Development of an artificial silk protein on the basis of a lacewing egg stalk protein

De elopmen o an a i icial silk p o ein on he basis o a lacewing egg s alk p o ein Disse a ion zu E langung des akademischen G ades Dok o de Na u wissenscha en An de Bay eu he G aduie enschule ü Ma hema ik und Na u wissenscha en de Uni e si ä Bay eu h o geleg on Diplom Biologe Felix Baue Bay eu h, Mai 2013 Die o liegende A bei wu de in de Zei on Juli / 2008 bis No embe /2013 in Bay eu h am Leh s uhl Bioma e ialien un e Be euung on He n P o esso D . Thomas Scheibel ange e ig . Volls ändige Abd uck de on de Bay eu he G aduie enschule ü Ma hema ik und Na u wissenscha en (BayNAT) de Uni e si ä Bay eu h genemig en Disse a ion zu E langung des akademischen G ades Dok o de Na u wissenscha en (D . e . na .) Disse a ion einge eich am: 17.05.2013 Zulassung du ch das Lei ungsg emium: 10.06.2013 Wissenscha liches Kolloquium: 14.11.2013 Am ie ende Di ek o : P o . D . F anz Xa e Schmid P ü ungsausschuss: P o . D . Thomas Scheibel (E s gu ach e ) PD D . S e an Geime (Zwei gu ach e ) P o . D . And eas Fe y (Vo si z) P o . D . Bi gi a Wöh l Con en I CONTENT 1. SUMMARY ................................................................................................................. 1 2. ZUSAMMENFASSUNG ................................................................................................ 3 3. INTRODUCTION.......................................................................................................... 7 Silk 7 3.1. 3.1.1. S uc u e ........................................................................................................................................... 8 3.1.2. My iapoda silk ................................................................................................................................... 9 3.1.3. Spide silk ........................................................................................................................................ 10 3.1.4. Insec silk ......................................................................................................................................... 11 3.1.4.1. Caddis ly silk ........................................................................................................................... 12 3.1.4.2. Lacewing silk ........................................................................................................................... 13 3.1.4.2.1. Cocoon silk ........................................................................................................................... 14 3.1.4.2.2. Egg s alk silk ......................................................................................................................... 14 3.1.4.2.3. P oduc ion o lacewing egg s alks ........................................................................................ 15 3.1.4.2.4. The colle e ial gland ............................................................................................................. 17 3.1.4.2.5. P o ein sequences/dope composi ion ................................................................................. 18 3.1.4.2.6. Mechanics o lacewing egg s alks ........................................................................................ 18 3.1.4.2.7. S uc u e .............................................................................................................................. 19 Recombinan p oduc ion o silk p o eins 20 3.2. Technical p ocessing o silk p o eins 21 3.3. Aims o he wo k 24 3.4. 4. OVERVIEW OF THE THESIS INCLUDING UNPUBLISHED DATA ..................................... 25 Mechanical analysis o na u al lacewing egg s alks and ib es o caddis lies 25 4.1. S uc u al analysis o lacewing egg s alk silk 26 4.2. Silk gland analysis o lacewings 28 4.3. Bio echnological p oduc ion o N[AS]8C, an a i icial lacewing egg s alk p o ein 30 4.4. Fib e/s alk o ma ion and analysis 33 4.5. Fu he p ocessing o a ecombinan lacewing p o ein 37 4.6. 4.6.1. Films ................................................................................................................................................ 37 4.6.2. Capsules .......................................................................................................................................... 39 Con en 4.6.3. Hyd ogels and oams ....................................................................................................................... 40 Cell cul u e on s uc u ed ilms 42 4.7. Indi idual con ibu ions o joined publica ions 46 4.8. 5. LITERATURE ............................................................................................................. 47 6. LIST OF ABBREVIATIONS ........................................................................................... 63 7. DEPENDENCE OF MECHANICAL PROPERTIES OF LACEWING EGG STALKS ON RELATIVE HUMIDITY ....................................................................................................................... 65 8. ARTIFICIAL EGG STALKS MADE OF A RECOMBINANTLY PRODUCED SILK PROTEIN ..... 75 9. CONTROLLABLE CELL ADHESION, GROWTH AND ORIENTATION ON LAYERED SILK PROTEIN FILMS ............................................................................................................... 83 10. LIST OF PUBLICATIONS AND PATENTS....................................................................... 91 11. ACKNOWLEDGEMENT .............................................................................................. 93 12. ERKLÄRUNG: ............................................................................................................ 95 Summa y 1 1. Summa y Silks a e widely used in ex ile indus y as clo hing and u nishings due o hei ensile s eng h, smoo hness, so ex u e, lus e, and d ape. Mos commonly silk o he mulbe y silkwo m Bombyx mo i (B. mo i) is used in such applica ions, howe e , silks e ol ed independen ly in many di e en a h opods o a ious pu poses.1 Du ing e olu ion he di e en silks we e op imised o hei ask-speci ic uses o e millions o yea s, e.g. adop ing di e en mechanical p ope ies. The mechanical p ope ies mainly de i e om he p o ein seconda y s uc u e and i s highe o de a angemen in silk ib es. Spide silk, o example, is known o i s ensile p ope ies su passing nylon, Ke la ®, silkwo m silk, and high- ensile s eel.2-5 Beyond hei mechanical p ope ies, some silks a e also epo ed o be biocompa ible and non-immunogenic.6 One bene icial ea u e o silk p o eins is he possibili y o p ocess hem in o a ious mo phologies.7, 8 Se e al o hese silk ea u es make hem in e es ing o ma e ial scien is s, in ending o p oduce silks wi h uneable p ope ies depending on he desi ed applica ion, anging om echnical ones such as high pe o mance ib es o medical ones such as d ug deli e y. This hesis deals wi h he cha ac e isa ion and ep oduc ion o a less explo ed silk, he lacewing egg s alk silk. Mechanical es ing e ealed a s ong dependence on he ela i e humidi y. In he d y s a e a 30% ela i e humidi y, he s alks a e qui e igid and b eak a an elonga ion o 2% whe eas a 70% and 100% ela i e humidi y hey elonga e up o 434%. This ex ension is accompanied by a seconda y s uc u e change om c oss- ß o pa allel-ß. The c oss-ß s uc u e in uns e ched s alks p o ides bending s i ness and igidi y o he s alk, and his bending s i ness ge s los when he s alks a e s e ched. In his hesis a model is p oposed which explains hese di e ences a a ious ela i e humidi y on he molecula le el, whe ein changes in he s eng h o hyd ogen bonds upon exposu e o wa e (a hyd ogen bond dono /accep o ) in combina ion wi h mul iple disulphide c oss-links (which a e no a ec ed by wa e ) ac oge he and a e esponsible o his beha iou . Summa y 2 Based on consensus sequences o published sequence da a (de i ed om MalXB2 an egg s alk p o ein o Mallada signa a (M. signa a)),9 an enginee ed egg s alk p o ein named N[AS]8C was ecombinan ly p oduced. To p oduce an a i icial s alk, a d ople o a solu ion o pu i ied N[AS]8C was placed on a subs a e, and weeze s we e used o pull ou a ib e. A e d ying, and pos ea men , he p ope ies o he a i icial s alks we e in es iga ed in compa ison o he na u al ones. Mechanical es ing e ealed simila beha iou a 30% ela i e humidi y, bu a 70% and 100% ela i e humidi y he a i icial s alks we e no as ex ensible as he na u al ones. This co esponds o he ac , ha no c oss-ß s uc u e was o med, and, he e o e, no ea angemen in o pa allel-ß s uc u e was possible. Subsequen ly, N[AS]8C was p ocessed in o non- ib ous mo phologies. I was possible o p oduce capsules, hyd ogels, oams, and ilms. The oams show an in e es ing mic o and nano s uc u e which di e s om ha o ecombinan spide silk. The ca i ies a e illed wi h a mesh o nano ib es building a 3D sca old. Films a e a mo phology wi h po en ial o applica ion in cell cul u e. Fib oblas a achmen on N[AS]8C ilms is qui e poo . The e o e, we ied o induce guided ib oblas g ow h on pa e ned p o ein ilms. A i s laye o he ilms was cas om n agCysC16-c(RGD K), an enginee ed spide silk p o ein coupled wi h he in eg in ecogni ion mo i RGD o p o ide a p o ein laye o which ib oblas s a ached well. The second p o ein laye was p oduced using a PDMS (polydime hylsiloxane) empla e and N[AS]8C. Fib oblas s g own on hese ilms adhe e only o he RGD modi ied spide silk and no o he N[AS]8C a eas. A second ea u e o such ilms is o o ien he ib oblas s on ilms wi h al e na ing lines o he wo p o eins. Such ilms migh be use ul o issue enginee ing o con ol cell adhesion and ge a s uc u ed cell pa e n. This is essen ial o many issues such as bones, muscles, and epi helia issue. The low cell adhesion p ope ies o N[AS]8C ilms migh be in e es ing o coa ings o applica ions whe e cell adhesion is no desi ed such as s en s o ca he e s. Zusammen assung 3 2. Zusammen assung Seide is wegen ih e Reiß es igkei , Glä e, weichen Tex u , ih es Glanzes und ih es Fal enwu s ein in de Tex ilindus ie wei e b ei e es Ma e ial ü Kleidungss ücke und Ein ich ungsgegens ände. Meis wi d ü solche Anwendungen die Seide des Maulbee spinne s Bombyx mo i (B. mo i) e wende . Seiden en wickel en sich unabhängig in ielen A h opoden und we den zu e schiedens en Zwecken e wende .1 Fü diese wu den sie sei Millionen on Jah en du ch die E olu ion op imie . Zum Beispiel haben Seiden un e schiedliche mechanische Eigenscha en en wickel , welche haup sächlich on de Sekundä s uk u de P o eine und ih e übe geo dne en Ano dnung in den Seiden äden abhängen. Spinnenseide is beispielsweise bekann ü ih e Zugdehnungseigenscha en, welche die on Nylon, Ke la ®, Seidenspinne seide und hoch es em S ahl übe agen.2-5 Da übe hinaus gel en iele Seiden als biokompa ibel und nich immunogen.6 Eine wei e e nü zliche Eigenscha on Seidenp o einen is de en Ve a bei ba kei in iele e schiedene Mo phologien.7, 8 Viele diese Eigenscha en machen Seiden in e essan ü Ma e ial o sche , welche Seide mi geziel beein lussba en Eigenscha en, abhängig on de e wünsch en Anwendung, p oduzie en wollen. Denkba e Anwendungen eichen on Hochleis ungs ase n ü echnische Anwendungen bis zu medizinischen Anwendungen, wie geziel e Pha mako he apie. Diese Disse a ion beschä ig sich mi de Cha ak e isie ung und de ekombinan en He s ellung eines wenig e o sch en Seidenp o eins aus Flo liegen Eie s ielen. Mechanische Tes s an Flo liegen Eie s ielen zeig en eine s a ke Abhängigkei de Dehnba kei on de Lu euch igkei . Im ockenen Zus and bei 30% ela i e Lu euch igkei sind die S iele bieges ei und b echen bei eine Dehnung on 2%, wohingegen sie bei 70% und 100% ela i e Lu euch igkei bis zu ca. 430% dehnba sind. Diese Dehnung ko elie mi eine Sekundä s uk u ände ung on c oss-ß zu pa allel-ß. Die c oss-ß S uk u in unges eck en S ielen wi d ü de en Bieges ei igkei e an wo lich gemach . Diese Bieges ei igkei geh e lo en, wenn die S iele ges eck we den. In diese A bei wi d ein Modell o geschlagen, welches die Un e schiede bei e schiedene Lu euch igkei au molekula e Ebene e klä . Ve an wo lich ü dieses Ve hal en sind Ände ungen in de Bindungsene gie on Zusammen assung 4 Wasse s o b ückenbindungen du ch die Anwesenhei on Wasse (Wasse s o b ücken Dono /Akzep o ) im Zusammenspiel mi Disul idbindungen, die nich du ch das Wasse beein luss we den. Basie end au Konsensussequenzen des e ö en lich en Eie s iel P o eins MalXB2 wu de das P o ein N[AS]8C, eine küns lich kons uie e Va ian e des P o eins, bio echnologisch he ges ell .9 Um einen küns lichen S iel zu p oduzie en wu de ein T op en eine Lösung on ge einig em N[AS]8C au einen Un e g und au geb ach und anschließend aus dem T op en mi hil e eine Pinze e Fäden gezogen. Nach dem T ocknen und Nachbehandeln de Fäden wu den die Eigenscha en de küns lichen sowie de na ü lichen S iele un e such . Mechanische Analysen zeig en ähnliche Eigenscha en de na ü lichen und küns lichen Eie s iele bei 30% ela i e Lu euch igkei , wohingegen die küns lichen S iele bei 70% und 100% wenige dehnba wa en als die na ü lichen. Dies s imm mi de Ta sache übe ein, dass in den küns lichen S ielen keine c oss-ß S uk u gebilde wu de und somi keine Um o mung on c oss-ß zu pa allel-ß s a inden konn e. In einem wei e en Sch i wu de N[AS]8C in wei e e Mo phologien e a bei e . Es wa möglich Kapseln, Hyd ogele, Schäume und Filme he zus ellen. Die Schäume zeigen eine in e essan e Mik o- und Nano-S uk u ie ung, die sich on Spinnenseiden-Schäumen un e scheide . Die Po en sind on Nano-Fase n du chzogen, die ein 3D Ne zwe k bilden. Diese Schäume könn en ü wei e e Un e suchungen und Anwendungen als Fil e ma e ial ode Zellkul u -Ge üs in e essan sein. Eine Mo phologie mi Po en ial zu Anwendung in de Zellkul u sind Filme. Da Fib oblas en schlech au Filmen aus N[AS]8C adhä ie en, wu de e such , Fib oblas en geziel au einem gemus e en Film wachsen zu lassen. Die G undschich wu de aus n agCysC16-c(RGD K), eine echnisch e zeug en Va ian e eines Spinnenseidenp o eins, an welches die In eg in-E kennungssequenz RGD gekoppel is , gegossen. Au g und diese E kennungssequenz adhä ie en Fib oblas en gu au diesen Filmen. Eine zwei e Schich aus N[AS]8C wu de mi hil e eine PDMS (Polydime hylsiloxan) Maske au geb ach . Fib oblas en adhä ie en au solchen gemus e en Filmen nu au den n agCysC16-c(RGD K) und nich au den mi N[AS]8C bedeck en Be eichen. Eine wei e e Besonde hei diese Filme is die Zusammen assung 5 Möglichkei , du ch Applika ion des zwei en Films in S ei en o m, Fib oblas en ausge ich e wachsen zu lassen. Solche Filme könn en ü „Tissue enginee ing“ genu z we den, um Zelladhäsion zu kon ollie en und eine s uk u ie e Zellaus ich ung zu e hal en. Dies is essen iell ü iele na ü liche Gewebe wie Knochen, Muskeln, und Epi helgewebe. Des Wei e en könn en die schwachen Zelladhäsions-Eigenscha en on N[AS]8C Filmen ü Beschich ungen, bei denen Zelladhäsion nich e wünsch is , wie beispielsweise S en s ode Ka he e n, in e essan sein. In oduc ion 12 helical s uc u e, and hese silks a e used o building cocoons.21, 28, 49 The second ype is a c oss-ß silk, which is used in egg s alks and is sec e ed by he colle e ial gland. 3.1.4.1. Caddis ly silk Caddis ly la ae a e commonly used by ecologis s o in es iga e he wa e quali y o i e s and lakes. They a e lis ed in he insec o de T ichop e a and a e Holome abola, which means hey unde go me amo phosis wi h la ae and adul s showing a o ally di e en habi us. The adul lies a ach hei eggs unde wa e o close abo e he wa e su ace. The la ae depend on wa e as habi a s whe e hey unde go i e o se en la al s ages. The la ae eed on algae, lea s, and small wa e insec s, depending on hei species and a e di ided in o wo g oups he e uci o m (casemaking) and he campodei o m ( ee-li ing and ne spinning) la ae (see Figu e 2).78 The e udi o m la ae build ound cases by gluing oge he a ious ma e ials such as s ones, sand, o small o ganic ma e ial by he use o a silken h ead. The ne spinning campodei o m la ae use silk o build small ne s o ca ch small wa e insec s and build a silken e ea o hide om p eda o s (see Figu e 2).78 La ae o bo h g oups use silk o pupa e in a cocoon.79 Caddis ly silk is p oduced in he labial glands which a e homologue o he labial glands o bu e lies and he e o e he silk mo phology and composi ion is compa able o lepidop e an silk. Two la ened ibbon like ib es a e glued oge he by se icin-like glue.80 The silk is composed o homologues o hea y chain and ligh chain ib oins bu no P25 was de ec ed so a .20, 81 The caddis ly hea y chain ib oin has some di e ences o hose o Lepidop e a, showing a high con en o bulky and basic amino acid esidues, a low alanine con en , and a epea ing mo i con aining phospho yla ed se ines which could p o ide c osslinking by Ca2+-ions.82 An addi ional p o ein, N -1, wi h a high con en o cys eine is hough o c osslink he p o eins by disulphide bonds, p o iding wa e insolubili y o he ib es.83, 84 In oduc ion 13 Figu e 2: A and B: Caddis ly cases buil om li le s ones; C and D: Caddis ly ne s spun in an a i icial su ounding. Scale ba s: 0.7 cm. 3.1.4.2. Lacewing silk G een lacewing la ae a e known o ea ing aphids which hey a e b ed o comme cially. Lacewings such as Ch ysopa ca nea (C. ca nea) (Neu op e a: Ch ysopidae) a e Holome abola. No mally insec s a e known o p oduce only one silk ype, bu lacewings p oduce wo.9 The la ae (Figu e 3 C) p oduce a cocoon be o e me amo phosis (Figu e 3 D), while adul emales (Figu e 3 A) use a second ype o a silken s alk o p o ec hei eggs om p eda o s (Figu e 3 B).85-87 In oduc ion 14 Figu e 3: Li ecycle o lacewings comp ising an adul lacewing (A), eggs on s alks (B), la a (C), and pupa ed la a (D). 3.1.4.2.1. Cocoon silk The cocoon o lacewing la ae comp ises wo laye s.21, 28, 88 Fi s he la a sec e es a silken h ead om he malphigian ubules and deposi s a loosely wo en cocoon. La e he la a deposi s an inne laye o lipids o p o ec i sel om wa e loss du ing me amo phosis. The silken h eads ha e diame e s o abou 2 µm, a e composed o one p o ein which is 49 kDa in size, and a e ich in alanine.21 The p o ein adop s an α-helical s uc u e bu su p isingly i does no assemble in o coiled coils as o he α-helical silks do.21 3.1.4.2.2. Egg s alk silk Female lacewings a ach hei eggs o silken s alks o p o ec he eggs om p eda o s such as an s o hei own la ae.87 The s alk is d awn om a sec e ed p o ein d ople p oduced in he colle e ial gland. An amino acid composi ion analysis e ealed high le els o se ine (41%), glycine (24%) and alanine (20%).89 In es iga ions In oduc ion 15 o a c-DNA lib a y o he lacewing (Mallada signa a (M. signa a)) colle e ial gland yielded clones encoding wo p o eins: MalXB1 (86 kDa) and MalXB2 (55 kDa), bo h con aining a highly epe i i e cen al s uc u e (o e 70%) which has a 16 amino acid pe iodici y.9 I was p oposed ha eigh amino acid long ß-s ands old in o a egula an ipa allel c oss-ß s uc u e which is s acked in ib e- and side chain-di ec ion. This s uc u e con e s in o a pa allel-ß s uc u e upon s e ching.47, 90 Fu he he s alks o some lacewing species such as Ce aeoch ysa smi hi a e epo ed o be coa ed by d ople s con aining a y acids, an es e , and a ious s aigh -chain aldehydes. This luid was shown o p o ec he eggs om p eda o s such as an s.86 3.1.4.2.3. P oduc ion o lacewing egg s alks The p oduc ion o egg s alks seems o be qui e simple compa ed o he complex spinning mechanisms o spide s whe e shea o ces, pH change, and ion exchange play impo an oles.54, 91-100 The emale lacewing i s aps he abdomen a ew imes on he su ace (Figu e 4 A-C), deposi s a d ople o spinning solu ion om he colle e ial gland on a su ace (Figu e 4 D), dips he end o an egg in o he solu ion, and aises i s abdomen o d aw a ib e be ween he d ople and he egg (Figu e 4 E- G).60 The ly s ays in his posi ion o 10 (~10% RH) o 35 seconds (~70% RH) o le he s alk d y and inally lea es.101 In oduc ion 16 Figu e 4: Lacewing p oducing an egg s alk: A-C: The lacewing aps i s abdomen se e al imes on he subs a e; D: A d ople o dope is deposi ed on he su ace and he egg is p essed in o he d ople ; E- G: The abdomen is pulled away om he subs a e and a s alk is d awn. G: The ly holds i s abdomen up un il he s alk is ha dened (app oxima ely 10-35 seconds depending on he ela i e humidi y); H: The s alk is inished. In oduc ion 17 3.1.4.2.4. The colle e ial gland The o igin o he lacewing egg s alk p o eins is he colle e ial gland o emale lacewings which is loca ed in he do sal egion o he six h o eigh h body segmen and has a sac-like shape. The exi o he gland joins he allopian ube (Figu e 5).102 Figu e 5: Schema ic o ganisa ion o he emale lacewings geni als. A: Colle e ial gland; B: Fallopian ube; C: Recep aculum seminis; D: Pai ed o a y; E: O a ioles; F: Egg. Lucas and Rudall showed by ansmission elec on mic oscopy ha sec e ion o he colle e ial gland o Ch ysopa la a, which was dilu ed wi h wa e , comp ised ib illa s uc u es. These s uc u es did no solubilise du ing dilu ion.26 The ib ils a e an assembly o a ound 20-30 molecules in side chain di ec ion (abou 13 nm) and ha e a hickness o app oxima ely 2.5 nm which is he hickness o he old o eigh amino acids p oposed by Geddes e al. and leng hs o abou 670 nm.26, 47 This p eassembly in he spinning dope has been discussed o be a key equi emen o a c oss-ß s uc u e in he s alk.26 In oduc ion 18 3.1.4.2.5. P o ein sequences/dope composi ion Egg s alks o M. signa a a e composed o a leas wo p o eins. The unde lying c-DNA was ex ac ed om a c-DNA lib a y o colle e ial gland cells.9 The wo genes encoding he p o eins (MalXB1 and MalXB2) a e ound o be exp essed in a a io o 7:1.9 Bo h p o eins consis o a highly epe i i e co e domain wi h a epea o 16 amino acids lanked by non- epe i i e e minal domains. MalXB1 addi ionally has a non- epe i i e cen al domain. MalXB1 is nega i ely cha ged wi h 17 acidic and 9 basic amino acid esidues, whe eas MalXB2 is posi i ely cha ged wi h 11 acidic and 31 basic amino acids. An in e es ing ac is he p esence o se en (MalXB1) and i e (MalXB2) cys eine esidues, which a e mos ly si ua ed in he non- epe i i e e minal domains. 3.1.4.2.6. Mechanics o lacewing egg s alks Mechanical es s on egg s alks we e unde aken by Hepbu n e al. (C. ca nea) and Weisman e al. (M. signa a).9, 103 S ess s ain measu emen s a 65% ela i e humidi y e ealed ex ensibili ies o ~249% and 381% and a ensile s eng h o ~375 MPa and 310 MPa. Measu emen s unde wa e showed highe ex ensibili y (502%/~560%) while s eng h was educed (186 MPa/~250 MPa). Weisman e al. measu ed he la e al s i ness o egg s alks by scanning p obe mic oscopy. Due o he lack o some physical cons an s o he sys em he modulus is epo ed in ela ion o B. mo i silk. The measu emen s e ealed 70% highe modulus alues o he egg s alks. Fo he calcula ion o he bending s i ness, whe e he shape o a c oss sec ion is aken in o accoun , he egg s alks ha e h ee imes highe alues ( ound shape o he s alks in compa ison o he ilobal shape o B. mo i silk).9 The high ex ensibili y and high la e al s i ness migh be ela ed o he c oss-ß s uc u e and disulphide c oss linking. In oduc ion 19 3.1.4.2.7. S uc u e The seconda y s uc u e o egg s alk p o eins was in es iga ed by Pa ke and Rudall.90 X- ay di ac ion pa e ns showed c oss-ß s uc u e wi h ß-s ands unning pe pendicula o he ib e axis, and we e exci ingly he i s de ec ed na u al p o ein wi h his s uc u e. 9, 90 1968 Geddes e al. p oposed a s uc u al model o explain he X- ay di ac ion pa e n.47 The s alk is buil up by 25 Å hick micelles being sepa a ed by a iable sized in e micelle spacing o 15 ± 4 Å (Figu e 6). The longes dimension o he micelle is o ien ed pa allel o he ib e axis. The ß-s ands a e p edic ed o ha e a leng h o eigh amino acids whe eo he i s wo and las wo amino acids o m a ß- u n. In each u n one o he wo cen al amino acids has o be a glycine.47 This p edic ion was con i med by Weisman e al. o MalXB1 and MalXB2 whe e one glycine esidue is ound pe u n in he epe i i e domain. They u he ound ha cha ged (Lysine) and bigge (>124 g/mol) amino acids a e si ua ed in he cen al u n egions.9 In MalXB2 wo o he ou cen al posi ions o he ß-s ands a e alanine esidues. Figu e 6: C oss-ß micelle o a lacewing egg s alk. In oduc ion 20 Recombinan p oduc ion o silk p o eins 3.2. In con as o spide s i is possible o a m lacewings which a e u ilised on a comme cial scale as a biological pes con ol.104 To ob ain lacewing silk ei he lies ha e o be killed, he silk gland dissec ed, and he p o ein ex ac ed, o single egg s alks ha e o be ha es ed, he eggs emo ed, he s alks solubilised, ollowed by ex ac ion o he p o eins. Bo h a e ime consuming and do no lead o la ge quan i ies o p o ein. Fu he mo e, bo h me hods yield an unde ined mix u e o he p o eins and o he molecules such as he ela i ely low molecula weigh egg de ence molecules men ioned be o e, which a e p esen in he s alk. Recombinan p oduc ion o lacewing silk p o eins in hos o ganisms such as bac e ia o yeas s is a possibili y o ob ain easonable quan i ies o pu e p o eins wi h consis en quali y, which is an essen ial equi emen o he use o he p o eins in indus ial applica ions. An ad an age is ha single domains o he p o eins can be p oduced, and hei con ibu ion o he assembly o he p o ein can be analysed. Ano he ad an age o ecombinan silk p oduc ion is he possibili y o al e he p ope ies o he p o eins. Fo example, he subs i u ion o poly-alanine ich modules (c ys alline leading o s i ness and s eng h) o coiled coil o ming modules (mo e ex ensible) migh lead o a mo e elas ic ib e a e spinning o he p o ein. Addi ion o signal pep ides such as he in eg in ecogni ion mo i e RGD o cell pene a ing pep ides o silk p o eins leads o new unc ions o he p o eins such as imp o ed cell adhesion.105-107 Commonly epo ed p oblems occu ing du ing he ecombinan p oduc ion o silk p o eins a e low yields and unca ed e sions o he p o eins. The easons he e o a e he size o silk p o eins (p o ein yield dec eases a p o ein sizes abo e 100 kDa in Esche ichia coli (E. coli)),108 and hei highly epe i i e cha ac e (leads o undesi able ecombina ion). Va ious a emp s ha e been made o o e come hese p oblems, o example op imisa ion o he gene sequence (less epe i i e genes due o codon a ia ion/adap ion o he codon usage o he hos o ganism), hos enginee ing (changes in he me abolism o he hos o ganism), and a modula app oach leading o sho ened e sions o he p o eins.108-115 In oduc ion 21 Many companies such as AMSilk GmbH (Ge many), BASF AG (Ge many), Commonweal h Scien i ic and Indus ial Resea ch O ganiza ion (Aus alia), and Spibe AB (Sweden) a e in e es ed in ecombinan p oduc ion o silk p o eins o indus ial applica ions. Technical p ocessing o silk p o eins 3.3. One pa icula ly a ac i e ea u e o silk p o eins is he possibili y o p oduce di e en mo phologies in addi ion o ib es. Fo o he silk p o eins, he p oduc ion o ib es, pa icles, capsules, hyd ogels, oams, ilms, and coa ings has been epo ed, wi h possible applica ions anging om d ug deli e y o high ech ex iles. Alle gan Inc. (USA), AMSilk GmbH (Ge many), Commonweal h Scien i ic and Indus ial Resea ch O ganiza ion (Aus alia), Ek eino Labo a o ies Inc. (USA), Neu o ex L d. (England), O hox L d. (England), Ox o d Bioma e ials L d. (England), Spibe AB (Sweden), Spin ec Enginee ing GmbH (Ge many), Su u ox L d. (England), and Vaxess Technologies Inc. (USA) wo k on p ocessing silk p o eins in o ma ke able p oduc s such as condui s o ne e egene a ion, coa ings o b eas implan s, su u es, meniscal ca ilage-, bone-, join epai , and he mo s able accines. In his hesis some o he men ioned mo phologies ha e been p oduced ou o ecombinan lacewing silk, and a e he e o e in oduced b ie ly. Fib es Fo he p ocessing o silk p o eins ( ecombinan as well as egene a ed na u al silk) in o ib es he e a e a ew echniques such as hand d awing, we spinning,116-125 mic o luidic spinning,92, 126 o elec o spinning.127-131 In his hesis he ib es we e hand d awn. Hand d awing ib es equi es a highly concen a ed p o ein solu ion which can be ei he aqueous o non-aqueous (Hexa luo oisop opanol (HFIP) o Hexa luo oace one ihyd a e (HFA) ha e been used). To p oduce a ib e, a d ople o silk solu ion is deposi ed on a su ace and a ib e is pulled ou o he d ople by using weeze s, and subsequen ly O e iew o he hesis including unpublished da a 28 A e ea angemen o he s ands a 100% ela i e humidi y, new hyd ogen bonds can be o med. This esul s in an ex ension o he s alk o up o 500%. Finally he ß-s ands s a slipping on each o he by a s ick-slip mechanism desc ibed by Ke en e al. be o e he s alk up u es.187 In con as a 30% ela i e humidi y he hyd ogen bonds ha e highe bond ene gy due o he lack o in e ac ing wa e molecules. The e o e, in one laye o he s alk he sum o hyd ogen bonds has highe bond ene gy han he indi idual disulphide bonds which c osslink he p o eins in he s alk. The disulphide bonds b eak and he s alk up u es wi hou b eakage o he hyd ogen bonds along he en i e leng h o he s alk. Silk gland analysis o lacewings 4.3. Colle e ial glands o emale C. ca nea we e dissec ed unde a 95 mM sodium chlo ide (NaCl) solu ion and incuba ed h ee imes in ixing bu e (100 mM HEPES (4-(2- hyd oxye hyl)-1-pipe azinee hanesul onic acid), 2.5% glu a aldehyde, 2% o maldehyde, pH 7.2) (one hou , wo hou s, 18 hou s in he idge espec i ely). A e wa ds he glands we e washed h ee imes wi h wash bu e (100 mM HEPES, pH 7.2) ollowed by incuba ion in 1% osmium e oxide ( /w in wa e ) o one hou a 4 °C and h ee imes washing in wa e . A e embedding he glands in 1% aga hey we e dehyd a ed using inc easing concen a ions o e hanol (30%, 50%, 70%, 90%, and wo imes 100%). E hanol was subs i u ed by incuba ing he embedded gland in a 1:1 mix u e o e hanol and p opylene oxide (15 minu es), ollowed by incuba ion wo imes in pu e p opylene oxide (12 hou s a -20°C). Finally he samples we e embedded in glycid e he 100 using s anda d p ocedu es. The samples we e cu wi h a diamond kni e equipped ul amic o ome in o ul a hin sec ions, which we e moun ed on coppe g ids. A e s aining wi h u anyl ace a e and lead ci a e hey we e imaged using a ansmission elec on mic oscope (TEM). Two o ou cell laye s we e de ec ed depending on he posi ion in he gland. In gene al he gland cells con ain many mi ochond ia and a e illed wi h ough endoplasmic e iculum (Figu e 9). In some cells esicles wi h ib illa s uc u es a e isible (Figu e 9 D-F). The ib ils ha e diame e s o abou 5 nm and leng hs o abou 200 nm. This may be ela ed o egg s alk p o eins o ming eigh amino acid ß-s ands. Such a s uc u e would ha e a hickness o 2.5 nm. Lucas and Rudall showed simila s uc u es in dilu ed dope om a O e iew o he hesis including unpublished da a 29 Ch ysopa la a colle e ial gland.26 Such a p e-s uc u ed silk solu ion migh be c i ical o achie e a c oss-ß s uc u e. The ib ils migh be o ien ed by shea o ces du ing he egg s alk p oduc ion, and he cys eines migh c oss-link he ib ils o gi e a s able egg s alk s uc u e (Figu e 15). Figu e 9: TEM images o c oss sec ions o a lacewing colle e ial gland; A: Silk gland issue and silk dope (a ows); B: Cells wi h Nucleus (n), mi ochond ia (m), and silk dope; C: Inhomogenei y o he silk solu ion; D, E and F: Cells a e illed wi h ough endoplasmic e iculum ( e ) and p o ein illed esicles wi h ib illa s uc u es ( ). O e iew o he hesis including unpublished da a 30 Figu e 10: Sil e s ained SDS-page o silk solu ion s o ed in he colle e ial gland o a emale lacewing (C. ca nea). M: p o ein ma ke ; G: silk gland ex ac . SDS-PAGE analysis o he silk dope o a emale C. ca nea e ealed i e dis inc bands (Figu e 10). Su p isingly his a e h ee mo e han Weismann e al. ga he ed om hei c-DNA lib a y o M. signa a.9 One eason o his migh be he quali y o hei c-DNA lib a y whe e only one clone encoding MalXB2 and se en clones encoding MalXB1 could be ound. Bio echnological p oduc ion o N[AS]8C, an 4.4. a i icial lacewing egg s alk p o ein Due o he low a ailabili y o lacewing egg s alk p o ein om na u al sou ces, i is an impo an s ep o p oduce he p o eins in a di e en manne , o use in bo h basic esea ch and echnical p ocessing. He e we de eloped an a i icial e sion o MalXB2, an egg s alk p o ein o M. signa a and p oduced i ecombinan ly in E. coli bac e ia. MalXB2 consis s o non- epe i i e amino- and ca boxy- e minal domains. These we e used as modules N (amino- e minal module) and C (ca boxy- e minal module) o he a i icial P o ein N[AS]8C (Figu e 11). To ensu e a p ope s e ic build-up o he p o ein ( he e minal domains in he na u al bluep in ace o di e en sides o he c oss-ß shee s o he epe i i e pa (Figu e 6)) he ca boxy- e minal module s a s wi h he las eigh amino O e iew o he hesis including unpublished da a 31 acids o he epe i i e pa o MalXB2 (Figu e 11). De ailed analysis o he amino acid sequence o he epe i i e pa o MalXB2 showed a epea e e y 48 amino acids, which was called AS module and be e i s o he o iginal sequence han a 16 amino acid epea p oposed by Weismann e al.. 9 The s a ing o he 48 amino acid epea was chosen o s a wi h a glycine which is ele an o he use o a cloning s a egy de eloped by Hümme ich.109 The modules we e ansla ed o E. coli op imised nucleo ide sequences and an addi ional nucleo ide iple (GGC) was added o he end o he N- and AS module (Due o he cloning echnique. These nucleo ides ge los du ing mul ime isa ion/cloning). The cloning echnique allows a seamless mul ime isa ion o he AS modules and subsequen linkage o he N- and C module (Figu e 11 C). As a leng h o he epe i i e pa we chose eigh AS modules o mimic he o iginal leng h. A e wa ds he N[AS]8C nucleo ide sequence was cloned in o a pET 28a ec o . E.coli BL21 (DE3) cells we e ans o med wi h he plasmid and we e cul i a ed in a 2.5 L e men e . A e induc ion and exp ession o he a i icial gene, he bac e ia could be ha es ed and a pu i ica ion s a egy o he p o ein had o be de eloped. The bac e ia we e lysed by an u ea/ hiou ea bu e ollowed by pH dec ease o 4. Finally he p o ein was pu i ied wi hou he use o columns by ac iona ed ammonium sulpha e p ecipi a ion, and a e washing wi h wa e , lyophilised and s o ed a -20°C. Pu i y was es ed by SDS-PAGE and mass spec ome y (Figu e 12). O e iew o he hesis including unpublished da a 32 Figu e 11: A: Sequence and o ganisa ion o MalXB2, a na u al egg s alk p o ein; B: The h ee modules de i ed om MalXB2; C: Schema ic build-up o an a i icial lacewing egg s alk p o ein - N[AS]8C. The ull leng h p o ein N[AS]8C has a molecula weigh o 53 kDa including de ec ion and pu i ica ions ags and is compa able in size o he na u ally occu ing a ian MalXB2 (55 kDa). O e iew o he hesis including unpublished da a 33 Figu e 12: SDS-PAGE (A) and mass spec ome y (B) o N[AS]8C. The lowe molecula weigh peaks a e mul iple cha ged N[AS]8C molecules. Adap ed wi h pe mission om Angewand e Chemie In e na ional edi ion (2012, 51, 6521-4). Copy igh 2012 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim. Fib e/s alk o ma ion and analysis 4.5. To p oduce ib es ou o N[AS]8C, we used a p ocess close o he na u al one. Due o he low solubili y o he p o ein in wa e we chose HFA as he sol en . The ola ili y o HFA is bene icial o as d ying o he gene a ed ib es. 10% w/ o lyophilised N[AS]8C was dissol ed in HFA o p oduce a solu ion om which ib es could be spun. A small d ople (~1-2 µL) was pipe ed on a su ace. Now weeze s we e dipped in o he d ople , mimicking he lacewing egg s alk p oduc ion, and we e subsequen ly pulled ou o d aw a ib e. The end connec ed o he weeze s was ans e ed o a in oil suppo (Figu e 13 A). A e d ying, he in oil suppo was mo ed close o he d ople o educe ension in he ib es and a oid up u e du ing pos ea men . By applying 60°C and 70% ela i e humidi y o e nigh he ß-shee con en inc eased om 20% o 32% which is close o he alues o na u al egg s alks wi h 40% ß- shee con en and he ib es u ned wa e insoluble. O e iew o he hesis including unpublished da a 34 Figu e 13: Compa ison o na u al and a i icial s alks. A) schema ic pic u e o he s alk p oduc ion 1) s alk d awn om he dope o in oil using weeze s; 2) elaxa ion o he s alk by mo ing o he in oil; 3) he s alk con ac s du ing pos - ea men a 60°C and 70% ela i e humidi y; B) pic u e o an a i icial egg s alk wi h in oil on op (le ) and a na u al egg s alk wi h an egg on op ( igh ). Rep in ed wi h pe mission om Angewand e Chemie In e na ional edi ion (2012, 51, 6521-4). Copy igh 2012 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim. The a i icial s alks a e s i , like he na u al ones (Figu e 13 B). Analysis o he a i icial s alks showed simila mechanical p ope ies o he na u al ones a 30% ela i e humidi y wi h 2% ex ensibili y o he na u al ones compa ed o 5% o he a i icial ones (Table 3). E en he ensile s eng h is nea ly equal wi h 55 MPa compa ed o 68 MPa o he na u al s alks. Ne e heless he e a e di e ences a 70% ela i e humidi y whe e he ex ensibili y o he na u al s alks o 210% could no be obse ed o he a i icial ones. A i icial s alks show, as well as he na u al lacewing egg s alks, bi e ingence unde a pola ised mic oscope, meaning he e a e o de ed s uc u es in he s alk (Figu e 14). Ne e heless, no c oss-ß s uc u e could be de ec ed by X- ay di ac ion o by pola ised FTIR (Fou ie ans o m in a ed spec oscopy) measu emen s. This migh be a eason o he di e ences in mechanical p ope ies a 70% ela i e humidi y. In he absence o c oss- ß s uc u e no ansi ion o pa allel-ß s uc u e is possible. This means he s alks will no be ha ex ensible and he mechanical p ope ies will di e . O e iew o he hesis including unpublished da a 35 Table 3: Tensile es ing o na u al (C. ca nea) and a i icial egg s alks. Expe imen s we e ca ied ou a 30% and 70% ela i e humidi y a 22°C. Rep in ed wi h pe mission om Angewand e Chemie In e na ional edi ion (2012, 51, 6521-4). Copy igh 2012 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim. Figu e 14: Compa ison o bi e ingence o na u al and a i icial s alks. A) Mic oscopic pic u es o a na u al lacewing egg s alk. B) Mic oscopic pic u es o an a i icial egg s alk. Top: b igh ield; bo om: wi h c ossed pola ise s; scale ba s: 50 µm. Rep in ed wi h pe mission om Angewand e Chemie In e na ional edi ion (2012, 51, 6521-4). Copy igh 2012 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim. Ex ensibili y [%] S eng h σmax [MPa] Young’s modulus [MPa] Toughness [MJ/m³] Li e a u e alues (M. signa a and C. spec) 65% RH9 381 310 65% RH103 249 ~375 Na u al egg s alk 30% RH 2 ± 1 68 ± 19 5,777 ± 1,257 1.2 ± 0.72 70% RH 210 ± 100 155 ± 75 3,175 ± 1,016 87 ± 49 A i icial egg s alk 30% RH 5 ± 2 55 ± 14 2,330 ± 850 1.76 ± 0.9 70% RH 6 ± 3 25 ± 11 1,012 ± 252 1.09 ± 0.59 O e iew o he hesis including unpublished da a 36 A possibili y o sol e his p oblem in he u u e is o use an aqueous spinning solu ion wi h p e-s uc u ed p o eins, as seen o na u al dope (Figu e 9), which migh o ien due o shea o ces du ing he p oduc ion p ocess and c osslink o each o he by disulphide bonds (Figu e 15).26 Figu e 15: Model o a p e-s uc u ed silk solu ion which assembles upon shea o ces. O e iew o he hesis including unpublished da a 37 Fu he p ocessing o a ecombinan lacewing 4.6. p o ein Nex he p ocessing o N[AS]8C in o di e en mo phologies was es ed as desc ibed o o he silk p o eins. 4.6.1. Films In o de o ob ain ilms (Figu e 16) 1% (w/ ) N[AS]8C was dissol ed in HFA o o mic acid. Bo h solu ions we e cas on polys y ene. A e d ying, he ilms we e ei he peeled o he subs a e o i s pos - ea ed by imme sion in me hanol o o e -nigh ea men a 60°C and 50% ela i e humidi y. Seconda y s uc u e was analysed using FTIR. Fo SEM he samples we e glued on aluminium s ubs and we e spu e -coa ed wi h pla inum. Images we e ob ained using a Zeiss 1530. Table 4: Seconda y s uc u e o ilms cas om HFA and o mic acid. The HFA ilms we e pos - ea ed wi h me hanol o a 60°C and 50% RH o e nigh . HFA Fo mic acid As cas Un ea ed a e 4 days 60°C 50% RH o e nigh MeOH ea ed As cas ß shee s 6% 19% 42% 50% 59% Alpha-helices 21% 12% 9% 7% 6% Tu ns 11% 26% 18% 22% 14% Random coils 58% 39% 23% 20% 20% Side chains/ agg ega ed s ands 4% 4% 8% 1% 1% Films cas om o mic acid (Figu e 16 B, D, F) we e mo e b i le han he ones om HFA, and wa e insoluble di ec ly a e d ying. They had a ß-shee con en o app oxima ely 59% (Table 4). In con as , ilms cas om HFA (Figu e 16 A, C, E) we e wa e soluble and, he e o e, had o be pos - ea ed. Wi hou pos - ea men hey had a ß-shee con en o 6% which inc eased due o aging o a ound 19% (a e 4 days). O e nigh hea ea men o he ilms a 60°C and 50% ela i e humidi y led o a ß-shee con en o a ound 42%. Sligh ly highe alues could be ob ained by ea ing he ilm wi h O e iew o he hesis including unpublished da a 44 non-adhe en . 94% o he cells g ew on he n agCysC16-c(RGD K) s ipes (Table 5 and Figu e 23). Su p isingly ib oblas s g own on s uc u ed con ol ilms o unmodi ied eADF4(C16) wi h N[AS]8C as a op laye and s uc u ed ilm only ou o eADF4(C16) showed he same endency o dis ibu ion wi h 92% espec i ely 85% o he ib oblas s in he g oo es (Table 5 and Figu e 23) bu wi h much lowe o e all cell numbe on he solely eADF4(C16) ilms. Figu e 23: BALB/3T3 ib oblas s g own on s uc u ed ilms. A: O ien a ion o ib oblas s g own on pa e ned ilms made o di e en p o ein combina ions (g ound laye p o ein/ idge p o ein) as depic ed by he colou code a e 48 hou s o incuba ion; B: Fluo escence mic oscopy o calcein AM (Calcein ace oxyme hyl es e ) s ained cells, g own on a ilm wi h n agCysC16-c(RGD K) as g ound laye and N[AS]8C as idges; C and D: ligh mic oscopic image a e 48 hou s o incuba ion using n agCysC16-c(RGD K) as g ound laye wi h N[AS]8C as idges (C) and eADF4(C16) as g ound laye wi h N[AS]8C as idges (D). Adap ed by pe mission o The Royal Socie y o Chemis y. O e iew o he hesis including unpublished da a 45 The o ien a ion o he ib oblas s was mos p onounced o ilms ou o eADF4(C16) wi h 80.6% o he cells being o ien ed in an angle o ±7.5° o he s uc u es axis whe eas only 52.1% o he ib oblas s on n agCysC16-c(RGD K)/N[AS]8C we e in his ange (Figu e 23 A). This indica es he impo ance o he s uc u e o o ien a e he cells while he ma e ials/p o eins p ope ies in luenced hei loca ion. E en a e 96 hou s o cul i a ion he ib oblas s s ayed mos ly on he n agCysC16- c(RGD K) p o ein s ipes and p oli e a ed well o a high cell densi y (Figu e 24 A). Such high cell densi ies a e necessa y o many issue cul u e expe imen s, such as di e encia ion o myoblas s in o myo ubes. C2C12 myoblas s showed he same endency as he ib oblas s, bu we we e no able o coun cells and measu e hei o ien a ion o he sca old due o hei abili y o o m myo ubes and highe cell densi y (Figu e 24 B). Figu e 24: A: BALB/3T3 ib oblas s g own on s uc u ed ilms using eADF4(C16) as g ound laye and N[AS]8C as idges a e 96 hou s o incuba ion; B: C2C12 myoblas s g own on eADF4(C16)/N[AS]8C ilms a e 48 hou s o incuba ion. Rep oduced by pe mission o The Royal Socie y o Chemis y. Such ilms migh be used in/as sca olds o issue enginee ing o issues, whe e an o de ed s uc u e o he cells and high selec i e cell densi y is o ad an age. Skele al muscles as well as bone o epi helial cul u es migh be possible applica ions. O e iew o he hesis including unpublished da a 46 Indi idual con ibu ions o join publica ions 4.8. Chap e 7 Chap e 6 is ep in ed wi h pe mission om Biomac omolecules (2012, 13, 3730-5). Copy igh 2012 Ame ican Chemical Socie y. “Dependence o Mechanical P ope ies o Lacewing Egg S alks on Rela i e Humidi y” By Felix Baue , Luca Be ine i, Admi Masic, and Thomas Scheibel I ca ied ou all o he measu emen s excep he RAMAN measu emen s. Luca Be ine i and Admi Masic pe o med he RAMAN measu emen s and co ec ed he manusc ip . Felix Baue and Thomas Scheibel w o e he manusc ip . Chap e 8 Chap e 7 is ep oduced wi h pe mission om Angewand e Chemie (2012, 51, 6521-4). Copy igh 2012 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim. “A i icial Egg S alks Made o a Recombinan ly P oduced Silk P o ein“ By Felix Baue and Thomas Scheibel I ca ied ou all he design, cloning, e men a ion, pu i ica ion and measu emen s. Felix Baue and Thomas Scheibel w o e he manusc ip . Chap e 9 Chap e 8 is ep oduced by pe mission o The Royal Socie y o Chemis y. This wo k is submi ed o publica ion: Bioma e ials Science (2013, 1,1244-9). “Con ollable cell adhesion, g ow h and o ien a ion on laye ed silk p o ein ilms” By Felix Baue , S e anie Wohl ab, and Thomas Scheibel. I de eloped he echnique o ab ica e he s iped p o ein ilms, and ca ied ou he image analysis a e cell cul u e. S e anie Wohl ab ca ied ou he cell cul u e expe imen s. Felix Baue , S e anie Wohl ab and Thomas Scheibel w o e he manusc ip . Li e a u e 47 5. Li e a u e 1. Su he land, T. D.; Young, J. H.; Weisman, S.; Hayashi, C. Y.; Me i , D. J., Insec silk: One name, many ma e ials. Annual Re iew o En omology 2010, 55, 171-188. 2. 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P oceedings o he Na ional Academy o Sciences o he Uni ed S a es o Ame ica 2004, 101, (8), 2287-92. 190. U y, D. W.; Pa anaik, A., Elas ic p o ein-based ma e ials in issue econs uc ion. Annals o he New Yo k Academy o Sciences 1997, 831, 32-46. Lis o abb e ia ions 63 6. Lis o abb e ia ions A. diadema us A aneus diadema us a.u. A bi a y uni B. mo i Bombyx mo i C. ca nea Ch ysopa ca nea Da Dal on E. coli Esche ichia coli FTIR Fou ie ans o m in a ed spec oscopy HEPES 4-(2-hyd oxye hyl)-1-pipe azinee hanesul onic acid HFA Hexa luo oace one ihyd a e HFIP Hexa luo oisop opanol M. signa a Mallada signa a MeOH Me hanol PDMS Polydime hylsiloxane RGD A ginine-glycine-aspa ic acid RH Rela i e humidi y SDS-PAGE Sodium dodecyl sul a e polyac ylamide gel elec opho esis SEM Scanning elec on mic oscopy TEM T ansmission elec on mic oscope T is 2-Amino-2-(hyd oxyme hyl)-p opan-1,3-diol w/ Mass/ olume Dependence o Mechanical P ope ies o Lacewing Egg S alks on Rela i e Humidi y 65 7. Dependence o Mechanical P ope ies o Lacewing Egg S alks on Rela i e Humidi y Dependence o Mechanical P ope ies o Lacewing Egg S alks on Rela i e Humidi y 66 Dependence o Mechanical P ope ies o Lacewing Egg S alks on Rela i e Humidi y 67 Dependence o Mechanical P ope ies o Lacewing Egg S alks on Rela i e Humidi y 68 Dependence o Mechanical P ope ies o Lacewing Egg S alks on Rela i e Humidi y 69 A i icial Egg S alks Made o a Recombinan ly P oduced Silk P o ein 76 A i icial Egg S alks Made o a Recombinan ly P oduced Silk P o ein 77 A i icial Egg S alks Made o a Recombinan ly P oduced Silk P o ein 78 A i icial Egg S alks Made o a Recombinan ly P oduced Silk P o ein 79 A i icial Egg S alks Made o a Recombinan ly P oduced Silk P o ein 80 A i icial Egg S alks Made o a Recombinan ly P oduced Silk P o ein 81 A i icial Egg S alks Made o a Recombinan ly P oduced Silk P o ein 82 Con ollable cell adhesion, g ow h and o ien a ion on laye ed silk p o ein ilms 83 9. Con ollable cell adhesion, g ow h and o ien a ion on laye ed silk p o ein ilms Con ollable cell adhesion, g ow h and o ien a ion on laye ed silk p o ein ilms 84 Con ollable cell adhesion, g ow h and o ien a ion on laye ed silk p o ein ilms 85 Acknowledgemen 93 11. Acknowledgemen Danksagung Diese A bei wu de on Juli 2008 bis Mai 2013 am Leh s uhl Bioma e ialien de Uni e si ä Bay eu h un e Be euung on P o . D . Thomas Scheibel du chge üh . Ihm möch e ich ganz he zlich ü die Möglichkei danken, dieses spannende Thema mi ielen F eihei en und Möglichkei en zu bea bei en. P o . D . Thomas Scheibel, P o . D . –Ing. Volke Al s ä und D . S e an Geime danke ich ü die Übe nahme meines Men o a s. Fü die Einweisung und Hil en sowie Au nahmen am REM und TEM danke ich D . Raine De sch, Johannes Diehl, Claudia Blüm, sowie D . S e an Geime und K is in Schach . Admi Masic und Luca Be ine i danke ich ü die Zusammena bei bei de Publika ion und die Messungen am Raman. S e anie Wohl ab will ich ü die gu e Koope a ion bei de A bei ü unse e Ve ö en lichung danken. P o . Louise Se pell und Kyle Mo is gil mein Dank ü ih e Bemühungen mi de Au nahme on Rön genbeugungsspek en, auch wenn diese nich den Weg in diese Disse a ion ge unden haben. U e Kuhn danke ich ü die TGA und DSC Messungen. Annema ie Heiduk danke ich ü die Un e s ü zung bei den Zugmessungen an Köche liegenseide im Rahmen ih e P ojek a bei . Na ü lich möch e ich mich auch bei meinen Kollegen bedanken: - Susanne Sch amm ü ih e imme eundliche und au mun e nde A . - John ü die Hil es ellungen in de An angszei und die lus igen Abende nach dem Kondi- T aining mi iso onischem Spo ge änk. - Lukas, Ma in, And ew ü das Ko ek u lesen de Pape und Diskussionen und Tipps wenn mal P obleme au a en. - Klonmas e Ande l ü die Ausbildung als sein Padawan. - Ande l und Johnobo ü ih e Hil e und Un e s ü zung beim Fe men ie en. - Den S ammkunden im BBB. Acknowledgemen 94 - Den Fußballe n ü die lus ige Ablenkung auch in s essigen Zei en. - Den Kelle kinde n. - Bei meinen Bü okollegen Da id, G ego , K is ina, Anja, Ma kus, Aniela, Elena und Claudia da ü , dass sie mich und meine Launen e agen haben und wi imme eine lus ige Zei mi einande ha en. Zu gu e Le z möch e ich meinen El e n danken, dass sie mich übe all die Zei un e s ü z haben und mi imme den Rücken ei gehal en haben. E klä ung 95 12. E klä ung: Hie mi e klä e ich eidess a lich, dass ich die o liegende A bei selbs ändig e ass , und keine ande en als die angegebenen Quellen und Hil smi el benu z habe. Fe ne e klä e ich, dass ich wede an de Uni e si ä Bay eu h noch ande wei ig mi ode ohne E olg e such habe, eine Disse a ion einzu eichen ode eine Dok o p ü ung abzulegen. Des Wei e en e klä e ich, dass ich wede bishe noch in Zukun Hil e on gewe blichen P omo ionsbe a e n bzw. – e mi le n in Ansp uch genommen habe bzw. nehmen we de. Bay eu h im Mai 2013 …………………………. Felix Baue