 Gene expression and recombinant production .................................................. 28 1.2.4 Purification ................................................................................................................... 29 1.3 Protein - interactions ..................................................................................................... 31 1.3.1 Physical ELR interactions .......................................................................................... 32 1.3.2 Ionic interactions ........................................................................................................ 32 1.3.3 Hydrophobic interaction of amphiphilic blocks and graft copolymers .......... 33 1.3.4 Intermolecular interaction of protein secondary structures ........................... 33 1.4 Chemical Modifications of ELRs .................................................................................. 36 1.4.1 Functionalization of ELRs and Covalent Cross-linked ELR Hydrogels .......... 36 1.5 ELR structures and applications ................................................................................... 38 1.5.1 Hydrogels ..................................................................................................................... 39 1.5.2 ELRs as drug carriers ................................................................................................. 40 4 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS 1.6.1 Cholesterols role in cell membranes ......................................................................41 1.6.2 Cholesterol modified materials ................................................................................41 1.7 Role of Proteases .............................................................................................................44 1.7.1 Peptidase Classification ..............................................................................................44 1.7.2 Role of Proteases in Matrix Remodeling and Related diseases............................46 1.7.2 Serine Proteases ..........................................................................................................49 1.7.3 MMPs ..............................................................................................................................50 1.7.4 Cleavable peptide bonds ............................................................................................52 1.7.5 Involvement of Peptidase in tissue regeneration .................................................54 1.7.6 Disease related to peptidases ...................................................................................58 1.7.7 Zymographic Methods of Protease Detection .....................................................60 2 Hypothesis ..............................................................................................................................69 3 Materials & Methods ............................................................................................................73 3.1 Materials ....................................................................................................................................73 3.1.1 Chemical Reagents. .........................................................................................................73 3.1.2 Glass ware & other materials.......................................................................................74 3.1.3 Molecular biology materials. .....................................................................................74 3.1.4 Other reagents .............................................................................................................79 3.1.5 Bacterial Strain .............................................................................................................79 3.1.6 Bacterial culture media ..............................................................................................79 3.1.7 Buffers ............................................................................................................................79 3.1.8 Elastin-like recombinamers .......................................................................................80 3.1.9 Cell lines and culture conditions .............................................................................82 3.2 Methods..............................................................................................................................83 3.2.1 DNA agarose gel electrophoresis ...............................................................................83 3.2.2 Plasmid purification .........................................................................................................84 3.2.3 DNA digestion with restriction enzymes ..............................................................84 3.2.4 DNA dephosphorylation ...........................................................................................84 3.2.5 DNA separation and extraction from agarose gel ..............................................84 3.2.6 Ligation of genes ..........................................................................................................85 3.2.7 Cloning on the pDrive/ p7 vector ...........................................................................85 3.2.8 Transformation of competent cells .........................................................................85 3.2.9 Glycerol stock preparation .......................................................................................87 3.2.10 Biopolymers expression and purification ..........................................................87 3.2.11 SDS Polyacrylamide gel electrophoresis ...........................................................89 3.2.12 Modification of ELRs ..............................................................................................91 5 Table of Content 3.2.13 Detection of Trypsin in ELR gels ........................................................................ 93 3.2.15 Optimization of screening parameters ............................................................. 95 3.2.16 Screening of MMPs ................................................................................................ 95 3.2.17 Critical Micelle Concentration ........................................................................... 96 3.2.18 Dynamic light scattering ....................................................................................... 96 3.2.19 Zeta potential.......................................................................................................... 97 3.2.20 Circular Dichroism ................................................................................................ 97 3.2.21 Viscosity measurements ....................................................................................... 98 3.2.22 Rheological determination of low-temperature gelation ............................. 98 3.2.23 Contact Angle ......................................................................................................... 99 3.2.24 Transmission electron microscopy TEM .......................................................... 99 3.2.25 Scanning electron microscopy SEM ................................................................. 100 3.2.26 Spectrophotometric cloud point determination .......................................... 100 3.2.27 Cell Culture ........................................................................................................... 100 3.2.28 FACS and Confocal Microscopy ....................................................................... 101 4 Results and Discussion ..................................................................................................... 107 4.1 CHAPTER 1: Development of a novel screening method for proteases based on proteolytically degradable ELR-acrylamide hydrogels ........................................................ 107 4.1.1 ELR IK-HEX gene Synthesis ....................................................................................... 111 ELRs expression and purification ........................................................................................ 115 4.1.2 Methacrylation of ELRs ............................................................................................... 117 4.1.3 WORKING PRINCIPLE OF THE METHOD ........................................................ 120 4.1.4 ELR in gel zymography ................................................................................................ 122 4.1.5 ELR in-well zymography (IWZ) ................................................................................ 126 4.2 CHAPTER 2: Chemical Modification of ELRs with Cholesterol and Structural Mechanistics of Cholesteryl Group Mediated UCST Gelation ........................................ 135 4.2.1 Chemical Modification of ELRs ................................................................................. 139 4.2.2 Characterization in solution ...................................................................................... 142 4.2.3 Characterization of gelation ...................................................................................... 152 4.2.4 Evolution of macrostructures below the UCST ................................................... 158 4.3 CHAPTER 3: ELR – Cell interaction triggered by hydrophobic modification ....... 169 4.3.1 ELR Modification .......................................................................................................... 173 4.3.2 Characterization in solution ...................................................................................... 177 4.3.3 Cytotoxicity of ELR particles .................................................................................... 179 4.3.4 ELR – cell Interactions ................................................................................................ 181 5 Conclusions ......................................................................................................................... 201 6 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS 5.1 ELR Zymography .................................................................................................................. 201 5.2 Structural deductions of cholesterol modified ELRs ................................................... 202 5.3 Cellular interactions with Cholesteryl-ELRs ................................................................. 204 6 References ........................................................................................................................... 209 7 Appendix .................................................................................................................................. III 7.1 Supporting Information .......................................................................................................... III 7.1.1 ABBREVIATIONS ............................................................................................................ X 7.1.2 TABLE OF STANDARD AMINO ACID ABBREVIATIONS ............................. XIII 7.1.3 List of Tables ............................................................................................................. XIV 7.1.4 List of Figures ............................................................................................................. XV 7.1.3 LIST OF ARTICLES AND PATENTS ORIGINATED FROM THE WORK OF THIS PhD THESIS ................................................................................................................ XXII 7 Table of Content INTRODUCTION 16 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS the formation of β-turns (Figure 1C) which in the polypeptide result in βspirals formed by consecutive β-turns117. As a consequence, the hydrophobic groups are exposed to the solvent phase leading to phase separation. Due to the hydrophilicity of the ELRs the segregated phase might still retain more than 60% of water by weight and has viscoelastic properties118. Figure 1: a) Structural changes of VPGVG oligopeptide below and above their LCST. b) Water in the clathrate-type structured state. c) Type II β -turn in the VPGVG pentapeptides119. In most proteins an increase in temperature causes loss of order (unfolding – denaturation), remarkably ELRs behave in a distinct way. Nonetheless, taking enthalpic and entropic events into account, this abnormality does not contradict the second law of thermodynamics. The loss of clathrate interactions, is compensated by approximately just one third with Van der Waals forces of the hydrophobic aggregation of the 17 Biomimetic Materials polypeptides119, therefore the folding is not driven by enthalpy. With respect to entropy, the order in the polypeptide is increased with folding, which as well is unfavorable. But considering the whole system, the rupture of water clathrate structures does increase the entropy of the whole system by the release of water molecules into a disordered “free” state. This process of folding and unfolding water is completely reversible in water. 1.1.2.2 Factors to influence ELR properties It has been proven that the amino acid sequence has a great influence on the LCST of ELRs120. Substitutions of the amino acid at the fourth position (Xaa) of the pentamer modify the LCST, to an extent that depends on the polarity of the amino acid side chain. The transition temperature of an ELR sequence based on (VPGXG)n can be controlled and adjusted to the desired applications by the selection of X (hydrophobic AA decrease, hydrophilic AA increase Tt)120, the segment length n (longer ELR sequences have lower Tt)121,122, concentration (higher concentrations of ELR decrease Tt)121, and by extrinsic factors like pH48, salt concentration, or solvents123–125. Intrinsic Factors Manipulation of the X position and the chain length have the biggest potential to tailor the Tt121,122. The substitution of the X position affects the Tt with respect to the hydrophobicity of the inserted AA (Figure 2)126. Trp > Tyr > Phe > Leu  Ile  Met > Val > Ala > Gly 18 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS Figure 2: Dependence of Tt on the guest residue in poly penta-peptide of the structure poly[fv (VPGVG), fx (VPGXG)]. fv and fx represent the mole fractions of the respective co-monomers (fv + fx = 1)127. As a rule of thumb an increase in the polarity, causes an increase in Tt and a decrease in ΔHT. Incorporation of an acid or a basic amino-acid (acid: Glu, Asp; basic: Lys, Arg, Asn or Gln) are also pH sensitive, and the ionization degree of those amino-acids will have an important effect on the Tt as can be seen in Figure 2. In this sense, above its characteristic pKa (for acidic AA), or below its characteristic pkb(for basic AA), , the residue is charged, resulting in an increasing of the mean polarity, which leads to an increase in the Tt127. The effect of chain length is of interest for very short oligopeptides, where the Tt decreases with increasing chain length, but can be neglected for polypeptides composed by more than 100 pentapeptides according to Meyer et al.128. 19 Biomimetic Materials Figure 3: Tt as a function of the number of pentapeptides for three different types of ELRs at 25µM in PBS. Reproduced from Meyer et. al.128. It is worth to mention, that none of the three intrinsic factors, (concentration, guest residue and chain length) should be considered independently in order to alter Tt, since the conjunction of the three together will determine the final Tt. Extrinsic Factors The effect of different salt anions and their concentration on the Tt of ELRs has been extensively studied by Cho et al.49 as depicted in Figure 4. The effect of salt concentration on ELRs can be best explained with the Hofmeister series129,130, anions to the left of Cl- (kosmotropes) have a strong tendency to salt out polymers/ proteins from solution, as well as cations to the right of Na+. “Salting in” ions (chaotropes) provoke opposite effects. 20 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS Figure 4: Effect of Hofmeister anions on the transition temperature of ELRs. Figure adopted from 49. CO32- > SO42- > S3O32- > H2PO4- > F- > Cl- > Br- > NO3- > I- > SCNNH4+ > Cs+ > Rb+ > K+ > Na+ > Li+ > Ca2+ > Mg2+ It is believed, that kosmotropic anions polarize the water molecules which are involved in hydrogen bonding to the amide group of the ELR employed. The direct interaction between the anions and the water molecules, weakens ELR-water interaction, causing a decrease in Tt and solubility. Whereas, chaotropic anions directly interact with amide moieties by ion binding, causing a salting-in effect129,130. The most commonly used salt is NaCl which has been found to cause a significant concentration-dependent decrease in Tt and an increase in the transition enthalpy in ELR-water systems119. The effect on the thermal behavior of the ELR can be compared to an increase in the hydrophobicity 21 Biomimetic Materials of the recombinamer chain, fostering a better organization of the recombinamer in the folded state. Besides salt changes, pH changes can result in changes in the degree of ionization of the ELR, which is more pronounced when polar guest amino acids are present 48. Biotechnology provides us with a powerful set of tools that can be used to successfully control the physicochemical features of the amino acid side chains and their association131,132, or to include any protein based functionality like protease active sides, which become important when degradation of the scaffold has to be adjusted, e.g. to the growth rate of new tissue133. 22 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS 1.2 Biosynthesis of ELRs Biosynthesis of proteins uses the natural route of protein production by inserting a DNA fragment specific for the protein of interest into the protein expression chain of an organism. The very susceptible process of protein expression requires very close control of DNA manipulation, which is obtained by recombinant DNA techniques2. The manipulation of DNA, insertion into an expression vehicle (vector), the insertion into a producing organism and the purification of the final protein is a time and resource intense process, nonetheless it is without alternative for the generation of larger amino acid sequences, where synthetic peptide production reaches its limit. Despite the time and effort required for the synthesis of genetically encoded polypeptides and the restriction to natural amino acids, the use of genetic engineering to obtain recombinant proteins has several advantages over their synthetic counterparts. Bioproduction provides a facile route for the design of novel protein polymers composed of repetitive amino acid sequences or peptide blocks whose structural complexity imparts distinct mechanical, chemical or biological properties2. The accentuate reduction on the production costs, the time reduction in large scale bio-production, the greater control over product sequence, size and uniformity, higher yields and the possibility of achieving structural complexity into an assortment of bio-inspired materials with distinct mechanical, chemical or biological properties, summarize some of them. Related to the establishment of this recombinant technology for the creation of protein-based materials, a new term, namely recombinamer134, has been implemented to evoke the oligomeric and recombinant nature of protein polymers produced by genetic engineering techniques. Following this new 23 Biosynthesis of ELRs terminology, from now onwards, recombinant protein polymers will be referred to as recombinamers in the present work. The biosynthesis of a recombinamer can be drafted in four main steps, namely 1) design of the codifying gene for the protein, 2) obtaining the monomeric gene and construction of the multi-block, 3) transformation of a bacterial strain for expression with the selected positive plasmid and 4) bio-production of the recombinant polypeptide and subsequent purification135. 1.2.1 Design of the codifying gene At the gene design stage it proved necessary to try to overcome the contradiction that exists between the use of the most common and appropriate codons that identify highly repeated amino acids and the need not to overload or even impoverish the cell translation system to the point of collapse136,137. If heterologous prokaryotic systems are used, the problem arising from their limited use of the genetic code, which varies with species, must be taken into account. The problem arising from the creation of a gene formed by a long sequence that codes for multiple and/or small artificial fragments have also to be taken into account. Finally, the high recombination frequency commonly found when the exogenous DNA contains repeats of highly similar domains also has to be overcome138,139. This stage involves the use of automated DNA synthesizers together with recombinant DNA techniques. The production of polymers with multiple repeats necessarily involves the production of a gene that codes for them which, depending on the length of the sequence, may require the prior synthesis of a polynucleotide-based monomer that can be connected linearly in the correct direction135. 24 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS The production of sufficient amounts of the monomeric gene containing the correct sequence for producing the repeat nucleotide sequences that code for the various polypeptides required a culture of the appropriate clones and digestion of their plasmids. This allows for high yields of monomer required for the controlled ligation, concatenation or concatenamerisation reaction to obtain oligomerized genes in a simple manner140,141. Although this approach may appear to be wasteful in terms of time, in vivo synthesis guarantees the production of the correct sequence in large amounts and also offers additional advantages, such as control of the monomeric gene prior to oligomerisation and the ability to subsequently modify the sequence by either directed mutagenesis or by the creation of copolymers prior to oligomerisation. 1.2.2 Construction of the multi block Polymerisation of the monomeric nucleotide sequences may be undertaken by, but is not limited to, concatenation, in other words random ligation of the monomeric blocks; the iterative/recursive method, a step-by-step technique for preparing oligomers from monomers; or by the Seamless cloning method, a definition that refers to the possibility to select a specific sequence that is translated into the desired amino acid at the cut-off point140,141. 25 Biosynthesis of ELRs Figure 5: Condensed summary of recombinant gene oligomerization in a vector with 2 restriction enzymes. Adapted from142. Concatenation allows the synthesis of polymers from oligomers by the unidirectional, linear head-to-tail attachment of the DNA segments that make up the monomer, although such attachment is only possible if the ends of the segments are single-stranded, protuberant and cohesive amongst themselves but not with themselves. Thus, the single stranded head end of the monomer will be complementary to, and will only hybridise with, the tail end of another identical monomer. These ends cannot therefore be palindromic, as currently occurs when they are generated by the restriction endonucleases routinely used in genetic engineering, where the recognition and cleavage sites occur sequentially, but must be different143. 32 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS selection of the cross-link sites, which usually correspond to the lysine groups106,165. 1.3.1 Physical ELR interactions Several strategies can be applied to foster physical ELR interaction. Among those the following have been described. 1.3.2 Ionic interactions The ionic cross-linking motifs are based on ELR segments of opposite charge, or by introducing chelating capacities for multivalent cations into the ELR backbone that can be further controlled by salt concentrations. Peptide sequences of alternating charge and with complementary ionic sides have been shown to perform well to physically crosslink. They are classified in different moduli, depending on the size of the equally charged ionic blocks (1-4 aminoacids). Modulus I, – + – + – + –+; modulus II, – – + + – – + +; modulus III – – – + + +; and modulus IV – – – – + + + +. A modulus I sequence reported by Holmes et al.166 showed a salt induced in-situ gelation167. The complexation of ions is obtained by including glutamic and aspartic acid residues into the ELR sequence accompanied by the addition of Ca2+ ions, such hydrogels can be stabilized at room temperature and at physiological pH168 and react sensibly towards chelating agents which reduce the number of accessible Ca2+. Another approach by Yeo et al.169 was to add monosaccharides to the side chains of the ELRs which are capable to form stable complexes with potassium. Furthermore, blends of ELR/Chitosan have shown to form stable films in the presence of sodium ions170. 33 Protein - interactions 1.3.3 Hydrophobic interaction of amphiphilic blocks and graft copolymers Self-assembly of amphiphilic blocks and graft copolymers. Hydrogels can be obtained through aggregation among hydrophobic segments of multiblock copolymers of ELRs. The hydrophobic functionalities are provided by alkylrich amino acids like alanine (Ala), leucine (Leu), isoleucine (Ile), valine (Val), phenylalanine (Phe), tryptophan (Thp), tyrosine (Tyr) or methionine (Met). These ELR based amphiphilic blocks have been shown to be stable in vivo159,171,172. Furthermore, it is common to find protein polymers characterized by alternating blocks of essentially hydrophilic and essentially hydrophobic amino acids. The presence of chemically distinct segments within the same protein chain causes a mutual repulsion between the different blocks, which tend to segregate. Nevertheless, such segregation is constrained by the “forced cohabitation” of these blocks within the same molecule. As a result, they just separate into different domains, and as a consequence, periodic nanostructures are formed173. 1.3.4 Intermolecular interaction of protein secondary structures The structure design offers the possibility to include almost any sequences into the ELR backbone that are able to form intramolecular interacting secondary structures, limiting factors here are, to retain the ELR temperature transition, and to take into account that complex folding might not be realized by a recombinant approach. 34 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS One bioinspired approach was to include natural silk sequences that are known to form intramolecular betasheets that self-assemble into crystalline regions and have unique mechanical properties174–178. These silk-based materials can be used to form a variety of structures like hydrogels, films, 3D porous matrices and submicron to macroscale fibers179. Furthermore, silk scaffolds have been proven to be biocompatible and performed well invivo180–182. In terms of the silkworm (Bombyx mori) a repetitive hydrophobic sequence of GAGAGS has been identified to be responsible for the strength of silk fibers183. These blocks are stabilized by hydrophilic compartments in the protein and a complex mixture of stabilizing proteins184. Fernandéz- Colino et al. have shown that the GAGAGS sequence can be successfully integrated into an ELR, facilitating in-situ gelation of the material and its uses as an injectable system159. A more complex crosslinking has been realized by the use of coiled-coil induced oligomerization through leucine-zipper motifs, which include sequences that are known to develop well defined secondary structures185– 187. Leucine zippers are characterized by heptad repeating peptide-units that form a distorted α-helix designated as “abcdefg”, where the “a” and “d” positions are occupied by hydrophobic residues such as leucine, whereas “b”, “c”, “e”, “f”, “g” usually displays a hydrophilic nature188. The oligomerization of different α-helices is primarily driven by the hydrophobic interactions189–192, and partly by electrostatic interactions193–195 and the number of heptad repeats190,196,197. The potential extrapolation of these domains to the creation of bioinspired domains has been explored in a much lower extension than it is the case for elastin or silk domains, which involves also a lower number of studies available regarding their biocompatibility. However, its human origin as well as the conserved nature of the sequence of these domains alleviate any 35 Protein - interactions concerns regarding its biocompatibility. To further support this notion, in vivo implantation of the leucine zipper based scaffolds in a mouse model has been recently reported, and no foreign body reaction to the scaffold was detected198. Protein based physical cross-links in nature are ubiquitous and it can be assumed that was has been reported to date is merely just the tip of the iceberg. 36 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS 1.4 Chemical Modifications of ELRs 1.4.1 Functionalization of ELRs and Covalent Cross-linked ELR Hydrogels Posttranslational modification in ELRs is obtained by the reaction of the functional groups in the protein backbone. Which in ELRs can be included by active amino acids into the X position of the (VPGXG) sequence, thus maintaining the pH and temperature sensitivity of the ELRs199,200. These active amino acids facilitate on the one hand for cross-linking reactions, and on the other for chemical modifications. Concentration, molecular weight and lysine content of ELRs are key parameters for hydrogel formation. Below a critical concentration201, the hydrogel network is not formed due to the lack of inter-molecular contacts. ELRs with high molecular weight are more prone to establish an elevated number of inter-molecular contacts that promote network formation. ELRs with a high content in lysine are the most used to prepare hydrogel networks, because of the suitability of the amino group of lysine to form covalent bonds between ELRs chains. Furthermore, relevant features of the hydrogel, such as gelation time, network pore size, stiffness and degradability, can be narrowly controlled through the nature and the concentration of the cross-linker agent. Labile chemical linkages can also be formed in order to be broken under physiological conditions, either enzymatically or chemically202. As a drawback, chemical cross-linking usually requires organic solvents and reagents that have to be exhaustively removed for biomedical applications, after synthesis of the network. Chemical cross linking can be obtained through the following mechanisms: 1) radical polymerization (acrylates), 2) coupling of complementary groups 37 Chemical Modifications of ELRs (click, reaction, Michael-addition203, condensation204), 3) high energy irradiation205, or 4) enzymatical cross-linking (transglutaminase)206–208. Typical functionalization motifs are fluorescent probes or bioactive sequences which include adhesion sites, inhibitors, antibodies and anchor, or signaling molecules. Permanent or chemical hydrogels are covalently cross-linked networks and may contain clusters spread within regions of the low cross-linking density and high water swelling. The free chain ends also cause defects in the gel and do not contribute to the elasticity of the networks. Both aqueous and organic media can be used to form ELRs networks; the cross-linking in an organic solvent rendering hydrogels with a more uniform structure due to the absence of transitions. Conversely, in water the behavior of the ELRs molecules is governed by the LCST105. Some organic solvents, such as trissuccinimidyl aminotriacetate, can react with the lysine residues of different ELRs chains to form a network. The cross-linking confers the hydrogel with structural stability, being insoluble in water even upon cooling. Also intermolecular crosslinks between proteins can be obtained by genipin, as it was shown for an ELR/fibronectin hybrid209. The chemical cross-linking strategy has some important advantages, for instance the covalent bonds avoid hydrogel network dilution and prevent components diffusing out from the place where the hydrogel is implanted. Click-chemistry has been of growing interest in the past 5 years to form ELRs networks avoiding the use of organic solvents210,211. It can be used to fuse ELRs with different bioactivity together212, or even form hybrid systems of the ELRs in an in-situ gelating manner. This method has further been used for the creation of nonthrombogenic stents213. 38 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS 1.5 ELR structures and applications Based on the design of ELRs and that nods of interactions incorporated, ELRs can form a variety of structures from nanoparticle to hydrogels (Figure 8), which allow for a variety of structure related applications. Figure 8: The diversity of ELR structures and their sequential origin. Amphiphilic ELRs consist of blocks of different polarity, which have the ability to form micelles in solution, here the hydrophobic block is embedded in the core and the hydrophilic block(s) form the corona. The formation of stable nanoparticles requires a relatively high molecular weight of 48 pentapeptides, and the particle size and molecular weight for a comparable ELR are directly related to214. The ITT of the ELRs depending on the characteristics of the micelles, can either lead to coalescence of micelles into lyotrophic gels112, to polydisperse microparticles215, or swelling and deswelling of the micelles, accompanied by size changes. Typical sizes of ELR 39 ELR structures and applications nanoparticles reported are usually in the range of 10 to 100124,216–220, but due to the temperature sensitivity and related agglomeration, also micro sized particles have been reported215,221. The approaches to influence ELR micelles are similar to those affecting the Tt of ELRs, like changing concentration112, the addition of surfactants222, salt concentrations124,218,220 and pH218. In general the shape of micelles is round, but with specific designed ELRs consisting of different amphiphilic blocks, micelles with a cylindrical shape have been obtained223. The rearrangement typically occurs over the critical micelle temperature (CMT) and is driven by a change in the secondary structure of the hydrophobic core, the cylindrical shape change further seems to enhance the cellular uptake of the particles214. Micelle solutions further have been used to generate thin coatings with nano topographic, or anti-fouling properties224. 1.5.1 Hydrogels Due to the ELRs origin, inspired by the extracellular matrix (ECM) protein elastin, most research is focused on the generation of matrices. In tissue engineering, where the predominant matrices in the past 40 years were hydrogels which already made it to application in the human body151, their high water content, the resulting mechanical properties, and stimuliresponsiveness are a good model for natural tissue152,153. To resemble the ECM physical properties (elasticity, stiffnes), the nano-topography and the presence of signaling molecules, protease active and adhesion sites. For the generation of hydrogels from linear ELRs, cross-linking points must be considered. They can be either of covalent or physical nature and their position along the ELR backbone can be fully controlled. Polypeptide-based block-copolypeptides for example manage to self-assemble into stable 40 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS hydrogels155, which can be further stabilized when flanked by protein segments with coiled-coil secondary structure111,156, another approach is the incorporation of recombinant segments of elastin, silk or collagen157–159. 1.5.2 ELRs as drug carriers ELRs have several advantages over other macromolecules as drug delivery carriers. They can be utilized in an active (by hyperthermia induced phase transition), or passive (by controlling the molecular weight) approach. Moreover, the recombinant nature of ELRs allows for the insertion of specific sequences that enhance tumor targeting. It was shown that an anticancer drug that has been conjugated to an ELR is administered, and focal hyperthermia applied, the thermoresponsive properties and enhanced permeability and retention effects of the ELP facilitate drug aggregation within tumor tissues225. Further supported by studies by by Chilkoti et al. using ELRs for targeting solid tumors by applications of hyperthermia to the tumor region, demonstrating a twofold increase in ELR accumulation within a tumor upon application of hyperthermia, as compared to treated with the ELR construct alone226,227. This adjustable ELR accumulation rates may be exploited to reduce drug cargo doses without impairing anti-cancer efficacy. It should be noted that besides the control over accumulation rates, ELRs are eliminated more quickly and with fewer side effects from the untreated regions that have not been subjected to hyperthermia. 41 ELR structures and applications 1.6 Cholesterol 1.6.1 Cholesterols role in cell membranes One of the most abundant molecules in the membrane is cholesterol, which due to the hydrophobicity lays inside the membrane. Cholesterol is responsible for membrane integrity and cellular signaling and makes up to 20% of the membrane's mass63. Cholesterol, among the many lipid constituents of mammalian cell membranes, is a key regulator of membrane fluidity and contributes to the formation of caveolae, and in maintaining caveolae microdomain64,65. Caveolae are specialized domains of the plasma membrane that are found in most cell types66. Nevertheless, they are more abundant in cells like adipocytes, endothelial cells, fibroblasts, and muscle cells67. Besides the influence of membrane fluidity, cholesterol is important for cellular structure and function, it is essential in locomotion and serves as a metabolic precursor for several signaling molecules, including oxysterols68, steroids228 and bile acids65,69,70. The intrisc property of cholesterol to form liquid crystal like domains (lipid rafts) in the cell membrane makes cholesterol an interesting group for the interaction cholesterol modified materials with cells. 1.6.2 Cholesterol modified materials A new class of materials, has been obtained by the hydrophobic modification of hydrophilic polymers with cholesteryl groups, generating polymer amphiphiles that self-assemble into nanoparticles in aqueous solutions. The underlying mechanism was described in cholesterol modified pullulan as a 48 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS Figure 11: Cascade of peptidase pro-form activations by peptidase mediated cleavage244. The inactive precursor of a peptidase that is activated by proteolytic cleavage, is called zymogen, or proenzyme. In contrast to phosphorylation phenomena, no energy source is needed, and activation can also occur extracellularly, it is noteworthy, that this form of activation is irreversible, the enzyme might be deactivated by other mechanisms, but cannot return to its initial uncleaved form244. Besides the secretion in a non-active form, there are several other regulatory functions in tissue, to avoid the false activity of peptidases. The coordinated control of ECM degradation on the cell surface involves three crucial elements: secreted proteases and their inhibitors, surface protease receptors and integral membrane proteases245. The very prominent role in regulation play inhibitors of peptidases, which are derived from plasma, or cells in the tissues. About 10% of the proteins in plasma are peptidase inhibitors, which are in general class specific, besides α2 macroglobulin, an inhibitor capable to act across the peptidase classes. The balance between peptidase and their inhibitors depends on several factors, including production rates of proteinases and inhibitors, and their 49 Role of Proteases secretion. Production levels of the proteinases and inhibitors within the cells are controlled mainly by their gene expression. Activation processes of proMMPs and membrane anchoring of their activities have been established by extensive experimental work. The presence of plasminogen activator (PA) inhibitors in the ECM is supposed to influence the extent of local PA and plasmin-mediated proteolysis. It was found, that the PA inhibitor is homogeneously distributed under fibroblast and fibrosarcoma cells246,247 and closely associated with the pericellular space of endothelial cells248–250, controlling the remodeling of the membrane locally. 1.7.2 Serine Proteases Serine peptidases induce hydrolysis of peptides via a serine group; they include the largest number of peptidases. The main ECM degrading proteases include neutrophil derived elastase, cathepsin G, proteinase 3, plasma derived plasminogen, kallikrein, and the plasmin and plasminogen activators like tissue-type plasmin activator (tPA) and urokinase-type plasminogen activator (uPA).The role of these proteases is best studied in endothelial cells, fibroblasts, chondrocytes or tumor cells243. Plasmin activators are secreted immediately after synthesis, thus the enzymes are found to be extracellular251. The synthesize occurs in the liver and is secreted to the plasma, due to the binding capacity to cells, proximity related to local control of cellular response can be exerted252,253. tPA and uPA play a key role in the activation of plasminogens for fibrinolysis and tPA in particular induce clearance fibrin from the blood and blood clots254. Serine proteases are crucial for a cascade of zymogen conversions, in which the activated form of one clotting factor catalyzes the activation of the next 50 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS precursor (compare Figure 11 proenzymes). One of the most studied zymogens is fibrinogen and its role in blood clotting. Fibrinogen is a highly soluble molecule in the plasma, and is converted into the insoluble fibrin by the hydrolysis of several arginine bonds causing its precipitation in the final step of blood clotting, a process which is induced by the serine proteases thrombin. As well as being responsible for the clotting of blood, serine proteases are also involved in the lysis of the blood clots and the remodeling, mediated by uPA and tPA plasminogen activation. Although zymogen activation is irreversible, specific inhibitors like anti-thrombin III function as modulators of peptidase activity244. Plasmin can degrade most of the matrix components laminin, fibronectin, and possibly type V collagen)255,256, but not types I, II, and III collagens. Thus, serine proteases are partly complementary to collagenases (MMP1, 8, 13). Elastase and cathepsin G are capable of elastin cleavage and cleave the telopeptide region of fibrillar collagen (types I, II, III, IV, VI, VIII, IX, X, and XI). Moreover, they can degrade ECM components such as fibronectin, laminin, and aggrecan. It was further reported, that serine proteases play a role in the activation of latent collagenases and other MMPs and thereby participate in the complete degradation of ECM257–260, as well as in the inactivation of peptidase inhibitors like α2 antiplasmin, α1 antichymotrypsin, and tissue inhibitors of metalloproteinases (TIMPs)261,262. 1.7.3 MMPs The second largest group of peptidases involved in tissue remodeling are MMPs; MMPs degrade most of the components of the ECM, like collagen, gelatin, aggrecan, tenascin, fibrinogen, elastin, laminin, osteonectin, or fibrin. MMPs possess several common characteristics: they are secreted in a latent form and require activation for proteolytic activity, they contain a heavy 51 Role of Proteases metal ion, and require Ca2+ for proteolytic activity, they degrade ECM components, and are a target to TIMPs262,263. Most secreted-type MMPs, including collagenases, gelatinases, and stromelysins, are composed of three basic domains; a propeptide, a catalytic domain, and a hemopexin-like domain, with a prepended hydrophobic signal domain. The propeptide domain includes a free cysteine that occupies a coordination spot on the zinc atom, hampering the catalytic hydrolysis of peptides. The propetide region is a target to other peptidases, by hydrolysis/activation of the propetide, the active site is released from the cysteine and fully functional. The catalytic domain includes the metal ion (mainly zinc) complexed by a histidine containing sequence. C-terminal hemopexin-like domain, interacts with ECM components via a proline-rich hinge region, and determines the MMPs substrate specifity. In the process of collagen remodeling, peptidases like collagenases (MMP1, 8, 13) are crucial for the degradation of the collagen. Collagenases are enzymes that are secreted by fibroblasts, neutrophils, or macrophages264,265, and that clip the collagen molecule at a specific site. The activated collagenases specifically degrade the native triple helix of type I, II or III collagen, by making a single, sequence-specific266. The action of the collagenase on collagen further results in “unwinding” of the helical structure to produce a molecule that is now susceptible to proteolytic cleavage by other proteinases and gelatinases243,267. ECM molecules like Proteoglycans, laminin and fibronectin are more susceptible to stromelysin (MMP3; MMP10) degradation268, but stromelysins are less capable of degrading type IV collagen and elastin. Moreover, MMPs are involved in the regulation of actin microfilament system269 and in lung remodeling (MMP9)270. Some MMPs are anchored to the membrane of cells, allowing for cell-directed cleavage of ECM. These MMPs located in the cell membrane differ in their c-terminal anchorage region, and can be classified into three groups: glycophosphatidylinositol (GPI) - linked MMPs; 52 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS transmembrane-type 1; and type 2 MMPs. Type I transmembrane-type MMPs (MTx-MMPs) include MMP-14-16271,272 and MMP-24273, capable of degrading collagens, gelatins, aggrecans, FN, Ln, fibrin, tenascin, nidogens, and proteoglycans 274–277. GPI linked MMPs include MMP-17 and MMP-25, involved in the degradation of gelatin and fibrin/fibrinogen278,279. So far just one type-2 transmembrane type MMP has been discovered, MMP23, which is expressed in reproducible organs of males and females, but the exact function remains to be discovered280,281. 1.7.4 Cleavable peptide bonds Current research in tissue engineering materials is challenged by a paucity of renewable sources, which include functional and immunologically compatible cells; appropriate biomaterials with desired mechanical, chemical and biological properties; and the inability to generate, vascularized tissues282. In the past ten years, the field has advanced tremendously catalyzed by the achievments in the related fields of biology, material science, chemistry and engineering. The lack of immunologically compatible cells might be covered by the discovery of induced pluripotent stem cells (iPSCs), paving the way for more sophisticated applications in personalized medicine283,284. Research has begun in detecting the proteolytic sequences in natural proteins, especially in ECM proteins. These groups were the first candidates for tissue engineering construct, by using short specific peptides as crosslinkers of natural or synthetic hydrogels. More recently, peptide libraries were designed to discover new proteolytic groups that were not or not yet found in natural proteins. The specificity of cleavage is mainly focused on the detection and or the targeted delivery of drugs in cancer, 53 Role of Proteases but can also be used for the targeted degradation and more specific regeneration of tissue engineering constructs. The number of proteolytic groups is steadily increasing, thus is their use for degradable hydrogels. A simple one component synthetic polymers might be the material of choice in terms of mechanical stability, and emerging techniques such as spatial patterning285,286, two-photon polymerization287, 3D printing288–295, programmed self-assembly296,297 overcome the lack of defined sub micro structures in synthetic polymers. These techniques allow the generation of complex biological structures with integrated vasculature and multiple cells or extracellular matrix (ECM) types at high spatial resolution. The material stiffness in TE targets several key functions, such as stem cell differentiation, enabling new ways of controlling cell phenotypes using physical cues298–300. The limitations of synthetic polymers are mainly related to the lack of biochemical cues like growth factors and cytokines to be presented with improved bioavailability and bioactivity301,302. The mimicking of the extracellular matrix, with all its mechanical and biological complexities, also demands proteolytically cleavable segments303,304. To be effective mimics of extracellular matrix molecules like fibrin and collagen, an artificial extracellular matrix analogue would have to be proteolytically degradable, just like the natural extracellular matrix305,306. Here the kinetics of degradation depends on the application, e.g. the release of small and large molecules in targeted treatments demands a degradation rate that fosters the controlled release and diffusion of the molecules, whereas encapsulation for immunoisolation of cells ideally does not degrade. In tissue regeneration application the rate of degradation should match the neo tissue formation rate307. Further challenges involving the design of future hydrogels are related to the understanding of the foreign bodies and the body’s immune surveillance 54 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS system282,308,309. By now biomaterials are designed to be invisible for the immune system or to induce a weak inflammatory response to initiate the regeneration cascades. A better understanding of the body’s immune system might enable further cues triggering the immune system for more controlled regeneration. 1.7.5 Involvement of Peptidase in tissue regeneration The general role of peptidases in protein degradation, is crucial for tissue regeneration. Peptidases are especially involved in vascularization, osteogenesis, neurogenesis and wound healing. 1.7.5.1 Vascularization Vascularization is crucial for all types of tissues and blood vessels are needed for the supply with nutrients. Peptidases might not be the key player in vascularization, but they complete the complex event cascades new blood vessel generation. Growth factors (GFs) like aFGF, bFGF, VEGF, TGFs are important for the cellular signaling related to vascularization, but there is also a need for proteases, that degrade and penetrate the ECM, for the invasion of blood vessels into the tissue. It has been shown, that the presence of growth factors affects the peptidase expression of endothelial cells in vitro, raising the issue of their importancs in vascularization. The cells responded to bFGF with an increased synthesis of uPA and collagenases310– 312. Further, it was shown, that endothelial cells invade ECM proteins like fibrin and type I collagen in the presence of tPA, resulting in blood vessellike tubes313,314, accompanied by increased collagenases (type I and IV), PA , 55 Role of Proteases and stromelysin activity310,311,315,316. Even though the MMPs are secreted in their latent form, their activity is regulated by the plasmin activation, which itself has been activated by the coordinately secreted serine proteases like uPA and tPA. The interdependency of peptidases-mediated vasculatization in the signaling cascade is highly controlled and further self-regulated by the presence of several peptidase inhibitors. The main inhibitiors synthesized by endothelial cells are plasminogen activator inhibitor-1 (PAI-1)310,317, and tissue inhibitors of metalloproteinases TIMPs315, which directly target plasminogen activators (uPA, tPA), and MMPs (collagenases and stromelysin). The mechanisms of peptidase activation and inhibition are most likely controlled by the localization of peptidases and inhibitors. Peptidases are frequently cell surface associated, whereas TIMP is secreted to interstitial fluids and PAI-1 is secreted to the ECM. It has been proposed that the localization of proteases to cell surfaces may provide several advantages for more invasive neovascularization, including concentrations of proenzyme and activator enzymes. It is supposed, that the rate of enzyme activation is enhanced, by the protection from inactivation by secreted or ECM-associated inhibitors, giving regional specifity to enzymatic activity, directing proteolysis and angiogenesis310. 1.7.5.2 Osteogenesis The regulation of matrix (re)modeling and degradation, by growth factors and peptidases is essential in bone development. Intramembranous and endochondral bone formed during embryogenesis, is subsequently remodeled in response to use, stress, or injury. Similar to vascularization events, a cascade of complex processes is involved, which include matrix deposition and resorption, cell recruitment and differentiation and related 56 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS expression of signaling molecules and proteases. Growth factors of the TGF- b family play a pivotal role in the regulation of bone matrix synthesis and degradation, as well as bone morphogenic proteins (BMPs), crucial for the formation of new, and the remodeling of the existing bone. The complexity of the process is troublesome. The identification of cell types and their function is complicated by differentiation events, and the interpretation of obtained in vivo results, aggravated by simultaneously occurring processes like angiogenesis and hematopoiesis267. The peptidases for bone remodeling are mediated by osteoblasts, which are the major source of bone collagenase, as has been shown by biochemical and immunohistochemical studies. Osteotrophic hormones such as parathyroid hormone (PTH) stimulate osteoblasts to produce procollagenase, PA, and TIMP and TGF-b318. TGF-b might stimulate the osteoblasts to produce type I collagen and fibronectin, and the parallel secretion of TIMP may prevent the collagenase mediatated degradation of newly synthesized matrix components267. PTH, which also stimulates PA accumulation in cultured osteoblasts319, may provide the means of activating collagenase in the resorption of bone. TGF-b and PA, may therefore also mediate the release of procollagenase from mineralized bone matrix and its conversion to an active form. Some of the TGF-b may remain latent and subsequently be released or incorporated in the remodeling process. The remodeling is induced by osteoclasts, they locally acidify the environment and secrete enzymes, TGF-b and PA. Thus, osteoblasts might be potentially stimulated by TGF-b released either from osteoclasts, or from the degraded bone matrix, and consequently release collagenases. The released collagenases can be activated by osteoclast derived PA, as a consequence bone resorption and synthesis a coupled in a feedback loop. 57 Role of Proteases 1.7.5.3 Neurogenesis In neurogenesis, neurite protrusion happens in a back-and-forth motion, making temporal contacts with ECM components. A similar process is observed in tumor cell invasion, where peptidases are involved, suggesting, that peptidases are most likely of general importance for the neurite outgrowth as well. This is corroborated by the fact, that approximately 50% of the neurons in the dorsal root ganglion secrete PA, with increased concentration at the growth cones320. The peptidase activity in the growth cone has been shown to be mediated by calcium ions321. Furthermore, the plasmin activated by PAs induces peptidase (MMPs, serinepeptidases) mediated degradation of the growth cone surrounding tissues. The dependence of neurite growth to ECM adhesion is supposed to be dependent on serine protease-like inhibitors, that are secreated by glial cells. The so called glia-derived nexin inactivates PA and thrombin322,323, which could stabilize the interaction of the growth cone with the ECM, resulting in an increasing neurite length. 1.7.5.4 Muscle injury In muscle injury, neutrophils and their secreted proteases are involved in the degradation, of debris and peptides. Their key function is phagocytosis and degradation of proteins, debris and foreign organisms, such as bacteria. This happens through the activation of peptidases and other antibiotic molecules, as well as the generation of toxic oxygen radicals. The neutrophil invasion is related to muscle usage324,325, the neutrophils are supposed to be phagocytic326, helping to degrade cellular debris, and other muscle damage related proteins327. Besides excessive use and injury, inflammation or 64 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS Table 1: Zymography used for clinical diagnostics and related zymogenic substrates. 65 Role of Proteases HYPOTHESIS 69 Hypothesis 2 Hypothesis A. The DNA recombinant technique enables total control over the design of ELRs, further it allows for the insertion of any kind of peptidic sequence. It is hypothesized that the use of a spatial arrangement of protein sequences will allow for the enzymatic degradation of these ELRs. Additionally, it is hypothesized, that the capability of degradation is possible to be detected by a zymographic method using ELRs as a novel substrate. B. Using ELR as a substrate for zymographic methods enables the opportunity to present only a short selective region to the enzymes. Thus it is hypothesized, that using ELRs for zymographic techniques allows for the design of a new method with potential for high throughput applications for protease detection. C. Cholesterol modified materials have been shown to exhibit strong intermolecular interactions. It is hypothesized, that these interactions can be translated into an ELR system to generate intermolecular forces and to trigger self-assembly of ELRs. D. Cholesterol rich ELRs are further hypothesized to enhance the interaction of ELRs with certain cell types involved in lipid uptake. This mechanism is hypothesized to assist in the coating of living cells with ELR proteins. MATERIAL AND METHODS 73 Materials & Methods 3 Materials & Methods 3.1 Materials 3.1.1 Chemical Reagents. All reagents employed in this work are listed in Table 2. Table 2: Reagents employed and corresponding suppliers. Reactive and Abbreviation Supplier 4-Dimethylaminopyridine (DMAP) Sigma Aldrich. Acetic acid Merck. Acetone Sigma Aldrich. Acrylamide/Bis-acrylamide Amresco. Agarose seakem. Cambrex. Alamar Blue Thermofisher Ammonium persulphate (APS). Sigma Aldrich. Ampicilin Apollo Scientific. Bromophenol blue Sigma Aldrich Calcium chloride (CaCl2) Sigma Aldrich. Chloridric acid Merck. Coomassie Brillant-blue R-250 Sigma Aldrich Diethyl ether Scharlau. Dimethyl sulfoxide (DMSO) Carlo Erba Disodium hydrogen phosphate (Na2HPO4) Fluka Ethanol Merck Ethylenediaminetetraacetic acid(EDTA) Sigma Aldrich Gelatin from porcine skin Sigma Aldrich Glicerol. Merck Isopropanol Invitrogen. Methanol. Sigma Aldrich. Methacrylic anhydride Sigma Aldrich N,N'-Dicyclohexylcarbodiimide (DCC) Sigma Aldrich. N,N-Dimethylformamide (DMF). Sigma Aldrich. 80 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS During the performance of the work presented on this thesis different buffers were employed: PBS (pH=7,4): 5mM Na2HPO4 pH=7,4, NaCl 140 mM TAE: 40 mM Tris-acetate, 1mM pH=8 EDTA TE (sonication buffer): 10 mM pH 8 Tris-base, 1 mM pH=8 EDTA, 1mM PMSF. TBS (washing buffer) :20 mM pH 8 Tris-base, 140 mM NaCl. Running buffer: Tris-base 25 mM pH=8,3, glicina 192 mM y SDS 0,1% (w/v). DNA loading buffer: 30% (v/v) glycerol, 0.1% (w/v) SDS, 0.05% (w/v) bromophenol blue (BPB), 50mM Tris pH 8, 0.05mM EDTA Protein loading buffer: Tris 1MpH 6.5 312.5 mM, SDS 10%(w/v), Glycerol ( v/v), β-Mercaptoethanol 25%(v/v), bromophenol blue (BPB) 2% (v/v). All the solutions were prepared using ultrapure ultrapure water (Millipore) 3.1.8 Elastin-like recombinamers All the elastin-like recombinamers (ELRs) employed in the development of this work have been synthesized in our laboratory (Grupo Bioforge) by DNA recombinant techniques. Those recombinamers listed in bold in . Table 6 were specifically designed and produced for the realization of this thesis. They were produced by Escherichia Coli fermentation and purified 81 3.1 Materials taking advantage of both the smart nature and the reversible thermodependent segregation showed by this kind of materials, by inverse transition cycling (ITC). The recombinamers employed, the abbreviation, molecular weight (Mw), and amino acid sequence are shown in . Table 6. Table 6: Composition and molecular weight of the ELbcRs and ELRs employed. Abbreviation Amino-acid sequence M W (Da) VKV MESLLP (((VPGVG) 2 VPGKG (VPGVG)2)24 V IK (ELR-Control) MESLLP (((VPGIG) 2 VPGKG (VPGIG)2)24 V 51969 IKHEX (ELR- Ela1) MESLLP (((VPGIG) 2 VPGKG (VPGIG)2)2 (VGVAPG)3)10 V 57856 PIT1(ELR-Ela1+FN) MESLLP ((VPGIG) 2 VPGKG (VPGIG) 2 EEIQIGHIPREDVDYHLYP (VPGIG)2 VPGKG (VPGIG)2 (VGVAPG)3)10V 80911 PIT2 (ELR-FN) MESLLP ((VPGIG) 2 VPGKG (VPGIG) 2 EEIQIGHIPREDVDYHLYP (VPGIG)2 VPGKG (VPGIG)2)10V 66494 82 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS 3.1.9 Cell lines and culture conditions Human umbilical vein endothelial cells (HUVECs, cc-2517) were purchased from Lonza (Lonza, Europe). Human aortic smooth muscle cells (HASMC, ref. CRL-1999) were purchased from the American Type Culture Collection (ATCC, USA). 3.1.9.1 Culture Media and Buffers Dulbecco’s Modified Eagle Medium, Medium 231, and smooth muscle growth supplement (SGMS) were purchased from Thermo Fisher Scientific, fetal bovine serum (FBS), penicillin streptomycin solution(P/S), trypsin- EDTA, DPBS, and Alamar Blue® were supplied by Invitrogen (USA), endothelial growth medium (EGM-2) was purchased from Lonza (Lonza, Europe). 83 Methods 3.2 Methods 3.2.1 DNA agarose gel electrophoresis DNA agarose gel electrophoresis is used to separate and check the appearance and size of DNA fragments form either a plasmid or from enzymatic digestion with endonucleases. Different concentrations (in 1x TAE), are applied according to the sizes of the DNA fragments and the kind of gel, analytical or preparative. The agarose is completely dissolved in hot water (70 – 90°C). After cooling down to 60°C the gel is casted in a horizontal tray with the desired comb. 0.20 volumes of 5x loading buffer are added to the samples. A fixed voltage, between 2 and 7 V/cm – according to each sample, is then applied. The electrophoresis is run having as references the color markers (Table 7). Last, the gel is stained for 10 to 30 minutes in a 1x GelRed solution, and the DNA bands are visualized by exposition to UV light in a Viber Lourmat, TFX-20M transilluminator. Table 7: Relation of linear DNA migration with the bromophenol blue (BFB). TAE 1x –BPB % Agarose 2900 0.30 1650 0.50 1000 0.75 500 1 370 1.25 200 1.75 150 2 84 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS 3.2.2 Plasmid purification The plasmids employed in this work were purified using “Quantum Prep Plasmid Mini, Midi o Maxiprep Kit” (Biorad) following the manufacturer’s instructions. DNA was eluted with ultrapure water. For applications were higher DNA concentration is required only half of the recommended elution volume is used and the elution water is used at 65°C. 3.2.3 DNA digestion with restriction enzymes Reaction conditions (temperature, concentration, time of reaction, buffer) for the digestion are supplied by the enzyme manufacturer. The rate of digestion is followed by DNA agarose gel electrophoresis 3.2.4 DNA dephosphorylation Dephosphorylation reaction conditions (temperature, time of reaction, buffer) are supplied by the phosphatase manufacturer. For the p7 expression vector two different consecutive phosphatases were used and the incubation time was extended to one hour. 3.2.5 DNA separation and extraction from agarose gel The target DNA band is first separated and visualized in an agarose gel of an appropriated concentration and stained with RedGel, secondly, the band 85 Methods is extracted from the gel with the help of a scalpel. The minimum quantity of agarose should be cut during band extraction. The purification of the fragment is carried out using the “QIAquick Gel Extraction Kit Protocol” (Qiagen), following the protocol indicated by the manufacturer. 3.2.6 Ligation of genes The reaction ligation is carried out in a final volume of 10-12µL by mixing the insert with the vector, in a molar relation from 1:1 to 5:1, and T4 DNA ligase as an enzyme with its corresponding buffer following the specifications indicated by the supplier. The reaction is conducted for 1 hour at room temperature or for 24 hours at 4°C. 3.2.7 Cloning on the pDrive/ p7 vector The ligation reaction is interrupted by the inactivation of the T4 DNA ligase by incubation for 10 minutes at 70°C. Once the ligation reaction is concluded, a certain quantity of it is used to transform competent cell as specified below. 3.2.8 Transformation of competent cells 3.2.8.1 Transformation of XL1 blue subcloning grade competent cells 86 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS This bacterial strain has an efficiency of ≥ 106 transformants per microgram of DNA. Plasmid DNA to be amplified by cloning is transformed in this bacterial strain following the protocol specified by the supplier. 3.2.8.2 Transformation of XL1 blue competent cells This bacterial strain has an efficiency of ≥ 108 transformants per microgram of DNA. Ligation products were transformed into this bacterial strain following the protocol specified by the supplier. 3.2.8.3 Transformation of BLR (DE3) competent cells This bacterial strain is transformed with the expression plasmid p7 following the method TSS reagent (“Transformation and Storage Solution”) A single colony, isolated and grown in an LB-agar plate, is used to inoculate 10mL of LB medium and is grown at 37ºC with shaking (250rpm), until reaching an OD600=0.3-0.4. At this point the metabolism and cell growth are stopped by incubation on ice for 5 minutes. The cell suspension is centrifuged at 3000rpm (1100Gx) for 10minutes at 4ºC. The supernatant is discarded and the pellet is re-suspended in 1mL of cold x1TSS solution. About 1-10ng of the plasmid in a final volume of 1-10µL is added to the mix. The cellular suspension plus the plasmidic DNA is kept on ice for one hour followed by a heat shock, exposition at 42ºC for 2 minutes. The heat shock is stopped by immersion on ice for 2 minutes. 1mL of warm LB is added and the suspension is incubated one hour at 37ºC with shaking (250rpm).Finally, 87 Methods 50-200µL of the transformation mix is plated in LB-agar plus the antibiotic plates that are incubated for 16-20 hours at 37ºC. 3.2.9 Glycerol stock preparation Positive clones, which are of special interest, are stored in glycerol stocks. The selected colonies were grown at 37°C with shacking (250rpm.) on LB or LB with 0.5% of glucose (for the expression strains), until reaching an OD600= 0.6-0.8. At this point 0.1 volumes of 80% sterile glycerol are added and the cells are kept at -80°C. 3.2.10 Biopolymers expression and purification Once the nucleotide sequences that encode for the whole biopolymers have been created, it must be expressed. To this end, the nucleotide sequence was transferred from the corresponding cloning vector to the specialised expression vector using conventional restriction enzymes. The expression vector p7 has been employed. p7 has been obtained in our laboratory by mutagenesis of pET25b (+) by Dr. Alessandra Girotti. The final constructions were transformed on the bacterial strain BLR (DE3) following the above mention protocol. ELRs expression starts inoculating the desired colony in liquid LB medium plus antibiotic at 37 ° C with orbital shaking (250 rpm.) for approximately 6 hours. This culture is used as inoculum for a fresh TB medium (plus antibiotic), in a volume ratio of 1:500, not exceeding 25% of the capacity of the Erlenmeyer used. 88 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS Expression of the recombinant polypeptides was initiated by inoculating an isolated colony containing the corresponding recombinant vector in liquid LB medium with the eventual resistance antibiotic for the strain and the acquired resistance antibiotic with the expression vector and 1% glucose. The bacterial culture was incubated at 37ºC, with orbital shaking at 250 rpm, for approximately 11 hours, then this culture broth was used to inoculate a fresh medium in a 1/30 ratio, preferably a 100-fold larger volume of TB medium with the same antibiotic and a concentration of 0.8% (v/v) glycerol, 0.2% (w/v) alpha-lactose and 0.05% (w/v) glucose. The mean volume in the Erlenmeyers did not exceed 20-25% of their capacity in order to ensure good oxygenation of the culture, and bacterial growth was continued under the same conditions until an optical density of around 0.6 at 600 nm had been achieved. At that point expression of the recombinant biopolymer was induced by adding isopropyl -beta-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, and the resulting culture was incubated at the appropriate temperature for the time required for each experiment. Once induction was complete, further growth and metabolism of the bacteria were inhibited by cooling to 4ºC. The cells were subsequently isolated by centrifuging for 10 min at 5000g and 4ºC, and washed twice with 100 mL/L of culture in Tris buffered saline (TBS; Tris-base 20 mM, NaCl 150 mM pH 8), the bacteria resuspended by vigorous stirring and/or pipetting and the mixture centrifuged again for 10 min at 5000g and 4ºC. The supernatant was then decanted and the sediment resuspended in 25 mL/L of culture with TE solution (Tris-base 20 mM, EDTA 1 mM pH 8) by vigorous stirring and/or pipetting. The resulting mixture was maintained at 4ºC and 10 Pg/mL of the protease inhibitor phenylmethylsulfonyl fluoride (PMSF) added. 89 Methods The bacteria were lysed by sonication in a Sonicator 3000 apparatus (Misonix, New York) for 6 cycles of 3.5 minutes, each consisting of pulses of 2 seconds every 5 seconds at a power of around 100 W. The sample was maintained on ice throughout this process in order to prevent heat-induced denaturation and precipitation of the proteins. Finally, it was centrifuged for 60 minutes at 15,000g and 4ºC, with the resulting supernatant containing the total soluble fraction and the sediment the total insoluble fraction. The total soluble fraction of the bacteria was acidified to pH 3.5 with hydrochloric acid diluted in water, maintaining the sample on ice and stirring. The resulting precipitate (mainly acid proteins and DNA) was then removed by centrifugation for 20 minutes at 15,000g and 4ºC. Depending on the polymer concerned, the saline concentration and pH of the supernatant were adjusted. The biopolymers were purified by taking advantage of the smart nature of ELRs and their inverse transition. Purification of the recombinant biopolymers from the soluble fraction of E. coli involves successive heating/precipitation and cooling/resuspension stages. Selective precipitation was performed by heating the sample to 70 ºC for two hours. The precipitate was then separated by centrifuging for 20 minutes at 15,000g and 40 ºC, and solubilized in 2 mL/L of ultrapure water at 4 ºC whilst stirring for 12 hours. Each purification stage was checked by polyacrylamide electrophoresis in the presence of SDS. Once the polymer was considered sufficiently pure, it was dialysed against ultrapure water and lyophilized. After the final solubilisation, the dissolved polymer was dialysed against ultrapure water at 4 ºC, then lyophilised and stored at -20 ºC until use. 3.2.11 SDS Polyacrylamide gel electrophoresis 96 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS 3.2.17 Critical Micelle Concentration The critical micelle concentration (CMC) of the different ELR solutions in Milli-Q water was determined from surface tension measurements derived from a drop shape analysis using the pendant drop technique390. The changes in the shape of the resulting drop at the air/water interface upon increasing the ELR concentration previously stabilized at 5.0, 15, or 35°C, for 5 min, from a blank solution of Milli-Q water to 50 μM ELR, were monitored using the SCA 20 software of the Data Physics OCA20 instrument, which outlined and scaled the profile hanging from a straight precision dosing needle. Drops (4 μL at 0.5 μL/sec) were infused using a 500 μL Gastight® Hamilton syringe. The pendant drop image formed at the tip of the needle was captured and analyzed digitally. Five drops per condition were measured and multiple comparisons between groups were performed using a two-way ANOVA (**** p<0.0001; **p<0.001). 3.2.18 Dynamic light scattering The average size distribution of the modified ELRs was determined by DLS using a Zetasizer Nano apparatus (Malvern Instruments, Worcestershire, UK), configured with a 173° scattering angle and equipped with a HeNe laser (633 nm) with an output power of 10 mW. Solutions (15 µM) of the ELRCTA modifications were dissolved in ultrapure water, and filtered (0.45μm PVDF syringe filter) prior to analysis. Samples were incubated for 10 minutes at 5, 10, 15, 20, 25, 30 or 35°C prior to measurement. Each sample was measured in triplicate and multiple comparisons between groups were performed using a one-way ANOVA (**** p<0.0001) with respect to the 97 Methods high polydispersity of the samples (0.15-0.5). Plotted values correspond to mean volume intensities. 3.2.19 Zeta potential The particle velocity distribution (electrophoretic-mobility) was determined by Laser Doppler Velocimetry and the zeta potential (ZP) was calculated for freshly prepared and filtered samples using a Zetasizer Nano Series (Malvern, UK). Default settings on the Zetasizer were used, in other words the dielectric constant, refractive index and viscosity were assumed to be the same as for water, and the Smoluchowski approximation was used. Determinations were carried out in a temperature range from 10 to 35°C by performing 10 readings. 3.2.20 Circular Dichroism Circular dichroism (CD) spectra were recorded using a Jasco J-1500 spectrometer in a stream of nitrogen in the range 190-250 nm. The temperature of each sample was varied from 5 to 35°C in steps of 5°C. The sample was equilibrated at 15°C for 10 min prior to each different temperature measurement and kept for 2 minutes at the target temperature. Sampling was carried out at points every 1 nm with a total of 15 accumulations per measurement. Samples were dissolved in ultrapure water at 3.3 µM and measured in a quartz cuvette (d=0.1 cm). Data were smoothed using a 15pt Savitky-Golay filter. Deconvolution of the substructure was performed on smoothed curves using the BeStSel web server391 to yield eight spectral components: helix (regular and distorted), irregular helix, anti-parallel (left-handed, relaxed and right-handed), parallel, 98 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS turns, and others. The boundaries for parallel and anti-parallel strand arrangements were set to 0° and 23°, respectively. 3.2.21 Viscosity measurements Samples were dissolved to a concentration of 0.75 mM at a temperature of 15°C and measured with an AR2000 (TA Instruments). Data were recorded using TRIOS software ( v4.1.1.33073). Samples were normalized between measurements by equilibration at 15°C for at least 5 minutes before measurement. Viscosity measurements were performed in triplicate at 5, 15, 25, and 35°C. Samples were conditioned with a pre-shear of 0.1 rad/s for 60 s, followed by a flow ramp from 1 to 1000 s-1. Samples at 35°C were only measured up to 300 s-1 to avoid disruption of the sample above the LCST. 3.2.22 Rheological determination of low-temperature gelation Samples were dissolved to a concentration of 0.75 mM at a temperature of 15°C and oscillatory shear measurements were performed using constant stress and an AR2000 apparatus (TA Instruments). Data were recorded using TRIOS software (v4.1.1.33073). A cooling and heating cycle was performed, cooling from RT to 2°C, followed by a heating step to 50°C above the LCST. The evolution of the storage and loss moduli was recorded. 99 Methods 3.2.23 Contact Angle Contact angle measurements were performed using solvent films cast from 0.75 mM aqueous solution after drying at 40°C overnight and incubation at the specified temperature for 5 minutes on a Peltier plate prior to CA measurement. For each condition, 5 drops of ultrapure water were analyzed using a Data Physics OCA20 system instrument. The drop profile images for the advancing and receding drops during micro-syringe dispensation were recorded using an adapted CCD video camera. The three-phase contact line of the liquid drop was made to advance or retreat by adding or withdrawing the liquid and the advancing (θa) and receding (θr) contact angles were measured in the stable phase of advance or recoil on both sides of the drop. 3.2.24 Transmission electron microscopy TEM Samples were prepared by placing a drop of the solution on a carbon-coated copper grid and, when needed, stained with uranyl acetate solution (1.0 wt %), followed by water evaporation. Samples were incubated for 1, 3, 7 and 28 days at a concentration of 125 µM at 4 or 37°C. C300Cu Carbon coated TEM grids were loaded with the sample by incubation for 90 s, followed by staining with uranyl acetate, as required. Images were taken using a Tecnai Thermoionic T20 microscope operated at 200kV. 100 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS 3.2.25 Scanning electron microscopy SEM Samples were incubated for 1, 3, 7 and 28 days at a concentration of 0.25 or 1.25 mM and 4 or 37°C. At specific time-points, samples were cryofractured in liquid N2 and lyophilized. Dried samples were mounted and coated with a 20 nm layer of carbon using the Leica EM ACE200 vacuum coater. Imaging of the ELR substrates was performed using an FEI Quanta 200 FEG instrument in low vacuum mode with a carbon coating. SEM images were employed to determine substrate characteristics and thickness as well as to check surface homogeneity. SEM captures were analyzed using the ImageJ software. 3.2.26 Spectrophotometric cloud point determination The absorbance of the ELR solutions was recorded using a Cary Series UV- Vis spectrophotometer (Agilent Technologies, USA). The ELR solutions were prepared in PBS (125 µM) and recorded using the CaryWinUV Thermal Application (version 4.20(468) over a temperature interval ranging from 5 to 40 ºC at a heating rate of 1ºC/min. 3.2.27 Cell Culture HUVECs were cultured in EGM-2 supplemented with 1% P/S and HASMCs were cultured in Medium 231 supplemented with SGMS and 1% P/S. Cells were incubated at 37 °C under 5% CO2 and their medium was replaced every two days. 101 Methods The relative number of metabolically active cells was evaluated using the Alamar Blue® assay according to the manufacturer’s guidelines. The culture was incubated in a 10% Alamar Blue solution for 3 h at 37 °C and under a 5% CO2 atmosphere. Cells were cultured in triplicate with medium containing 375 nM, or 750 nM of the non-fluorescent substrates, for 1, 3 and 7 days respectively. The datasets were normalized to cells of the same cell type and at the same time point grown on TCPS. A two-way ANOVA multiple Comparison was performed using the Holm-Sidak multiple comparison test. ****: p< 0.0001; ***: p < = 0.001; **: p< =0.01; *: p<0.05. 3.2.28 FACS and Confocal Microscopy A total of 250,000 cultured cells per condition were incubated for 24 h. Followed by incubation in serum-free media containing ELRCTA (375 nM) for 18h. After incubation, cells were washed several times with cold DPBS to remove unbound ELRs, then trypsinized and resuspended in cold DPBS. Flow cytometry analysis was performed to assess the amount of ELRCTA- positive cells. A total of 50,000 events were detected in the cytometer (Gallios flow cytometer, Beckman-Coulter). Quantification was performed using Kaluza software v1.2 (Beckman-Coulter), and for better quantification the fluorescent channel was presented on a logical scale with a linear region from -2 to 15. Samples used for FACS quantification were visualized using a Leica SP5 confocal microscope (Leica Microsystems, Heidelberg) with a 63x lens. For better contrast in the merged image, the red channel was displayed in magenta. Z-slicing depth on selected cells was set to 0.4 µm. 102 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS Cells prepared for FACS were cultured in 96-well plates to track ELRCTA in living cells. Thus, 3000 cells incubated with ELRCTA were imaged for 18 h at 0.56 mfps. Bright-field and fluorescence microscopy were performed using a NIKON Eclipse Ti fluorescence microscope equipped with a digital camera system (Digital sight DS-2MBWc) (Nikon, Japan). 103 Methods RESULTS AND DISCUSSION 112 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS (pUC57) containing the sequences encoding the IKHEX domain was provided by GenScript. Starting from the amino acid sequence of interest, the DNA sequence is translated by using the expression codons of the corresponding amino acids. The sequence is than reduced to a fragment ~200 bp, further restriction sites for the restriction enzymes EcoRI and EarI are include to the front and the end of the sequence, enabling the extraction of the sequence of interest from the vector. For IKHEX this resulted in a final sequence of 241 bp (sequence of interest plus restriction sites), with a total length of 204 bp, which was provided by GenScript embedded in the plasmid cloning vector pUC57. Figure 15: DNA sequence of the 204 base pairs (white region) and the corresponding amino acid sequence. For the multiplication of the sequence, the sequence was introduced into a cloning vector (pDrive), which can be opened by SapI and EarI digestion. EarI digestion openes the vector before and after the sequence of interest, to remove it from the vector and SapI digestion opens the vector behind 113 CHAPTER 1: Development of a novel screening method for proteases based on proteolytically degradable ELR-acrylamide hydrogels the sequence of interest. The introduction of the sequence into the vector is obtained by ligation of the open pDrive together with the Ear1 digested sequence of interest using T4 ligase. For the introduction of consecutive sequence blocks, ligation of a SapI digested pDrive containing the 204 bp sequence of interest is performed together with the EarI digested sequence of interest. Increasing the concentration of the insert against the concentration of the vector leads to the introduction of multiple fragments into the vector. This resulted in sequences of the length (204)x+1 base pairs, where x is the number of inserted sequences. This process of ligation was repeated until the final sequence of (204)10 base pairs was obtained. This sequence was then removed from the cloning vector by EarI digestion and transferred into an expression vector. Due to the design of the expression vector for a better expression of the protein of interest, the sequence MESSLP is added before the sequence of interest, and due to the use of EarI digestion a V (grey shadows in Figure 15) is added to the end of the sequence of interest resulting in a final sequence of 2061 base pairs. 114 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS Figure 16: Agarose gels of the insert IkHex of 204 basepairs. Left gel corresponds to the unimer, as received from the supplier after Ear1 digestion. Right gel corresponds to the decamer (204)10, obtained after several steps of ligation of the fragment. The sequences at each step were validated using automated dideoxy DNA sequencing with fluorescent ddNTP and agarose gel electrophoresis. Once the desired constructions were obtained and their sequences controlled their released inserts after enzymatic digestion with EarI and DpnI, which is used in order to avoid the appearance of fragments with similar size to the segment of interest, were subcloned in the modified pET 25b+ vector pET 7 expression vector. These resulting vectors were used to transform E. coli expression strains as described in paragraph 1.2.3. 115 CHAPTER 1: Development of a novel screening method for proteases based on proteolytically degradable ELR-acrylamide hydrogels ELRs expression and purification The final gene constructions were transformed on the bacterial strain BLR (DE3) following the above mention protocol. The expression of the recombinamers was made as previously described The ELRs purification protocol was made based on the ITT (refer to paragraph 1.2.4) consisting on three sequentially rounds of inverse transition cycling, which rendered highly pure and monodisperse polymers, as corroborated by SDS-PAGE. Figure 17: SDS-PAGE of ELRs to determine molecular weights. M= marker, 1 = ELR-Control, 2= ELR-Ela, 3=ELR-FN, 4=ELR-Ela+FN The electrophoretic pattern showed an excellent level of purity for all the ELRs with little degradational products in ELR-ELA1 and ELR-FN, which are due to the proteolytically sensitive nature of these ELRs. No contaminant proteins form E.coli were found. In order to confirm the recombinamer’s molecular weight and amino acid composition, the samples underwent MALDI-TOF and amino acid analysis. 116 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS Table 11: Theoretical and experimental molecularweight of ELRs and the corresponding amino acid sequence, obtained by amino acid quantification. ELR-Ela+FN ELR-FN ELR-Ela ELR-control Mw theo 80911.76 66494.83 57856.45 51969.82 Mw MALDI 80503 66177 57401 51623 Amino acids The o. Exp. The o. Exp. The o. Exp. The o. Exp. Ala (A) 30 31,31 0 0 30 33,15 0 0,00 Arg (R) 10 10,38 10 11,04 0 0,00 0 0,00 Asn (N) 0 0,00 0 0,0 0 0,00 0 0,00 Asp (D) 20 21,23 20 21,21 0 0,00 0 0,00 Cys (C) 0 0,00 0 0,00 0 0,00 0 0,00 Gln (Q) 10 44,03 10 43,79 0 0,00 0 0,00 Glu (E) 31 31 1 1,83 1 1,04 Gly (G) 270 270,43 210 213,54 260 262,68 240 250,86 His (H) 20 16,86 20 16,18 0 0,00 0 0,00 Ile (I) 110 107,51 110 106,87 80 80,25 96 94,52 Leu (L) 12 11,85 12 11,98 2 2,91 2 2,12 Lys (K) 20 17,60 20 16,99 20 15,75 24 19,46 Met (M) 1 0,75 1 0,98 1 0,06 1 0,88 Phe (F) 0 0,00 0 0,00 0 0,00 0 0,00 Pro (P) 151 151,78 121 131,11 131 131,51 121 121,78 Ser (S) 1 0,70 1 0,84 1 0,98 1 0,76 Thr (T) 0 0,00 0 0,00 0 0,00 0 0,00 Trp (W) 0 0,00 0 0,00 0 0,00 0 0,00 Tyr (Y) 20 21,00 20 20,80 0 0,00 0 0,00 Val (V) 171 172,19 111 113,30 161 160,57 121 120,82 Succesful expression of ELRs was performed by pre-expressional sequencation of the plasmid and NMR of the expressed proteins, moreover, the molecular weight of the expressed protein was correlated to theoretical mass by mass spectoscropy and SDS-PAGE (Figure 17). 117 CHAPTER 1: Development of a novel screening method for proteases based on proteolytically degradable ELR-acrylamide hydrogels 4.1.2 Methacrylation of ELRs ELRs have been succesfully modified with methacrylamide groups by modification of 40-55% of lysines present in the protein, as characterized by MALDI-TOF and NMR, results are shown in Table 12. Table 12: Results of characterization of ELR modification with methacrylate groups (MA) by NMR and MALDI-TOF. Spectra are shown below. %age of lysines modified (NMR) %age of lysines modified (MALDI-TOF) ELR-Control 54,5 42,5 ELR-Ela 46,0 54,1 ELR-FN 46,0 36,6 ELR-Ela+FN 45,5 42,5 118 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS Figure 18: Mass spectra obtained by MALDI-TOF. The left column presents the pure ELRs and the right column shows mass after modification with methacrylic anhydride. 119 CHAPTER 1: Development of a novel screening method for proteases based on proteolytically degradable ELR-acrylamide hydrogels Figure 19: NMR spectra of modified and unmodified ELRs used in this study. Methacrylate signals are isolated singuletts at 5.25 and 5.60 ppm. 120 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS 4.1.3 WORKING PRINCIPLE OF THE METHOD The present method uses the advantage of the spatial design of the ELR for the detection of proteases in a zymography-like manner. As a prerequisite, the ELR should consist of at least three domains, a degradable group (peptidase target), a mechanism of detection, and a stable cross-link to the acrylamide matrix. For better integrity with the matrix an additional block, which is not a target to most of the peptidases was implemented, as a spacer, to augment the molecular weight and to minimize sterical hindrance between cross-links and degradable groups. The process of detection is displayed in Figure 20A, in the first step, cross-linking motifs, in this study methacrylate groups, are added to a sufficient number of free lysins in the stainable region, and cross-linked to the substrate (acrylamide gel). The modification of lysines impairs stainability with coomassie BB as indicated by the grey boxes (Figure 20A), this is crucial since crosslinks will remain in the matrix gel and should not give a false positive signal, on the other hand a sufficient number of lysines has to remain unmodified for detection. In the next step the gel is incubated with an enzyme selective to the proteolytic group and stained after washing-off the degraded parts. This approach allows for calibration and quantification of protease activity. 121 CHAPTER 1: Development of a novel screening method for proteases based on proteolytically degradable ELR-acrylamide hydrogels Figure 20: Design of recombinant protein for protease detection and mechanism of zymographic detection. The first step is the genetic design of the ELR consisting of non-staining, stainable and functional, and degradable region. The next step is the introduction of cross-linking features on freelysins, which allow for cross-linking to the substrate and disable Coomassie BB detection of modified lysins. The ELR-substrate complex is than incubated with proteolytic enzymes and cleaved parts are washed off and the remaining gel is stained with Coomassie BB, and the efficiency of cleavage towards the presented group is monitored. 224 ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS Pluripotent Stem Cells. 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VI ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS 235,00 - 0,74 - 0,41 - 0,77 - 0,46 - 0,80 - 0,51 - 0,81 - 0,56 - 0,74 - 0,56 - 0,76 - 0,59 - 0,79 - 0,61 236,00 - 0,68 - 0,27 - 0,71 - 0,32 - 0,74 - 0,36 - 0,74 - 0,41 - 0,67 - 0,41 - 0,69 - 0,43 - 0,72 - 0,46 237,00 - 0,62 - 0,22 - 0,65 - 0,26 - 0,68 - 0,30 - 0,68 - 0,34 - 0,60 - 0,34 - 0,62 - 0,36 - 0,65 - 0,38 238,00 - 0,57 - 0,17 - 0,60 - 0,22 - 0,61 - 0,26 - 0,62 - 0,31 - 0,53 - 0,33 - 0,55 - 0,35 - 0,58 - 0,37 239,00 - 0,51 - 0,12 - 0,55 - 0,16 - 0,56 - 0,19 - 0,56 - 0,24 - 0,47 - 0,27 - 0,49 - 0,29 - 0,52 - 0,31 240,00 - 0,47 - 0,09 - 0,49 - 0,12 - 0,50 - 0,15 - 0,51 - 0,19 - 0,42 - 0,21 - 0,43 - 0,23 - 0,46 - 0,25 241,00 - 0,43 - 0,04 - 0,45 - 0,07 - 0,44 - 0,10 - 0,47 - 0,13 - 0,36 - 0,15 - 0,38 - 0,17 - 0,40 - 0,18 242,00 - 0,39 0,02 - 0,41 - 0,01 - 0,42 - 0,03 - 0,42 - 0,06 - 0,32 - 0,09 - 0,33 - 0,10 - 0,35 - 0,11 243,00 - 0,35 0,00 - 0,37 - 0,02 - 0,37 - 0,04 - 0,38 - 0,07 - 0,27 - 0,08 - 0,28 - 0,09 - 0,30 - 0,10 244,00 - 0,33 0,00 - 0,34 - 0,02 - 0,34 - 0,04 - 0,35 - 0,06 - 0,24 - 0,07 - 0,24 - 0,08 - 0,26 - 0,08 245,00 - 0,30 0,01 - 0,32 - 0,01 - 0,30 - 0,02 - 0,32 - 0,04 - 0,21 - 0,04 - 0,20 - 0,05 - 0,22 - 0,05 246,00 - 0,28 0,02 - 0,29 0,01 - 0,29 0,00 - 0,29 - 0,02 - 0,19 - 0,02 - 0,18 - 0,02 - 0,19 - 0,03 247,00 - 0,27 0,02 - 0,27 0,01 - 0,28 0,00 - 0,27 - 0,02 - 0,16 - 0,02 - 0,16 - 0,03 - 0,16 - 0,03 248,00 - 0,26 0,06 - 0,26 0,05 - 0,24 0,04 - 0,26 0,02 - 0,14 0,01 - 0,14 0,00 - 0,15 0,00 249,00 - 0,24 0,09 - 0,26 0,09 - 0,26 0,08 - 0,24 0,06 - 0,13 0,05 - 0,12 0,04 - 0,13 0,03 250,00 - 0,23 0,10 - 0,25 0,09 - 0,24 0,09 - 0,23 0,08 - 0,12 0,06 - 0,11 0,06 - 0,12 0,05 Temp. (°C) CTA1 Temp. (°C) 5 10 15 20 25 30 35 Waveln. (nm) Exp . Fit Exp . Fit Exp . Fit Exp . Fit Exp . Fit Exp . Fit Exp . Fit 190,00 - 2,91 - 2,61 - 2,66 - 2,42 - 2,39 - 2,12 - 2,13 - 1,95 - 1,36 - 1,26 - 1,17 - 1,05 - 1,04 - 0,92 191,00 - 3,20 - 3,06 - 2,96 - 2,84 - 2,49 - 2,45 - 2,42 - 2,28 - 1,49 - 1,44 - 1,23 - 1,18 - 1,06 - 1,02 192,00 - 3,54 - 3,52 - 3,27 - 3,24 - 2,86 - 2,81 - 2,63 - 2,60 - 1,68 - 1,66 - 1,33 - 1,37 - 1,17 - 1,18 193,00 - 3,77 - 3,95 - 3,54 - 3,62 - 3,02 - 3,15 - 2,80 - 2,92 - 1,95 - 1,89 - 1,53 - 1,58 - 1,37 - 1,37 194,00 - 4,10 - 4,29 - 3,65 - 3,92 - 3,26 - 3,44 - 3,02 - 3,18 - 2,00 - 2,12 - 1,76 - 1,79 - 1,38 - 1,56 195,00 - 4,26 - 4,44 - 3,99 - 4,03 - 3,44 - 3,56 - 3,23 - 3,28 - 2,03 - 2,21 - 1,74 - 1,87 - 1,55 - 1,63 196,00 - 4,51 - 4,45 - 3,99 - 4,03 - 3,54 - 3,57 - 3,19 - 3,28 - 2,21 - 2,22 - 1,87 - 1,89 - 1,69 - 1,63 197,00 - 4,51 - 4,41 - 3,94 - 3,98 - 3,67 - 3,54 - 3,39 - 3,26 - 2,26 - 2,24 - 1,92 - 1,90 - 1,65 - 1,64 198,00 - 4,41 - 4,29 - 4,14 - 3,88 - 3,57 - 3,43 - 3,25 - 3,15 - 2,31 - 2,15 - 1,97 - 1,80 - 1,64 - 1,54 199,00 - 4,39 - 4,18 - 3,98 - 3,78 - 3,47 - 3,34 - 3,22 - 3,09 - 2,22 - 2,12 - 1,88 - 1,77 - 1,65 - 1,52 200,00 - 4,08 - 4,06 - 3,64 - 3,67 - 3,30 - 3,24 - 3,11 - 3,02 - 2,17 - 2,11 - 1,87 - 1,75 - 1,60 - 1,52 201,00 - 3,79 - 3,71 - 3,48 - 3,34 - 3,02 - 2,94 - 2,79 - 2,74 - 2,01 - 1,90 - 1,58 - 1,57 - 1,47 - 1,35 202,00 - 3,47 - 3,43 - 3,09 - 3,10 - 2,76 - 2,72 - 2,61 - 2,55 - 1,85 - 1,80 - 1,46 - 1,48 - 1,30 - 1,29 203,00 - 3,16 - 3,15 - 2,82 - 2,85 - 2,50 - 2,51 - 2,39 - 2,37 - 1,64 - 1,69 - 1,44 - 1,40 - 1,22 - 1,22 204,00 - 2,83 - 2,82 - 2,59 - 2,56 - 2,23 - 2,26 - 2,16 - 2,13 - 1,57 - 1,53 - 1,26 - 1,28 - 1,13 - 1,12 205,00 - 2,50 - 2,51 - 2,31 - 2,28 - 2,01 - 2,01 - 1,80 - 1,90 - 1,32 - 1,36 - 1,11 - 1,14 - 0,92 - 1,00 206,00 - 2,17 - 2,21 - 1,95 - 2,02 - 1,73 - 1,78 - 1,61 - 1,69 - 1,12 - 1,21 - 0,98 - 1,02 - 0,84 - 0,90 207,00 - 1,92 - 2,01 - 1,71 - 1,84 - 1,53 - 1,63 - 1,47 - 1,55 - 0,98 - 1,09 - 0,80 - 0,91 - 0,73 - 0,81 208,00 - 1,68 - 1,81 - 1,54 - 1,67 - 1,38 - 1,48 - 1,33 - 1,42 - 0,92 - 1,00 - 0,75 - 0,84 - 0,61 - 0,76 209,00 - 1,51 - 1,67 - 1,43 - 1,56 - 1,27 - 1,40 - 1,22 - 1,35 - 0,86 - 0,96 - 0,70 - 0,82 - 0,67 - 0,76 210,00 - 1,40 - 1,54 - 1,32 - 1,48 - 1,25 - 1,36 - 1,18 - 1,34 - 0,82 - 1,00 - 0,68 - 0,87 - 0,66 - 0,83 VII 7.1 Supporting Information 211,00 - 1,29 - 1,42 - 1,29 - 1,37 - 1,17 - 1,28 - 1,14 - 1,27 - 0,85 - 0,96 - 0,72 - 0,85 - 0,69 - 0,82 212,00 - 1,25 - 1,32 - 1,24 - 1,28 - 1,13 - 1,22 - 1,18 - 1,22 - 0,84 - 0,94 - 0,74 - 0,84 - 0,74 - 0,83 213,00 - 1,25 - 1,24 - 1,24 - 1,22 - 1,14 - 1,17 - 1,14 - 1,19 - 0,91 - 0,93 - 0,81 - 0,85 - 0,79 - 0,85 214,00 - 1,25 - 1,24 - 1,16 - 1,22 - 1,18 - 1,19 - 1,21 - 1,23 - 0,92 - 0,99 - 0,87 - 0,92 - 0,87 - 0,93 215,00 - 1,24 - 1,22 - 1,18 - 1,22 - 1,23 - 1,20 - 1,26 - 1,25 - 1,01 - 1,02 - 0,93 - 0,97 - 0,92 - 0,98 216,00 - 1,23 - 1,21 - 1,24 - 1,22 - 1,20 - 1,21 - 1,30 - 1,27 - 1,08 - 1,06 - 1,00 - 1,01 - 1,05 - 1,04 217,00 - 1,25 - 1,22 - 1,30 - 1,24 - 1,29 - 1,23 - 1,32 - 1,30 - 1,12 - 1,09 - 1,11 - 1,06 - 1,13 - 1,09 218,00 - 1,26 - 1,22 - 1,31 - 1,24 - 1,28 - 1,24 - 1,39 - 1,33 - 1,20 - 1,12 - 1,19 - 1,10 - 1,23 - 1,14 219,00 - 1,29 - 1,19 - 1,35 - 1,23 - 1,35 - 1,23 - 1,45 - 1,33 - 1,22 - 1,13 - 1,25 - 1,12 - 1,28 - 1,17 220,00 - 1,31 - 1,22 - 1,34 - 1,26 - 1,32 - 1,26 - 1,47 - 1,38 - 1,26 - 1,18 - 1,28 - 1,18 - 1,36 - 1,23 221,00 - 1,29 - 1,20 - 1,34 - 1,25 - 1,35 - 1,25 - 1,48 - 1,38 - 1,32 - 1,19 - 1,31 - 1,19 - 1,40 - 1,25 222,00 - 1,24 - 1,20 - 1,32 - 1,26 - 1,35 - 1,26 - 1,49 - 1,39 - 1,32 - 1,22 - 1,34 - 1,23 - 1,38 - 1,29 223,00 - 1,25 - 1,21 - 1,32 - 1,28 - 1,34 - 1,28 - 1,50 - 1,43 - 1,35 - 1,26 - 1,36 - 1,28 - 1,43 - 1,35 224,00 - 1,22 - 1,21 - 1,29 - 1,29 - 1,30 - 1,29 - 1,45 - 1,45 - 1,32 - 1,30 - 1,36 - 1,32 - 1,43 - 1,40 225,00 - 1,18 - 1,21 - 1,25 - 1,29 - 1,30 - 1,31 - 1,42 - 1,47 - 1,30 - 1,35 - 1,34 - 1,38 - 1,39 - 1,45 226,00 - 1,13 - 1,21 - 1,22 - 1,30 - 1,21 - 1,32 - 1,37 - 1,48 - 1,29 - 1,38 - 1,30 - 1,41 - 1,38 - 1,49 227,00 - 1,09 - 1,18 - 1,19 - 1,27 - 1,21 - 1,29 - 1,33 - 1,45 - 1,27 - 1,37 - 1,29 - 1,41 - 1,38 - 1,48 228,00 - 1,03 - 1,14 - 1,12 - 1,23 - 1,14 - 1,25 - 1,27 - 1,41 - 1,21 - 1,34 - 1,24 - 1,39 - 1,30 - 1,46 229,00 - 0,99 - 1,05 - 1,06 - 1,13 - 1,08 - 1,16 - 1,22 - 1,31 - 1,14 - 1,27 - 1,20 - 1,31 - 1,24 - 1,38 230,00 - 0,91 - 0,96 - 1,00 - 1,05 - 1,00 - 1,07 - 1,16 - 1,22 - 1,11 - 1,19 - 1,12 - 1,24 - 1,18 - 1,30 231,00 - 0,85 - 0,88 - 0,91 - 0,97 - 0,94 - 1,00 - 1,08 - 1,13 - 1,02 - 1,12 - 1,08 - 1,16 - 1,10 - 1,22 232,00 - 0,78 - 0,77 - 0,85 - 0,85 - 0,87 - 0,88 - 0,99 - 1,00 - 0,96 - 1,00 - 0,99 - 1,04 - 1,04 - 1,09 233,00 - 0,71 - 0,66 - 0,78 - 0,74 - 0,80 - 0,76 - 0,89 - 0,87 - 0,89 - 0,88 - 0,91 - 0,91 - 0,98 - 0,95 234,00 - 0,66 - 0,55 - 0,71 - 0,61 - 0,72 - 0,63 - 0,82 - 0,72 - 0,81 - 0,73 - 0,84 - 0,76 - 0,91 - 0,80 235,00 - 0,58 - 0,43 - 0,63 - 0,49 - 0,66 - 0,51 - 0,74 - 0,58 - 0,74 - 0,59 - 0,77 - 0,61 - 0,79 - 0,64 236,00 - 0,53 - 0,31 - 0,59 - 0,36 - 0,59 - 0,37 - 0,68 - 0,43 - 0,69 - 0,43 - 0,74 - 0,45 - 0,76 - 0,48 237,00 - 0,46 - 0,25 - 0,53 - 0,29 - 0,53 - 0,31 - 0,62 - 0,35 - 0,63 - 0,36 - 0,67 - 0,38 - 0,70 - 0,40 238,00 - 0,44 - 0,25 - 0,49 - 0,29 - 0,49 - 0,30 - 0,53 - 0,34 - 0,57 - 0,36 - 0,59 - 0,37 - 0,62 - 0,39 239,00 - 0,38 - 0,20 - 0,42 - 0,23 - 0,42 - 0,25 - 0,50 - 0,28 - 0,51 - 0,30 - 0,55 - 0,31 - 0,54 - 0,33 240,00 - 0,32 - 0,16 - 0,36 - 0,19 - 0,37 - 0,20 - 0,43 - 0,23 - 0,45 - 0,24 - 0,48 - 0,26 - 0,50 - 0,27 241,00 - 0,26 - 0,11 - 0,31 - 0,13 - 0,35 - 0,14 - 0,39 - 0,17 - 0,39 - 0,18 - 0,41 - 0,19 - 0,42 - 0,20 242,00 - 0,24 - 0,05 - 0,29 - 0,07 - 0,28 - 0,08 - 0,30 - 0,09 - 0,31 - 0,10 - 0,37 - 0,11 - 0,35 - 0,12 243,00 - 0,19 - 0,05 - 0,22 - 0,07 - 0,24 - 0,08 - 0,26 - 0,09 - 0,28 - 0,10 - 0,29 - 0,11 - 0,31 - 0,11 244,00 - 0,18 - 0,04 - 0,19 - 0,06 - 0,21 - 0,06 - 0,25 - 0,07 - 0,23 - 0,08 - 0,26 - 0,09 - 0,26 - 0,09 245,00 - 0,16 - 0,03 - 0,16 - 0,04 - 0,17 - 0,04 - 0,19 - 0,05 - 0,22 - 0,05 - 0,23 - 0,06 - 0,23 - 0,06 246,00 - 0,14 - 0,01 - 0,16 - 0,01 - 0,17 - 0,02 - 0,20 - 0,02 - 0,20 - 0,03 - 0,21 - 0,03 - 0,21 - 0,03 247,00 - 0,12 - 0,01 - 0,13 - 0,01 - 0,13 - 0,02 - 0,17 - 0,03 - 0,16 - 0,04 - 0,19 - 0,04 - 0,19 - 0,04 248,00 - 0,08 0,02 - 0,08 0,02 - 0,13 0,01 - 0,12 0,00 - 0,15 - 0,01 - 0,15 - 0,02 - 0,15 - 0,02 249,00 - 0,10 0,06 - 0,10 0,05 - 0,13 0,04 - 0,15 0,04 - 0,13 0,02 - 0,15 0,02 - 0,15 0,02 250,00 - 0,09 0,07 - 0,09 0,06 - 0,11 0,06 - 0,10 0,06 - 0,08 0,04 - 0,11 0,04 - 0,11 0,04 VIII ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS CTA5 Temp. (°C) 5 10 15 20 25 30 35 Waveln. (nm) Exp . Fit Exp . Fit Exp . Fit Exp . Fit Exp . Fit Exp . Fit Exp . Fit 190,00 - 1,82 - 1,62 - 3,13 - 2,77 - 3,08 - 2,71 - 2,82 - 2,45 - 2,19 - 1,93 - 1,99 - 1,76 - 1,82 - 1,62 191,00 - 1,83 - 1,74 - 3,31 - 3,15 - 3,22 - 3,08 - 2,91 - 2,77 - 2,25 - 2,13 - 2,02 - 1,92 - 1,83 - 1,74 192,00 - 1,88 - 1,88 - 3,48 - 3,51 - 3,40 - 3,42 - 3,03 - 3,06 - 2,34 - 2,34 - 2,09 - 2,09 - 1,88 - 1,88 193,00 - 1,92 - 2,02 - 3,65 - 3,84 - 3,55 - 3,72 - 3,16 - 3,33 - 2,42 - 2,54 - 2,16 - 2,27 - 1,92 - 2,02 194,00 - 1,97 - 2,14 - 3,84 - 4,06 - 3,69 - 3,92 - 3,27 - 3,52 - 2,51 - 2,71 - 2,24 - 2,42 - 1,97 - 2,14 195,00 - 2,01 - 2,12 - 3,94 - 4,09 - 3,78 - 3,92 - 3,38 - 3,52 - 2,59 - 2,73 - 2,30 - 2,42 - 2,01 - 2,12 196,00 - 2,05 - 2,05 - 4,01 - 4,02 - 3,82 - 3,83 - 3,45 - 3,43 - 2,63 - 2,67 - 2,35 - 2,36 - 2,05 - 2,05 197,00 - 2,04 - 2,00 - 4,04 - 3,92 - 3,83 - 3,73 - 3,46 - 3,34 - 2,68 - 2,62 - 2,34 - 2,30 - 2,04 - 2,00 198,00 - 2,02 - 1,87 - 3,97 - 3,78 - 3,79 - 3,60 - 3,40 - 3,21 - 2,67 - 2,49 - 2,31 - 2,16 - 2,02 - 1,87 199,00 - 1,96 - 1,84 - 3,82 - 3,68 - 3,67 - 3,52 - 3,29 - 3,15 - 2,58 - 2,43 - 2,24 - 2,11 - 1,96 - 1,84 200,00 - 1,86 - 1,80 - 3,63 - 3,58 - 3,49 - 3,43 - 3,11 - 3,06 - 2,43 - 2,36 - 2,11 - 2,05 - 1,86 - 1,80 201,00 - 1,74 - 1,60 - 3,38 - 3,26 - 3,26 - 3,12 - 2,89 - 2,78 - 2,23 - 2,11 - 1,95 - 1,82 - 1,74 - 1,60 202,00 - 1,58 - 1,51 - 3,07 - 3,02 - 2,98 - 2,90 - 2,64 - 2,59 - 2,03 - 1,97 - 1,77 - 1,70 - 1,58 - 1,51 203,00 - 1,43 - 1,43 - 2,78 - 2,78 - 2,69 - 2,68 - 2,39 - 2,40 - 1,82 - 1,83 - 1,60 - 1,59 - 1,43 - 1,43 204,00 - 1,27 - 1,31 - 2,49 - 2,50 - 2,40 - 2,42 - 2,14 - 2,17 - 1,63 - 1,66 - 1,41 - 1,44 - 1,27 - 1,31 205,00 - 1,13 - 1,18 - 2,22 - 2,24 - 2,13 - 2,17 - 1,91 - 1,95 - 1,44 - 1,48 - 1,24 - 1,29 - 1,13 - 1,18 206,00 - 1,00 - 1,06 - 1,95 - 1,99 - 1,87 - 1,93 - 1,70 - 1,74 - 1,27 - 1,32 - 1,09 - 1,16 - 1,00 - 1,06 207,00 - 0,90 - 0,99 - 1,73 - 1,83 - 1,67 - 1,78 - 1,53 - 1,62 - 1,12 - 1,23 - 0,97 - 1,08 - 0,90 - 0,99 208,00 - 0,82 - 0,92 - 1,54 - 1,66 - 1,49 - 1,64 - 1,37 - 1,49 - 1,00 - 1,13 - 0,89 - 1,00 - 0,82 - 0,92 209,00 - 0,78 - 0,91 - 1,39 - 1,56 - 1,37 - 1,54 - 1,26 - 1,42 - 0,94 - 1,09 - 0,85 - 0,98 - 0,78 - 0,91 210,00 - 0,78 - 0,98 - 1,30 - 1,46 - 1,30 - 1,48 - 1,20 - 1,40 - 0,92 - 1,11 - 0,84 - 1,03 - 0,78 - 0,98 211,00 - 0,81 - 0,96 - 1,24 - 1,36 - 1,25 - 1,38 - 1,17 - 1,32 - 0,92 - 1,06 - 0,86 - 1,00 - 0,81 - 0,96 212,00 - 0,86 - 0,96 - 1,20 - 1,27 - 1,24 - 1,30 - 1,18 - 1,26 - 0,94 - 1,04 - 0,89 - 0,99 - 0,86 - 0,96 213,00 - 0,93 - 0,97 - 1,19 - 1,20 - 1,24 - 1,25 - 1,21 - 1,22 - 0,98 - 1,02 - 0,94 - 0,99 - 0,93 - 0,97 214,00 - 0,99 - 1,04 - 1,19 - 1,21 - 1,26 - 1,26 - 1,24 - 1,25 - 1,03 - 1,07 - 1,00 - 1,05 - 0,99 - 1,04 215,00 - 1,06 - 1,08 - 1,21 - 1,21 - 1,29 - 1,26 - 1,29 - 1,27 - 1,09 - 1,10 - 1,07 - 1,09 - 1,06 - 1,08 216,00 - 1,14 - 1,12 - 1,24 - 1,21 - 1,32 - 1,27 - 1,34 - 1,29 - 1,15 - 1,12 - 1,14 - 1,12 - 1,14 - 1,12 217,00 - 1,21 - 1,17 - 1,28 - 1,22 - 1,35 - 1,30 - 1,39 - 1,33 - 1,21 - 1,16 - 1,21 - 1,16 - 1,21 - 1,17 218,00 - 1,28 - 1,21 - 1,30 - 1,23 - 1,37 - 1,31 - 1,44 - 1,35 - 1,26 - 1,18 - 1,27 - 1,20 - 1,28 - 1,21 219,00 - 1,35 - 1,23 - 1,32 - 1,21 - 1,40 - 1,29 - 1,48 - 1,34 - 1,30 - 1,18 - 1,32 - 1,20 - 1,35 - 1,23 220,00 - 1,40 - 1,28 - 1,33 - 1,23 - 1,41 - 1,33 - 1,49 - 1,39 - 1,33 - 1,22 - 1,36 - 1,25 - 1,40 - 1,28 221,00 - 1,43 - 1,29 - 1,32 - 1,22 - 1,42 - 1,32 - 1,50 - 1,39 - 1,34 - 1,22 - 1,39 - 1,26 - 1,43 - 1,29 222,00 - 1,45 - 1,32 - 1,30 - 1,23 - 1,41 - 1,33 - 1,50 - 1,41 - 1,35 - 1,24 - 1,40 - 1,28 - 1,45 - 1,32 223,00 - 1,45 - 1,37 - 1,29 - 1,26 - 1,40 - 1,36 - 1,49 - 1,45 - 1,35 - 1,28 - 1,39 - 1,32 - 1,45 - 1,37 224,00 - 1,44 - 1,40 - 1,26 - 1,26 - 1,37 - 1,37 - 1,47 - 1,46 - 1,33 - 1,30 - 1,38 - 1,34 - 1,44 - 1,40 225,00 - 1,41 - 1,44 - 1,23 - 1,27 - 1,34 - 1,37 - 1,43 - 1,48 - 1,31 - 1,33 - 1,36 - 1,38 - 1,41 - 1,44 226,00 - 1,38 - 1,47 - 1,19 - 1,27 - 1,30 - 1,38 - 1,39 - 1,49 - 1,27 - 1,35 - 1,31 - 1,40 - 1,38 - 1,47 IX 7.1 Supporting Information 227,00 - 1,33 - 1,45 - 1,14 - 1,24 - 1,24 - 1,35 - 1,33 - 1,46 - 1,22 - 1,33 - 1,26 - 1,38 - 1,33 - 1,45 228,00 - 1,27 - 1,42 - 1,09 - 1,20 - 1,18 - 1,30 - 1,28 - 1,41 - 1,17 - 1,30 - 1,21 - 1,35 - 1,27 - 1,42 229,00 - 1,21 - 1,32 - 1,03 - 1,11 - 1,12 - 1,20 - 1,21 - 1,30 - 1,11 - 1,21 - 1,15 - 1,25 - 1,21 - 1,32 230,00 - 1,15 - 1,24 - 0,97 - 1,02 - 1,05 - 1,11 - 1,14 - 1,21 - 1,04 - 1,13 - 1,08 - 1,17 - 1,15 - 1,24 231,00 - 1,07 - 1,16 - 0,91 - 0,94 - 0,98 - 1,02 - 1,07 - 1,12 - 0,98 - 1,05 - 1,01 - 1,09 - 1,07 - 1,16 232,00 - 1,00 - 1,04 - 0,84 - 0,84 - 0,91 - 0,91 - 0,99 - 0,99 - 0,91 - 0,94 - 0,93 - 0,98 - 1,00 - 1,04 233,00 - 0,92 - 0,91 - 0,77 - 0,72 - 0,84 - 0,78 - 0,91 - 0,86 - 0,84 - 0,82 - 0,86 - 0,85 - 0,92 - 0,91 234,00 - 0,85 - 0,76 - 0,70 - 0,60 - 0,76 - 0,65 - 0,84 - 0,72 - 0,77 - 0,68 - 0,79 - 0,71 - 0,85 - 0,76 235,00 - 0,77 - 0,61 - 0,64 - 0,48 - 0,69 - 0,52 - 0,76 - 0,58 - 0,70 - 0,55 - 0,72 - 0,57 - 0,77 - 0,61 236,00 - 0,70 - 0,45 - 0,58 - 0,35 - 0,62 - 0,38 - 0,69 - 0,43 - 0,63 - 0,40 - 0,66 - 0,42 - 0,70 - 0,45 237,00 - 0,64 - 0,38 - 0,52 - 0,29 - 0,56 - 0,32 - 0,63 - 0,35 - 0,57 - 0,33 - 0,59 - 0,35 - 0,64 - 0,38 238,00 - 0,57 - 0,37 - 0,47 - 0,28 - 0,50 - 0,30 - 0,56 - 0,34 - 0,50 - 0,32 - 0,52 - 0,34 - 0,57 - 0,37 239,00 - 0,51 - 0,31 - 0,42 - 0,22 - 0,44 - 0,24 - 0,50 - 0,28 - 0,45 - 0,27 - 0,46 - 0,28 - 0,51 - 0,31 240,00 - 0,45 - 0,25 - 0,36 - 0,18 - 0,39 - 0,19 - 0,44 - 0,22 - 0,39 - 0,22 - 0,40 - 0,23 - 0,45 - 0,25 241,00 - 0,39 - 0,19 - 0,32 - 0,12 - 0,35 - 0,14 - 0,39 - 0,16 - 0,34 - 0,16 - 0,35 - 0,17 - 0,39 - 0,19 242,00 - 0,33 - 0,11 - 0,28 - 0,06 - 0,31 - 0,07 - 0,35 - 0,09 - 0,29 - 0,09 - 0,31 - 0,10 - 0,33 - 0,11 243,00 - 0,28 - 0,10 - 0,24 - 0,06 - 0,26 - 0,07 - 0,30 - 0,09 - 0,25 - 0,09 - 0,26 - 0,09 - 0,28 - 0,10 244,00 - 0,25 - 0,08 - 0,21 - 0,06 - 0,24 - 0,06 - 0,27 - 0,07 - 0,21 - 0,07 - 0,23 - 0,07 - 0,25 - 0,08 245,00 - 0,21 - 0,05 - 0,18 - 0,03 - 0,20 - 0,04 - 0,23 - 0,04 - 0,18 - 0,04 - 0,19 - 0,04 - 0,21 - 0,05 246,00 - 0,18 - 0,03 - 0,15 - 0,01 - 0,17 - 0,02 - 0,20 - 0,02 - 0,15 - 0,02 - 0,16 - 0,02 - 0,18 - 0,03 247,00 - 0,15 - 0,04 - 0,13 - 0,01 - 0,15 - 0,02 - 0,18 - 0,02 - 0,13 - 0,03 - 0,14 - 0,03 - 0,15 - 0,04 248,00 - 0,14 - 0,01 - 0,12 0,02 - 0,13 0,01 - 0,16 0,01 - 0,11 0,00 - 0,12 - 0,01 - 0,14 - 0,01 249,00 - 0,12 0,03 - 0,11 0,05 - 0,12 0,05 - 0,15 0,05 - 0,09 0,03 - 0,10 0,03 - 0,12 0,03 250,00 - 0,11 0,05 - 0,11 0,07 - 0,11 0,07 - 0,13 0,06 - 0,08 0,05 - 0,09 0,05 - 0,11 0,05 X ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS 7.1.1 ABBREVIATIONS AA: amino acid A: absorbance BMP: bone morphogenic protein CA: contact angle CD: circular dichroism CT: computed tomography CTA: Cholesteryl triethylenglykol azide DLS: Dynamic light scattering DSC: differential scanning calorimetry ECM: extracellular matrix ELbcR: elastin-like block co-recombinamer ELP: elastin-like protein ELR: elastin-like recombinamers ELRCTAx: ELR modified with x CTA groups FGF: fibroblast growth factor FN: Fibronectin FP: fluorescent protein FRET: Förster resonance energy transfer HASMCs: human aortic smooth muscle cells hMSCs: human mesenchymal stem cells XI 7.1 Supporting Information HPLC: high-performance liquid cromatography HUVECs: human umbilical vein endothelial cells IGZ: in-gel zymography ITC: inverse transition cycle ITT: inverse temperature transition IWZ: in-well zymography LCST: lower critical solution temperature MALDI-TOF: matrix-assisted laser desorption/ionization-time-of-flight MCST: multi-slice computed tomography MMP: matrix metalloproteinase MQ: milliQ ultra-pure water Mw: molecular weight NMR: nuclear magnetic resonance PA: plasminogen activator PBS: phosphate buffered saline pKa: dissociation constant of acids pKb: dissociation constant of bases PTH: parathyroid hormone ROI: region of interest SEM: scanning electron microscopy SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis SELR: silk-elastin-like recombinamers XII ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS TEM: transmission electron microscopy TGF-b: transforming growth factor beta TIMP: tissue inhibitors of metalloproteases TNFα: tumor necrosis factor α tPA: tissue plasminogen activator Try: Trypsin Tt: transition temperature uPA: urokinase plasminogen aktivator ε: extinction coefficient XIII 7.1 Supporting Information 7.1.2 TABLE OF STANDARD AMINO ACID ABBREVIATIONS Amino acid 3-letter code 1-letter code Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V XIV ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS List of Tables Table 1: Zymography used for clinical diagnostics and related zymogenic substrates. 64 Table 2: Reagents employed and corresponding suppliers. ____________________ 73 Table 3: Restriction enzymes used in this work and corresponding suppliers. _______ 75 Table 4: Other reagents used for this work. _______________________________ 79 Table 5:Bacterial strains used for this work. _______________________________ 79 Table 6: Composition and molecular weight of the ELbcRs and ELRs employed. ____ 81 Table 7: Relation of linear DNA migration with the bromophenol blue (BFB). ______ 83 Table 8: Optimum percentage of Acrylamide/Bisacrylamide according to protein size. 90 Table 9: Composition of the resolving and stacking gel in a gel 15%T ____________ 91 Table 10: Amino acid sequence of the different building blocks employed in this work. ______________________________________________________________ 111 Table 11: Theoretical and experimental molecularweight of ELRs and the corresponding amino acid sequence, obtained by amino acid quantification. _________________ 116 Table 12: Results of characterization of ELR modification with methacrylate groups (MA) by NMR and MALDI-TOF. Spectra are shown below. ___________________ 117 Table 13: Linearity of ELR-Calibration curve at different wavelengths of absorbance. 128 Table 14: Overview of NMR-signals and the related conversion of functional groups. 139 Table 15: Comparison of viscosities obtained from flow measurements at a shear rate of 60 s-1(Exp.) using parameters derived from the Bingham and Herschel-Bulkley models. _________________________________________________________ 153 Table 16: Overview of NMR signals and the related conversion of functional groups.P1 serves as internal reference, representing –CH3 and -CH2- protons of amino acid residues (G, E, L, K, M, P, S, V); P2 represents H2C-O(C=O)O- of the BCN linker; P3 represents C=C-H proton of the cholesteryl residue; P4 is an isolated signal from the ETERNEON fluorophore (structure not known).Complete spectra are provided as Supplementary Information. _________________________________________ 175 Table 17: Degree of modification, calculated from the mass analysis. ___________ 177 Table 18: Datapoints of BeStSel algorythm fit to CD data. ____________________ III XV List of Figures List of Figures Figure 1: a) Structural changes of VPGVG oligopeptide below and above their LCST. b) Water in clathrate-type structured state. c) Type II β -turn in the VPGVG pentapeptides119. __________________________________________________ 16 Figure 2: Dependence of Tt on the guest residue in poly penta-peptide of the structure poly[fv (VPGVG), fx (VPGXG)]. fv and fx represent the mole fractions of the respective co-monomers (fv + fx = 1)127. ________________________________________ 18 Figure 3: Tt as a function of the number of pentapeptides for three different types of ELRs at 25µM in PBS. Reproduced from Meyer et. al.128. ____________________ 19 Figure 4: Effect of Hofmeister anions on the transition temperature of ELRs. ______ 20 Figure 5: Condensed summary of recombinant gene oligomerization in a vector with 2 restriction enzymes. Adapted from142. __________________________________ 25 Figure 6: Illustration of the recognition sequences of Ear1 and Sap1 type IIS endonucleases. ___________________________________________________ 27 Figure 7: Ligation-based DNA assembly using restriction endonucleases. a) DNA assembly using conventional type II endonucleases requires a type II recognition site at the junction between the two starting pieces of DNA. The junction remains in the product, precluding the construction of arbitrary DNA sequences. b) DNA assembly using type IIS endonucleases, which allow the generation of overhangs of any arbitrary sequence, and thus sequence-independent construction. Reproduced from130. _____ 28 Figure 8: The diversity of ELR structures and their sequential origin. ____________ 38 Figure 9: Schematic illustration of the poly core model for cholesteryl stabilized particles. Reproduced from230. _______________________________________________ 42 Figure 10: Synonyms of peptidases and the differences in endo- and exopeptidases. The names in the box are commonly used terms for proteolytic enzymes. The differences in endo- and exopeptidases are, that endopeptidases preferentially act away from protein termini and exopeptidases act close to the C- or N-terminus (as indicated by the triangular shades)235. _______________________________________________ 46 Figure 11: Cascade of peptidase pro-form activations by peptidase mediated cleavage241. ______________________________________________________ 48 XXII ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS LIST OF ARTICLES AND PATENTS ORIGINATED FROM THE WORK OF THIS PhD THESIS J.C. Rodriguez‐Cabello, A. Ibáñez Fonseca, M. Alonso Rodrigo, L. Poocza, F. Cipriani, I. González De Torre, Encyclopedia of Polymer Science and Technology: Elastin-Like Polymers: Properties, Synthesis, and Applications, 2017, Wiley Publishing J.C. Rodríguez-Cabello, I. González De Torre, F. Cipriani, L. Poocza, Elastinlike materials for tissue regeneration and repair in Peptides and Proteins as Biomaterials for Tissue Regeneration and Repair, Dec 2017, Woodhead Publishing. L. Poocza, M. Alonso Rodrigo, J.C. Rodríguez-Cabello, Recombinant Biopolymer for Proteases Detection, Patent PCT/EP2019/068674 (Submitted: 04/18). L. Poocza, F. Cipriani, M. Alonso Rodrigo, J.C. Rodriguez‐Cabello, Hydrophobic cholesteryl moieties trigger substrate cell-membrane interaction of elastin-mimetic protein coatings in vitro, ACS OMEGA, 2019, 4, 6, 10818-27. S. Acosta, L. Poocza, L. Quintanilla Sierra J.C. Rodriguez-Cabello, Charge Distribution as a molecular modulator of the nanostructuration in intrinsically disordered proteins, small submitted November 2019. XXIII Other contributions L. Poocza, S. Acosta, M. Alonso Rodrigo, J.C. Rodriguez‐Cabello, Introduction of thermoreversible UCST gelation by hydrophobically hijacking LCST related secondary structure motifs in a model Elastin-like recombinamer, submitted November 2019. L. Poocza, M. Alonso Rodrigo, J.C. Rodriguez‐Cabello A fast, sensitive and cost-effective method for protease detection using non-fluorescent substrate, in preparation. F. Cipriani, L. Poocza, I. González de Torre, M. Alonso Rodrigo, José Carlos Rodríguez-Cabello, Selective Enzymatic Responsive Smart-ELRs with Allosteric Control of RNase A Activity, in preparation. Other contributions Oral presentations Kampleiter, L. Poocza, C. Richard, G. Hildebrand, K. Liefeith, J. C. Rodríguez-Cabello, C. González, A. Markus, C. Dullin, F. Alves, O. Hoffmann, Pre-clinical screening of novel biodegradable biomaterials for bone tissue engineering, 24th Scientific Congress of Austrian Pharmaceutical Society, Vienna, Austria, Sep. 2015. XXIV ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS L. Poocza, F. Cipriani, J.C. Rodíguez-Cabello, Elastin-like Recombiners (ELRs): From bioinspired Motifs to Biomedical Application, Ringberg Conference, Schloß Ringberg, Germany, May 2016. L. Poocza, P. de Almeida, F. Cipriani, I.G. de Torre, P. Kouwer, AE Rowan, JC Rodíguez-Cabello, Cross-linking of elastin-like recombinamers (ELRs) for biomedical application, 10th Young Researchers’ Retreat University of Osaka (YRR), Kyoto, Japan, Jul. 2016. L. Poocza, M. Alonso, J. C. Rodríguez-Cabello, Hydrophobic modification of ELRs for enhanced cell membrane interaction, Advanced Materials for Biomedical Application, Ghent, Belgium, Sep. 2017. L. Poocza, M. Alonso, J.C. Rodríguez-Cabello, Hydrophobic cholesteryl moieties trigger substrate cell-membrane interaction of elastin-mimetic protein coatings in vitro, European Elastin Meeting, Nijmegen, Netherlands, Jun. 2018. L. Poocza, F. Cipriani, M. Alonso, J.C. Rodríguez-Cabello, Hydrophobic cholesteryl moieties trigger substrate cell-membrane interaction of elastinmimetic protein coatings in vitro, AND, L. Poocza, F. Cipriani, M. Alonso, J.C. Rodríguez-Cabello, Specific Detection of Proteases using novel-protein substrates for Zymographic Methods, 5th TERMIS World Congress, Kyoto, Japan Sep. 2018. Poster presentations L. Poocza, P. de Almeida, F. Cipriani, P. Kouwer, A.E. Rowan, J.C. Rodíguez- Cabello, Strain-Stiffening Behaviour in PIC-ELR Hybrid Systems: Their Potential as Stimuli Responsive Scaffolds, Ringberg Conference, Schloß Ringberg, Germany, May 2016. XXV Other contributions L.Poocza, P. de Almeida, F. Cipriani, I.G. de Torre, PHJ Kouwer, AE Rowan, JC Rodíguez-Cabello, Cross-linking of elastin-like recombinamers (ELRs) for biomedical application, 10th Young Researchers’ Retreat University of Osaka (YRR), Osaka/Kyoto, Japan, Jul. 2016. L. Poocza, P. de Almeida, P. Kouwer, J.C. Rodíguez-Cabello Stress-Stiffening in PIC-ELR Hybrid Systems Trigger Myofibroblast Differentiation in vitro, Coatings and Sensors Business Case Workshop at Noviosense, Nijmegen, Netherlands, Apr. 2017. L Poocza, P de Almeida, F Cipriani, P Kouwer, JC Rodríguez-Cabello, Functional Hybrids of elastin-like recombinamers and polyisocyanates, 6th China-Europe Symposium on Biomaterials in Regenerative Medicine (CESB), Porto, Portugal, May 2017. L. Poocza, P. de Almeida, P. Kouwer, A.E. Rowan, J.C. Rodrìguez-Cabello, Introduction of strain-stiffening features into elastin-like recombinamers by isocyanate hybridization, European Chapter Meeting of the Tissue Engineering and Regenerative Medicine International Society (TERMIS), Davos, Switerland, Jun. 2017. L. Poocza, P. de Almeida, F. Cipriani, P. H. J. Kouwer, J. C. Rodrìguez- Cabello, Elastin-like Recombinamers and Polyisocyanates: A Functional Hybrid System for Tissue Engineering, 28th Annual Conference of the European Society for Biomaterials (ESB), Athens, Greece, Sep. 2017. L. Poocza, M. Alonso, JC Rodríguez-Cabello, Hydrophobic cholesteryl moieties trigger substrate cell-membrane interactions of elastin-mimetic protein coatings in vitro, Material for Life 2nd Karman Conference, Bergisch Gladbach, Germany, Apr. 2018. XXVI ACCESSING NEW BIOMEDICAL APPLICATIONS BY COMBINING GENETIC DESIGN AND CHEMICAL MODIFICATION OF ELASTIN-LIKE RECOMBINAMERS Courses and certifications - Time and Project Management. Bpmsat (Spain). - Statistical Validation and Experimental Design. Biostatech (Spain). - Leadership and coaching techniques. University of Valladolid (Spain). - Research ethics and integrity. Organized by BIOGEL project. - Mechanical properties of the biomaterials. Radboud University. Nijmegen (the Netherlands). - Biomimetic hydrogels. Organized by BIOGEL project. - Skills you really need when you apply for a job. Organized by BIOGEL project. - The Route to Academia: How to fail successfully. Organized by BIOGEL project. - Hydrogel Imaging. Austrian Institute of Technology (AIT). (Wien) Austria. - In vitro, ex vivo, and in vivo models. LifeTec Group. (Eindhoven) the Netherlands. - Synthesis of Biomaterials. University of Thessaloniki. Thessaloniki (Greece). - Critical thinking for public discourse. University of Amsterdam (The Netherlands). XXVII Other contributions 7Marie Curie Fellowship for Early Stage Research Training Attendance of the listed progress meetings with scientific communication: - Network Meeting at TPNBT, 14th December, 2015. Valladolid (Spain). - Network Meeting at Radboud University, 22th June, 2016. Nijmegen (the Netherlands). - Network Meeting at University of Valladolid, 12th December, 2016. Valladolid (Spain). - Network Meeting organized by the Centre for Research & Technology Hellas (CERTH), 14th June, 2017. Poros (Greece). - Network Meeting at Austrian Institute of Technology (AIT), 30th January, 2018. (Wien) Austria. - Network Meeting at DWI-Leibniz Institute for Interactive Materials, 25th July, 2018. (Aachen) Germany. - Network Meeting at DWI-Leibniz Institute for Interactive Materials, 14th December, 2018. Aachen (Germany). Teaching Supervision of the scientific training and mentorship of the bachelors thesis with the title “DESAROLLO DE UN MÉTODO ANALÏTICO A PARTIR DE BIOPOLIMEROS”of the student Cristina Merino Garotte at the University of Valladolid (Spain).