The Potential of Eukaryotic Cell-Free Systems as a Rapid Response to Novel Zoonotic Pathogens: Analysis of SARS-CoV-2 Viral Proteins [original]

The Potential of Eukaryotic Cell-Free

Systems as a Rapid Response to Novel

Zoonotic Pathogens: Analysis of

SARS-CoV-2 Viral Proteins

Franziska Ramm

, Srujan K. Dondapati

, Hoai Anh Trinh

, Dana Wenzel

Ruben M. Walter

, Anne Zemella

and Stefan Kubick

Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), Potsdam, Germany,

Institute of Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany,

Department of Applied Biochemistry, Institute

of Biotechnology, Technical University Berlin, Berlin, Germany,

Faculty of Health Sciences, Joint Faculty of the Brandenburg

University of Technology Cottbus–Senftenberg, The Brandenburg Medical School Theodor Fontane, The University of Potsdam,

Potsdam, Germany

The ongoing pandemic caused by the novel coronavirus (SARS-CoV-2) has led to more

than 445 million infections and the underlying disease, COVID-19, resulted in more than 6

million deaths worldwide. The scientiﬁc world is already predicting future zoonotic

diseases. Hence, rapid response systems are needed to tackle future epidemics and

pandemics. Here, we present the use of eukaryotic cell-free systems for the rapid

response to novel zoonotic diseases represented by SARS-CoV-2. Non-structural,

structural and accessory proteins encoded by SARS-CoV-2 were synthesized by cell-

free protein synthesis in a fast and efﬁcient manner. The inhibitory effect of the non-

structural protein 1 on protein synthesis could be shown in vitro. Structural proteins were

quantitatively detected by commercial antibodies, therefore facilitating cell-free systems for

the validation of available antibodies. The cytotoxic envelope protein was characterized in

electrophysiological planar lipid bilayer measurements. Hence, our study demonstrates

the potential of eukaryotic cell-free systems as a rapid response mechanism for the

synthesis, functional characterization and antibody validation against a viral pathogen.

Keywords: eukaryotic cell-free systems, SARS-CoV-2, viral proteins, rapid response, protein analytics, planar lipid

bilayer measurements

INTRODUCTION

Infectious diseases that are transferred from an animal to a human being, so-called zoonoses, can lead

to devastating health issues around the world as can be seen from the example of the severe acute

respiratory syndrome coronavirus type 2 (SARS-CoV-2). The close interaction with animals, such as

in agriculture and with domesticated animals (pets), the increasing consumption of different meats

as well as the intrusion of humans into the natural habitat of animals, causes a high risk for the

development of novel zoonoses that might lead to short lived disease outbreaks, epidemics or even

pandemics. Standard techniques such as the detection of viral antigens by polymerase chain reactions

(PCR) as well as rapid antigen tests accelerate prompt responses such as quarantines and shutting

down social contacts. Unfortunately, some pathogens, such as airborne viruses, are persistent and

have to be counteracted with vaccines and therapeutics. Consequently, the thorough characterization

of the virus itself and its mode of action, including the viral assembly, cell attack, pathogenesis of the

Edited by:

Simon J. Moore,

University of Kent, United Kingdom

Reviewed by:

Karen Marie Polizzi,

Imperial College London,

United Kingdom

Dong-Myung Kim,

Chungnam National University,

South Korea

*Correspondence:

Stefan Kubick

[email protected]aunhofer.de

Specialty section:

This article was submitted to

Synthetic Biology,

a section of the journal

Frontiers in Bioengineering and

Biotechnology

Received: 15 March 2022

Accepted: 01 April 2022

Published: 19 April 2022

Citation:

Ramm F, Dondapati SK, Trinh HA,

Wenzel D, Walter RM, Zemella A and

Kubick S (2022) The Potential of

Eukaryotic Cell-Free Systems as a

Rapid Response to Novel Zoonotic

Pathogens: Analysis of SARS-CoV-2

Viral Proteins.

Front. Bioeng. Biotechnol. 10:896751.

doi: 10.3389/fbioe.2022.896751

Frontiers in Bioengineering and Biotechnology | www.frontiersin.org April 2022 | Volume 10 | Article 8967511

ORIGINAL RESEARCH

published: 19 April 2022

doi: 10.3389/fbioe.2022.896751

underlying disease and characterization of the viral proteins are

necessary to tackle these tasks and to identify efﬁcient drugs. It is

essential to understand the different modes of action of the

individual proteins as each particular component plays a

speciﬁc role in the viral assembly, host infection and immune

invasion as well as viral replication. The novel coronavirus

expresses 10 different open reading frames (ORFs) encoding

the ORF1ab polyprotein, eight single ORFs corresponding to

eight single proteins as well as ORF7 which can be further

separated into ORF7a and b which encode two different

proteins (Figure 1A)(Kim et al., 2020;Wang et al., 2020;

Yoshimoto, 2020). The ORF1ab polyprotein encodes 16 non-

structural proteins (nsp) including proteases (nsp3 and nsp5), a

RNA-helicase (nsp13) and a RNA-dependent RNA-polymerase

(RdRp, nsp12) that are mainly responsible for viral replication

(Gao et al., 2020;Jang et al., 2020;Shin et al., 2020;Shu et al., 2020;

Yoshimoto, 2020;Zhang et al., 2020). This polyprotein is encoded

by the two ORFs ORF1a and ORF1b. A frameshift before the stop

codon in ORF1a facilitates the translation to be continued to

ORF1b and therefore resulting in the polyprotein ORF1ab (Kim

et al., 2020). The other ORFs can be divided into two major

classes, namely the structural proteins and the accessory proteins.

Structural proteins are well known and well characterized as these

proteins assemble to the viral capsid. The core structure of SARS-

CoV-2 virus is maintained by the ORF2 surface glycoprotein (S)

otherwise known as the Spike protein, the ORF4 envelope protein

(E), the ORF5 membrane glycoprotein (M) and the ORF9

nucleocapsid protein (N) (Figure 1A). The accessory proteins

of SARS-CoV-2 include membrane proteins like the putative ion

channel encoded by ORF3 (Kern et al., 2021), the type I

transmembrane protein coded by ORF7a (Rosenthal et al.,

2020) as well as the integral membrane protein ORF7b

FIGURE 1 | Cell-free synthesis of SARS-CoV-2 proteins in a CHO cell-free system. (A) Schematic ﬁgure showing the SARS-CoV-2 virus particle and its structure as

well as the coding sequences of the genome. (B) Qualitative analysis of cell-free synthesized SARS-CoV-2 proteins by autoradiography. Autoradiograph showing

C-leucine labeled proteins synthesized in a CHO system from the translation mixture (TM).

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Ramm et al. Analysis of SARS-CoV-2 Viral Proteins

(Schaecher et al., 2008). Accessory proteins ORF6 and ORF8 are

known to be involved in the host cell immune invasion and in

interferon signaling (Miorin et al., 2020;Flower et al., 2021;

Zhang et al., 2021). In some infections the protein encoded by

ORF10 is present whilst in others this protein cannot be found

which might be caused due to a read through of this ORF (Pancer

et al., 2020;Hassan et al., 2021). The proteins encoded by SARS-

CoV-2 and their characteristics are summarized in Table 1.

In order to characterize such a versatile set of proteins, a

system that can produce and characterize all kinds of proteins is

needed. Therefore, we present eukaryotic cell-free systems as a

promising methodology for the fast and efﬁcient synthesis and

characterization of viral proteins based on the example of SARS-

CoV-2 proteins. In cell-free protein synthesis a crude cell lysate

rather than viable, intact cells are used, allowing the synthesis of

“difﬁcult-to-express proteins”such as membrane proteins or

even cytotoxic proteins (Chalmeau et al., 2011;Orth et al.,

2011;Henrich et al., 2015;Thoring et al., 2017). With the

utilization of a cell lysate, the production of genetically

modiﬁed organisms becomes obsolete. As a result no high

laboratory safety standards are necessary for the cell-free

production of toxic and viral proteins. In order to avoid

cloning procedures for the generation of templates encoding

viral proteins, PCR templates can be used for cell-free

synthesis (Sawasaki et al., 2002). Thus, a fast screening of

different mutants is possible. Another advantage of some

eukaryotic cell-free systems are endogenous microsomal

vesicles derived from the endoplasmic reticulum (ER) that are

present in the lysate. These vesicles enable post-translational

modiﬁcations (PTMs) and are a natural surrounding for

membrane proteins (Brödel et al., 2014). Here, we

demonstrate that the whole set of viral proteins derived from

SARS-CoV-2 including membrane proteins, enzymes as well as

modulatory proteins can be synthesized in a lysate based on

Chinese hamster ovary (CHO) cells (Brödel et al., 2014;Thoring

et al., 2017). The synthesis of functional protein was veriﬁed by

cell-free synthesized nsp1 downregulating the in vitro synthesis of

a model protein and the demonstration of cytotoxic events as well

as single channel events in planar lipid bilayer measurements

induced by the cell-free synthesized envelope protein. Further,

the cell-free synthesized nucleocapsid protein was used to

demonstrate the possibility to quantitatively evaluate the

binding of commercially available antibodies. Taken together

eukaryotic cell-free systems, including but not limited to the

use of CHO lysate, can be applied to characterize viral proteins

and might facilitate the screening of antibodies as well as

pharmaceuticals and blockers against these viral proteins.

RESULTS

Viral pathogens such as SARS-CoV-2 induce cytotoxic effects

often associated with severe damage to the host cell. This might be

one of the major factors in the pathology and disease caused by

viruses. A valid system to characterize novel viral pathogens

should be able to synthesize and characterize structural as well

as non-structural proteins. Therefore, we used a eukaryotic cell-

free system to synthesize non-structural, structural and accessory

proteins encoded by SARS-CoV-2 (Figure 1A). Qualitative

analysis of viral proteins synthesized in a CHO cell-free

system showed that all viral proteins tested, could be

synthesized. Additionally, multimerization of proteins such as

ORF3 and ORF7a as well as ORF8 and ORF10 was visualized and

deﬁned cleavage products as seen for Spike proteins and

nucleocapsid protein were detected by autoradiography

(Figure 1B).

Quantitative analysis by hot TCA precipitation and

subsequent liquid scintillation veriﬁed the acquired data for

the qualitative analysis. Total protein yields for non-structural

proteins showed that these proteins were mainly present in a

soluble form as higher protein yields were detected in the

supernatant fraction (SN) compared to the microsomal

fraction (MF). Cell-free protein synthesis of the full length

Spike protein (ORF2/S) was conducted using a PCR template

which resulted in a lower template concentration used. This

reduced template concentration led to lower total protein

yields. Nonetheless, the high molecular weight protein could

be synthesized in an equal amount to the comparably small

TABLE 1 | SARS-CoV-2 viral proteins and their characteristics.

ORF Protein Short Form Function

1ab Nsp1 —Inhibition of protein translation

Nsp5 m

Pro

Protease

Nsp12 RdRp RNA-dependent RNA polymerase

Nsp13 RNA-Hel RNA-helicase

Nsp16 MT Methyltransferase

2 Surface glycoprotein Spike/S Binding of host cells

3ORF3 —Ion channel

4 Envelope E Facilitates assembly

5 Membrane protein M Interacts with E, S and N to form stable assembly

6ORF6 —Immune invasion

7 ORF7a —Virus-host interaction, the type I transmembrane protein

ORF7b —Virus-host interaction, integral membrane protein

8ORF8 —Immune invasion

9 Nucleocapsid protein N Genome packaging

10 ORF10 —Unclear

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Ramm et al. Analysis of SARS-CoV-2 Viral Proteins

envelope protein (ORF4/E). The initial data for the synthesis of

the accessory proteins showed that all of these proteins could be

synthesized as well. The immunomodulatory protein encoded by

ORF6 showed the overall lowest protein yield of 6 μg/ml while the

transmembrane protein encoded by ORF3 showed the highest

protein yields with 25 μg/ml (Figure 2A).

In order to increase the protein yields in a continuous-

exchange cell-free (CECF) system, one representative protein

of the three protein groups was synthesized for 24 h. The

nsp12 coding for an RNA-dependent RNA polymerase

(RdRp), the ORF5 membrane glycoprotein and the channel-

like ORF3 were chosen (Figure 2B). The protein yields from a

batch-based synthesis could be increased by about 20, 35 and 50

fold for ORF5, ORF3 and nsp12, respectively, in a 24 h CECF

reaction. Apparently, the nsp12 enzyme was not suitable for a

CECF reaction as the soluble protein aggregated in the MF which

suggests that a batch-based reaction was more suitable for this

enzyme. These data indicate that CFPS offers a platform for the

rapid synthesis and analysis of SARS-CoV-2 proteins. As each

protein showed different requirements for the cell-free synthesis,

the open cell-free system offers an easy way to adapt the synthesis

conditions to the need of each individual protein.

To further show the applicability of CFPS as a rapid response

system for viral pathogens, we analyzed the individual protein

groups and analyzed the functionality of proteins of interest. In a

ﬁrst step, cell-free synthesized nsp1 protein was characterized.

This protein is also known as the leader protein responsible for

the inhibition of host protein translation (Banerjee et al., 2020;

Thoms et al., 2020;Lapointe et al., 2021). It was further shown

that nsp1 did not decrease the translation of viral mRNA

(Banerjee et al., 2020), thus nsp1 was synthesized without any

alterations in the cell-free synthesis scheme. The nsp1 protein was

pre-synthesized in a cell-free manner and was added to the cell-

free synthesis of the model protein enhanced yellow ﬂuorescent

protein (eYFP). Subsequently, the ﬂuorescence intensity of eYFP

was measured during a 3 h synthesis time (schematic

representation in Figure 3A). The ﬂuorescence signal of eYFP

without the supplementation of nsp1 was set as a baseline value of

100%. All other data were normalized to this intensity. In an

initial experiment pre-synthesized nsp1 from a CECF reaction, a

volume equivalent NTC and ORF6 as a protein control were

added to the eYFP synthesis. These data showed that the addition

of nsp1 at concentrations of 1000 and 600 nM reduced the

ﬂuorescence intensity of eYFP in a concentration dependent

manner. 1000 nM of nsp1 led to a complete inhibition of

eYFP ﬂuorescence. Unfortunately, the addition of the NTC

and ORF6 at 600 nM showed interactions with the eYFP

ﬂuorescence. Nonetheless, 600 nM nsp1 induced the highest

FIGURE 2 | Quantitative analysis of cell-free synthesized SARS-CoV-2 proteins. Viral proteins were synthesized in CHO lysate in (A) batch-based reaction and (B) a

CECF reaction. The fold increase of total protein yield from a batch reaction to a CECF reaction is shown. Quantitative analysis of

C-labeled cell-free synthesized

proteins was performed by liquid scintillation counting. Standard deviations were calculated from triplicate analysis. The translation mixture (TM) was separated into the

soluble proteins in the supernatant (SN) and the microsomal fraction (MF). The template for the full length Spike protein was based on a PCR-template (*).

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Ramm et al. Analysis of SARS-CoV-2 Viral Proteins

ﬂuorescence decline (Supplementary Figure S1). Therefore, the

reaction conditions were optimized and a batch-based reaction

was used. Two concentrations (25 and 90 nM) of nsp1 protein

were added to the eYFP synthesis. The NTC was administered in

a volume equivalent to the highest nsp1 concentration. Starting

after about 20 min reaction time, the ﬂuorescence intensity of the

eYFP slowly decreased when nsp1 was supplemented to the

reaction but no speciﬁc effect could be seen for an NTC

supplementation (Figure 3B). A concentration of 90 nM of

nsp1 decreased the eYFP ﬂuorescence to less than 60%, while

25 nM nsp1 decreased the eYFP ﬂuorescence to about 75%. This

suggested a concentration dependent effect. Strikingly, the

inhibitory effect of nsp1 weakened over time so that the

ﬂuorescence intensity of eYFP increased again (Figure 3B).

Nonetheless, the translation inhibition effect of nsp1 could

be shown.

The characterization of non-structural viral proteins is

essential for a rapid response to a virus. Nonetheless, the virus

needs accessory proteins to stably infect the host. In SARS-CoV-2

these are known to trigger a variety of different interactions

within the host such as the involvement in the interferon

signaling pathway (Miorin et al., 2020;Yuen et al., 2020),

causing an immune invasion (Flower et al., 2021) or acting as

membranous channel-like proteins in order to disrupt the host`s

cell homeostasis (Yoshimoto, 2020;Kern et al., 2021). As shown

in Figure 1, all accessory proteins could be synthesized in a cell-

free manner and showed that cell-free protein synthesis can be a

tool to rapidly analyze the synthesis of such proteins, to identify

optimal synthesis conditions as well as to assess their solubility. In

the beginning of a pandemic, the role of such proteins is not yet

fully known, therefore we tested whether a signal peptide

interferes with a deﬁned protein or might even inhibit the

translation of such a protein. Accordingly, ORF3, ORF6,

ORF7b, ORF8, and ORF10 were synthesized with and without

a Melittin (Mel) signal peptide, which typically allows for the co-

translational translocation. As some proteins might inhibit the

protein translation machinery, the Mel signal peptide allows for

the translocation of the protein into the vesicles present in the

cell-free lysates, enabling protein translation. All constructs with

and without a signal peptide were fractionated into the soluble

proteins (SN) and the proteins in the microsomal fraction (MF).

In general, our data indicated that all ORFs were more stably

expressed in the presence of a signal peptide (Figure 4A). The

NCM-ORF3 construct was based on a PCR template which might

have resulted in a lower overall translation efﬁciency due to a

lower template concentration. Strikingly, ORF10 protein

translation could not be quantitatively detected without a

signal peptide (Figure 4A, red box). In the presence of a Mel

signal peptide, protein yields of 13.7 μg/ml were detected for

ORF10 suggesting a better translation initiation. ORF7a is known

to harbor a native signal peptide as it encodes a type I

transmembrane protein (Rosenthal et al., 2020). Comparing

the native signal peptide (Nat-SP) with the Mel signal peptide,

the native signal peptide showed a higher total amount of protein

FIGURE 3 | Functional analysis of nsp1. (A) Schematic scheme of nsp1 functionality assay. (B) Application of cell-free synthesized nsp1 at 90 and 25 nM to the cell-

free protein synthesis of the model protein eYFP. Data were normalized to eYFP ﬂuorescence signal. Standard deviation was calculated from two experiments.

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Ramm et al. Analysis of SARS-CoV-2 Viral Proteins

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