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. 2021 Apr 29;16(4):e0250610. doi: 10.1371/journal.pone.0250610

Fast and efficient purification of SARS-CoV-2 RNA dependent RNA polymerase complex expressed in Escherichia coli

Clément Madru 1,#, Ayten Dizkirici Tekpinar 1,2,#, Sandrine Rosario 1, Dariusz Czernecki 1,3, Sébastien Brûlé 4, Ludovic Sauguet 1,*, Marc Delarue 1,*
Editor: Filippo Prischi5
PMCID: PMC8084133  PMID: 33914787

Abstract

To stop the COVID-19 pandemic due to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which caused more than 2.5 million deaths to date, new antiviral molecules are urgently needed. The replication of SARS-CoV-2 requires the RNA-dependent RNA polymerase (RdRp), making RdRp an excellent target for antiviral agents. RdRp is a multi-subunit complex composed of 3 viral proteins named nsp7, nsp8 and nsp12 that ensure the ~30 kb RNA genome’s transcription and replication. The main strategies employed so far for the overproduction of RdRp consist of expressing and purifying the three subunits separately before assembling the complex in vitro. However, nsp12 shows limited solubility in bacterial expression systems and is often produced in insect cells. Here, we describe an alternative strategy to co-express the full SARS-CoV-2 RdRp in E. coli, using a single plasmid. Characterization of the purified recombinant SARS-CoV-2 RdRp shows that it forms a complex with the expected (nsp7)(nsp8)2(nsp12) stoichiometry. RNA polymerization activity was measured using primer-extension assays showing that the purified enzyme is functional. The purification protocol can be achieved in one single day, surpassing in speed all other published protocols. Our construct is ideally suited for screening RdRp and its variants against very large chemical compounds libraries and has been made available to the scientific community through the Addgene plasmid depository (Addgene ID: 165451).

Introduction

The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected millions of people with a death toll exceeding two million worldwide [1–4]. SARS-CoV-2 is an enveloped, single-stranded virus with a positive-sense RNA genome [5, 6]. One of the most promising druggable targets is the RNA-dependent RNA polymerase (RdRp), a central element of SARS-CoV-2 life cycle, responsible for the transcription and replication of the ~30kb genome [7–10]. RdRp from coronaviruses are error-prone enzymes [11], recognizing various modified nucleosides analogs (NAs) as substrate as well. Such NAs may disrupt viral RNA synthesis via chain termination, making them important candidates as anti-viral agents [12–14]. Early efforts to discover treatments were focused on evaluating the efficacy of already known NAs against SARS-CoV-2. Among them, Remdesivir was initially approved to treat Ebola [15] and received U.S. Food and Drug Administration (FDA) approval for COVID-19 treatment, while Favipiravir, currently licensed in Japan for use in the treatment of influenza virus [16], is being evaluated in clinical trials [17, 18]. Considering the global impact of the COVID-19 pandemic, the new variants appearance and the possibility of re-emergence of coronavirus infections in the future, there is an urgent need to develop new antiviral agents specifically targeting the RdRp involved in pivotal steps of SARS-CoV-2 pathogenesis.

The production of sufficient amounts of heterologous RdRp with a native structure and full biological activity is a prerequisite for the discovery, optimization and comprehensive evaluation of new drugs directed against SARS-CoV-2, including in High Throughput Screening (HTS) assays involving huge chemical libraries. RdRp is composed of 3 viral non-structural proteins (nsp) named nsp7, nsp8 and nsp12. The core component nsp12 hosts the catalytic polymerase activity, greatly enhanced by the two accessory subunits nsp7 and nsp8 [19, 20]. RdRp has been a subject of intensive structural biology efforts, yielding high resolutions cryo-EM structures of the RdRp in its apo form [21, 22], or bound with RNA [23–25], inhibitors [24–27], and to other factors [28–30]. These structures showed the nsp12 core bound to a heterodimer nsp7-nsp8 and an additional nsp8 at a different binding site. The two nsp8 copies expose long N-terminal alpha helices that slide along the exiting RNA to prevent premature dissociation [23]. The production of intact and correctly assembled (nsp7)(nsp8)2(nsp12) complexes is therefore required to get fully active and processive polymerase.

The main strategies employed so far for the overexpression of recombinant RdRp consists in expressing and purifying the catalytic nsp12 subunit and the accessory nsp7-nsp8 subunits separately before assembling the complex in vitro [11, 22, 24, 28–30]. However, while nsp7 and nsp8 express readily in Escherichia coli, nsp12 shows limited solubility in bacterial expression systems and is often produced in insect cells [22, 23, 25–27]. These approaches multiply the protein expression and purification steps, making RdRp isolation cost- and time-consuming. Here, we describe an alternative strategy to co-express the SARS-CoV-2 RdRp directly in E. coli, using a single plasmid. Characterization of the purified recombinant SARS-CoV-2 RdRp using analytical ultracentrifugation assays (AUC) revealed that it forms an heterotetramer with the expected stoichiometry. RNA polymerization activity was measured using primer-extension assays and showed that the purified enzyme is indeed functional. This approach provides a useful alternative to more expensive and complicated protein expression systems, and offers many practical advantages inherent to bacterial systems, such as easy generation of mutants and simple cultivation handling. Our fast, single-day purification protocol results in a stable and active complex that can be used in most protein biochemistry laboratories for drug screening as well as for functional studies.

Materials and methods

Optimization of recombinant RdRp expression

Cloning

The open reading frames (ORF) of the nsp7, nsp8 and nsp12 genes from SARS-CoV-2 virus (GenBank: MN908947.3) were optimized for expression in E. coli, synthesized commercially by geneArt (Thermo Fisher) and inserted into a modified pRSFDuet-1 (Novagen), resulting in pRSFDuet-1(14his-nsp8/nsp7)(nsp12) and the pRSFDuet-1(14his-nsp12)(nsp7/nsp8) (S1 and S2 Figs in S1 File).

Optimal inducer concentration

BL21 Star (DE3) strain from E. coli (Thermo Fisher) containing pRSFDuet-1(14His-nsp8/nsp7)(nsp12) was grown overnight in Lysogeny broth medium supplemented with 100 μg/mL kanamycin (LBK). A fresh culture was then inoculated (1:100) and incubated at 37°C, 180 rpm. When its optical density at 600 nm (OD600) reached 0.6, the culture was divided into 2 series of 6x10 mL in 50 mL tubes, and the induction was made individually with IPTG concentrations of 0, 0.05, 0.1, 0.2, 0.5 and 1 mM. After 3 hours at 37˚C and 20 hours at 20˚C, OD600 was measured and 1 mL aliquots were centrifuged. Cells were resuspended in 1X Lithium dodecyl sulfate (LDS) sample buffer (Invitrogen) supplemented with 10 mM dithiothreitol (DTT) at a concentration of 10 UOD600/mL. Samples were then vortexed for 30 sec, boiled for 5 min and centrifuged for 2 min at 20 000 g. 5 μL of supernatant were finally loaded on SDS-PAGE 4–20%.

Optimal post-induction temperature and post-incubation time

A culture of BL21-Star-(DE3)-pRSFDuet-1(14his-nsp8/nsp7)(nsp12) was grown at 37°C, 180 rpm, and split into 3x100 mL in 500 mL flasks at OD600 0.6. The recombinant protein expression was then induced by adding 0.05 mM IPTG at 20°C, 30°C and 37°C, 180rpm. OD600 were measured and 1 mL aliquots were taken after 2, 4, 6, 8 and 20 hours. Loading samples for SDS-PAGE analysis were prepared as described above.

Large scale production of recombinant RdRb

100 ng of pRSFDuet-1(14-his-nsp8/nsp7)(nsp12) were added to 50 μL chemically competent BL21 Star (DE3) and incubated for 30 min in ice. Cells were heat-shocked for 30 sec at 42°C and incubated in ice for 2 min. 900 μL of super optimal broth (SOC) medium were added and the mixture incubated at 37°C for 1 h. 200 mL of LBK were then inoculated with the transformation reaction in 1 L flask, and cells were grown at 37°C overnight with 180 rpm agitation. 30 mL of the overnight culture were inoculated (1:100) into 1 L of LBK in 5 L flasks and incubated at 37°C with 180 rpm agitation for a few hours until OD600 reaches 0.6. Flasks were then placed for 15 min at 4°C and recombinant protein expression was induced by adding 0.05 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) and incubating 20 hours at 20°C with 180 rpm agitation. Cells were harvested by centrifugation, washed once with fresh LB, and stored at -80˚C.

Purification of recombinant RdRp

Cells were resuspended in the HisTrap buffer A (50 mM Na-HEPES at pH 8, 500 mM NaCl, 10 mM imidazole) supplemented with complete EDTA-free protease inhibitors (Thermo Fisher) and 500 units of benzonase (Sigma) at 4˚C. Resuspended cells were then lysed by mechanical disruption with 3 passes through a pre-cooled cell disruptor (Constant System Limited) at 1.4 kPa, and the lysate was centrifuged at 20 000 g for 30 min at 4˚C. All the following steps described below were performed with chromatography columns from GE Healthcare connected to an ÄKTA pure system (GE Healthcare) at 4˚C. After centrifugation, the clear supernatant containing the RdRp complex was loaded onto a 5 mL HisTrap nickel affinity column (GE Healthcare). The column was then washed with 25 mL of 5% HisTrap buffer B (50 mM Na-HEPES pH 8, 500 mM NaCl, 500 mM imidazole). The complex was finally eluted using a 50 mL linear gradient of imidazole (5%-100% HisTrap buffer B). Fractions were analyzed by SDS-PAGE 4–20%, and DNA contamination was detected by measuring the ultraviolet (UV) absorption spectra and the ratio of absorbance at 260 nm vs 280 nm (A260/280). RdRp-containing HisTrap fractions were combined and 5-fold diluted in a 50mM Na-HEPES pH 8 solution before being loaded onto a 5 ml HiTrap Q HP anion exchange column (GE Healthcare), pre-equilibrated in the HiTrap Q buffer A (50 mM Na-HEPES pH 8, 150 mM NaCl). The column was washed with 25 mL of HiTrap Q buffer A while protein complex was eluted with a 50 mL linear gradient of NaCl realized by mixing HiTrap Q buffer A with HiTrap Q buffer B (50 mM Na-HEPES pH 8, 1 M NaCl). The purest fractions containing RdRp complex were combined and concentrated up-to 5 mg/mL using Amicon Ultra‐4 centrifugal filter units 30 000 NMWL (EMD Millipore). The purification final step involved a size-exclusion chromatography on a Superdex 200 10/300 equilibrated in S200 buffer (20 mM Na-HEPES pH 8, 300 mM NaCl, 1 mM MgCl2). RdRp containing fractions were then combined, concentrated up-to 3 mg/mL, flash-frozen in liquid nitrogen and stored at -80˚C.

Analytical ultracentrifugation assays

Sedimentation velocity experiment was performed with a Beckman Coulter Optima analytical ultracentrifuge (Beckman-Coulter, USA) with an An-60 Ti rotor at 20°C. The freshly purified RdRp complex at a concentration of 2.7 mg/ml was centrifuged at 35,000 rpm in 3-mm double-sector epoxy centerpieces. 100 scans were collected at 1 min intervals with a radial step size of 0.001cm. Detection of the protein complex as a function of radial position and time was performed by absorbance measurements at 250 nm, 280 nm and by interference detection. Profiles were analyzed using the continuous (s) distribution model of the software Sedfit [31]. The partial specific volume of the protein, of 0.733 was theoretically calculated in Sedfit. The buffer viscosity of 0.01046 Poise and the buffer density of 1.0128 were respectively determined with the Viscosizer TD (Malvern Panalytical, UK) and the DMA 5000 M (Anton Paar).

RNA primer extension assays

The RNA duplex was prepared by mixing a 40-mer RNA template (5’-CUAUCCCCAUGUGAUUUUAAUAGCUUCUUAGGAGAAUGAC-3’) corresponding to the 3’ end of SARS-CoV-2 genome with a 20-mer fluorescent RNA primer (5’-FAM-GUCAUUCUCCUAAGAAGCUA-3’) in water. The mix was then heated for 2 min to 70 °C and slowly cooled to room temperature. Primer extension assays were performed with 500 nM of purified RdRp complex in the presence of 50 μM NTPs and 50 nM RNA duplex, in a reaction buffer containing 20 mM Na-HEPES pH 8, 50 mM NaCl, and 3 mM MgCl2. The reaction was carried out in 150 μL at 37°C. After 30 sec, 1, 5, 10, 15, and 30 min, 10 μL were pipetted from the reaction mix and immediately diluted in 20 μL of formamide gel-loading buffer (10 mM EDTA, 0.1 mg/mL Xylene Cyanol, 0.1 mg/mL bromophenol blue, 95% formamide). Reactions were then stopped by boiling samples for 3 min at 90°C. 5 μL samples were loaded in 40 x 30 cm urea-polyacrylamide (8%) gel and separated for 3 hours at 1500V. Images were taken using a typhoon FLA 9000 (GE Healthcare). For the primer extension assay in presence of RdRp eluted from nickel affinity chromatography, the concentration of the native (nsp12)(nsp8)2(nsp7) could not be accurately determined due to an excess of nsp7 and nsp8. The fraction used for the activity assay showed an A280 of 8.0 after 1 hour of dialysis in S200 buffer.

Results and discussion

Optimization of recombinant RdRp expression

Recombinant vector construction

The nsp7, nsp8 and nsp12 genes were optimized for recombinant expression in E. coli, and inserted into a modified pRSFDuet-1 vector, allowing the expression of 14 histidine (14his) N-fused proteins. This vector encodes two multiple cloning sites (MCS1 and 2), each of which is preceded by a T7 promoter, lac operator, and ribosome binding site (RBS). Two different cloning strategies were employed to express the RdRp complex, giving two plasmids named pRSFDuet-1(14his-nsp12)(nsp7/nsp8) and pRSFDuet-1(14his-nsp8/nsp7)(nsp12) (Fig 1A). The pRSFDuet-1(14his-nsp12)(nsp7/nsp8) was first evaluated to achieve the production of recombinant RdRp. Early expression and purification trials yielded ~0.2 mg of recombinant RdRp per liter of culture. The purified complex was functionally active but partially proteolyzed, exhibiting a fourth band on SDS-PAGE analysis after four purification steps (S3 Fig in S1 File). This additional band corresponding to an approximately 12 kDa protein was identified as the globular C-terminal part of nsp8, showing that the N-terminal region of nsp8 (~80 amino-acids) is degraded during the purification process. This flexible region becomes ordered when the RNA duplex exits from the enzyme’s active site, making extensive interactions with the RNA molecule [23]. This proteolysis could not be suppressed despite all our efforts and optimizations. Therefore, we designed another construct, the pRSFDuet-1(14his-nsp8/nsp7)(nsp12), which was tested for RdRp production and showed promising results (Fig 1B). Indeed, only the three expected bands nsp7, nsp8 and nsp12 are observed in SDS-PAGE analysis from the first affinity purification step. Besides avoiding the purification of proteolyzed nsp8 by fusing the tag to the sensitive region, this specific arrangement also leads to an increased production of the catalytic subunit nsp12, often limited in bacterial system. Indeed, the mRNA transcripts of the second MSC2 can be generated independently of the “read-through” transcript from the first promoter. Purification assays using the pRSFDuet-1(14his-nsp8/nsp7)(nsp12) construct yield ~1 mg of pure full-length complex per liter of culture, thereby allowing a markedly improved RdRp production compared to the pRSFDuet-1(14His-nsp12)(nsp7/nsp8) construct.

Fig 1. Recombinant expression vectors.

Fig 1

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Top: ORF arrangement in pRSFDuet-1(14his-nsp8/nsp7)(nsp12) (A) and pRSFDuet-1(14his-nsp8/nsp7)(nsp12) (B) employed in this study. Nsp7, nsp8 and nsp12 ORFs were cloned in a modified pRSFDuet-1, allowing the expression of TEV-cleavable 14xhistidine (14his-TEV) N-fused protein. The vector encodes two multiple cloning sites (MCS1 and MCS2) each of which is preceded by a T7 promoter (T7), a lac operator (LacO) and a ribosome binding site. The fully annotated plasmid maps are given in S1 Fig in S1 File. Below: representative analysis by SDS-PAGE (4–20%) of the purified RdRp, purification pathway and yields are given for each plasmid.

Optimal inducer concentration, induction temperature and post-induction incubation time

The yield of BL21-Star-(DE3)-pRSFDuet-1(14his-nsp8/nsp7)(nsp12) cells was optimized for IPTG concentration, incubation time and temperature. First, the influence of inducer concentration was monitored by adding IPTG at OD600 0.6 to the final concentrations of 0.05, 0.1, 0.2, 0.5 and 1 mM. As depicted in Fig 2A, varying IPTG concentration did not significantly affect the recombinant expression level at 20˚C for 20 hours and 37˚C for 3 h. It was however set to the lowest value 0.05 mM, because of its negative effect on cell growth.

Fig 2. Optimization of the SARS-CoV-2 RdRp recombinant expression in E. coli.

Fig 2

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(A) RdRp recombinant expression levels and cell growth at various IPTG concentrations. IPTG concentrations of 0.05, 0.1, 0.2, 0.5, and 1 mM were added to the culture in the mid-exponential growth phase. After 3 hours incubation at 37°C (left) or 20 hours at 20°C (right), the total cell extracts were analyzed on SDS-PAGE (4–20%) and the cell biomass productions were determined. Error bars represent 1 SD (n = 3). (B) Comparison of RdRp production and cell growth at various post-induction incubation times and temperatures. Total cell lysates were analyzed on SDS PAGE (4–20%) and final biomass were determined after 2, 4, 6, 8 and 20 hours at 20˚C, 30˚C and 37˚C. Error bars represent 1 SD (n = 3).

Effects of post-induction temperature and post-induction incubation time on RdRp production were also investigated. After induction with 0.05 mM IPTG, cells were incubated at 20˚C, 30˚C and 37˚C for 2, 4, 6, 8 and 20 hours and lysed; the lysates were analyzed on SDS-PAGE (Fig 2B). 20 hours incubations at 20˚C and 30˚C have given highest ratios of recombinant RdRp in cell lysate per liter of culture. In order to choose the best incubation temperature, purification of recombinant RdRp from 20°C and 30°C E. coli cultures were performed using the same protocol (see below). While the final yields were similar, the complex purified from the 20°C-incubated cells appeared purer, with fewer host contaminants in eluted fractions from the first purification step (S4 Fig in S1 File).

Purification of recombinant RdRp

The purification protocol consists of three successive chromatographic steps, including nickel affinity, anion exchange, and size exclusion columns. Nsp8 is fused to an N-terminal 14his-tag, enabling large-scale purification of the recombinant RdRp using nickel affinity chromatography. As shown in Fig 3A, weakly bound proteins such as host proteins were mostly washed out using a low concentration of imidazole (30 mM), while the eluted fractions (75–400 mM) showed 3 bands on SDS-PAGE, at the range of 105 kDa, 25 kDa and 10 kDa, corresponding respectively to nsp12, 14his-nsp8 and nsp7. Relative band quantification revealed a large excess of the 14his-nsp8 and nsp7 accessory subunits compared to nsp12. An HiTrap Q ion exchange chromatography is thereby needed to remove the accessory subunits excess. The elution profile exhibits a unique peak containing the RdRp complex while small subunits alone flow through the column at pH 8 in 150 mM NaCl (Fig 3B). The final step of purification uses a size-exclusion chromatography step that results in one single peak (Fig 3C). An overall yield of 1.0 ± 0.2 mg of complex is obtained per liter of E. coli culture. The UV spectrum of the purified complex did not reveal DNA contamination with a A260/A280 ratio of 0.6. Depending on the applications, the N-terminal 14his-tag fused to nsp8 can be removed following TEV-protease cleavage. We recommend to do so after the HiTrapQ step. The RdRp could thus be incubated overnight with TEV-protease at 4˚C in the presence of 1mM DTT before the final size exclusion chromatography. However, we did not verify RdRp activity after cleavage in the present study. While this manuscript was under review, a method for producing tag-free SARS-CoV-2 RdRp in E. coli has been reported by Dangefield et al. [32]. Their approach requires many more steps than traditional purification of tagged proteins, but yields 7 mg of active enzyme per liter of culture.

Fig 3. Purification of the recombinant SARS-CoV-2 RdRp complex.

Fig 3

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Representative elution profiles and associated SDS-PAGE (4–20%) analysis from each purification step. (A) Nickel affinity chromatography. Elution was performed with an imidazole gradient (10–500 mM). (B) Anion exchange chromatography. Elution was performed with an NaCl gradient (150–1000 mM). Fractions containing nucleic acids are indicated with black arrows. (C) Size exclusion chromatography in 20 mM Na-HEPES pH 8, 300 mM NaCl, 1 mM MgCl2.

Biophysical and functional characterization of the purified RdRp complex

Analytical ultracentrifugation revealed that the purified RdRp is homogeneous, exhibiting a main peak at 280 nm, with a sedimentation coefficient of 6.2 S. The combination of the sedimentation coefficient and the integration of absoprtion at 250 nm, 280 nm as well as the interference signal allows to resolve the complex stoichiometry using our in-house protocols. The molecular mass of 160 kDa displayed by Sedfit and the peak integration analysis are fully compatible with the expected (nsp7)(nsp8)2(nsp12) stoichiometry with a theoretical molecular mass of 169 kDa including the two flexible 14His tags (4 kDa). Also, the frictional ratio of 1.55 suggests that the heterotetramer is slightly elongated (Fig 4A).

Fig 4. Biophysical and functional characterization of the recombinant SARS-CoV-2 RdRp complex.

Fig 4

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(A) Sedimentation distribution profile of recombinant RdRp complex by sedimentation velocity. The main peak at a concentration of 2.7 mg/mL (16.5 μM) shows a sedimentation coefficients of 6.2 S, compatible with the expected (nsp7)(nsp8)2(nsp12) stoichiometry. (B) RNA primer extension assay with the recombinant RdRp. The recombinant RdRp complex shows polymerase activity in vitro, extending a 20-mer primer strand labeled with a fluorescent dye at the 5′ end (FAM-primer). A 5’-labeled template has been also loaded as a control (FAM-template). (C) RNA primer extension assay with various purified RdRp samples. Reactions were carried out for 30 min with RdRp eluted from nickel affinity chromatography (Histrap), with purified RdRp kept at +4˚C (+4˚C), with purified RdRp flash-frozen in liquid nitrogen and stored at -20˚C (-20˚C), and with purified RdRp flash-frozen in liquid nitrogen with 50% glycerol and stored at -20˚C (-20˚C +gly).

In addition, an RNA primer extension assay was performed to verify whether the purified RdRp was active. The purified RdRp was incubated for 1 hour with a 40-mer RNA template mimicking the 3’-extremity of the viral genome, primed by a fluorescently-labeled 20-mer RNA. As shown in Fig 4B, the purified RdRp is active and mediates primer-dependent RNA elongation. Moreover, both fresh and thawed protein samples showed similar activity, suggesting that freezing does not affect enzyme activity (Fig 4C). Finally, we showed that the RdRp eluted from the nickel pseudo-affinity chromatography is already active (Fig 4C). Albeit containing an excess of the nsp7 and nsp8 subunits, active RdRP can be obtained from one single purification step. In particular, this approach offers many practical advantages for generating and screening RdRp variants. This could facilitate the first steps of the high throughput mutant characterization by shortening the purification time at the cost of sample purity.

In summary, the quality of the purified RdRp has been fully assessed using biophysical and biochemical assays. The sample has the expected stoichiometry, is homogeneous and is functionally active.

Conclusion

Motivated by E. coli’s broad accessibility, ease of culture, rapid growth rates and proven scalability, we developed an efficient expression and purification system for the SARS-CoV-2 RdRp complex in this bacterial host. Active RdRp can be immobilized and isolated from bacterial lysate by using one single purification step (nickel affinity), thus facilitating high-throughput screens and biochemical studies. Furthermore, the three-step purification protocol yields an intact and correctly assembled (nsp7)(nsp8)2(nsp12) complex that should be suitable for many applications, including structural studies. Our construction has been made available to the entire scientific community through the Addgene plasmid repository (Addgene ID: 165451) upon publication of this manuscript.

Supporting information

S1 File

(PDF)

Click here for additional data file. (598.8KB, pdf)

Acknowledgments

We would like to thank Dr. Bertrand Raynal (Molecular Biophysics Platform, C2RT, Institut Pasteur) for helping us with AUC data analysis. We wish also to thank Dr. Margarida Gomes for helpful advices for molecular biology.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was funded by the Institut Pasteur, and by an ANR JCJC grant ANR-17-CE11-0005-01. The post-doctoral fellowship of C.M is funded by the Pasteur-Roux-Cantarini fellowship from the Institut Pasteur. The fellowship of D.C was funded by Sorbonne University ED515.

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Decision Letter 0

Filippo Prischi

14 Sep 2020

PONE-D-20-24456

Fast and efficient purification of SARS-CoV-2 RNA dependent RNA polymerase complex expressed in Escherichia coli

PLOS ONE

Dear Dr. Sauguet,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Please find the reviewers' comments appended below. While all reviewers agree that the work presented in this paper could have a high impact and the potential to significantly facilitate future drug discovery studies on SARS-CoV-2, they have raised points that need to be addressed. Overall, I think very useful and detailed comments have been provided by the reviewers. 

==============================

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Filippo Prischi

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

Reviewer #3: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript by Madru et al. describe a new strategy to purify SARS-CoV-2 RdRp (nsp7/82/12) in E. coli and provide biochemical characterization to demonstrate the quality of the purified, recombinantly expressed RdRp. This study outlines a fast purification scheme for the RdRp which may have applications in general RdRp research and RdRp inhibitor drug discovery. The study uses a series of chromatographic steps to purify the RdRp followed by analytical centrifugation, negative-stain electron microscopy (EM), and primer extension assays to test the quality of the RdRp.

I do feel that a pipeline that would lead to a high yield of the holo-RdRp ((nsp7/82/12) expressed in bacteria would greatly facilitate the efforts many researches trying to screen for inhibitors, so the significance is high and I appreciate's this group's intent. However, the data here do not support that the authors do indeed get a higher yield of protein that the other protocols using bacterial expression data. Indeed, 100 µg from 1.5 L seems rather low and the methods used to purify are not that different from already published methods.

Major critiques

1. The purification described in the paper is largely similar to that described in:

Chen J, Malone B, Llewellyn E, et al. Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex [published online ahead of print, 2020 Jul 28]. Cell. 2020;S0092-8674(20)30941-7. doi:10.1016/j.cell.2020.07.033

2. Authors do not reference the appropriate papers and citations are misplaced throughout the manuscript. The following are a few examples out of many where citations are lacking, incorrect, or misplaced:

i. Page 3 - “So far, Remdesivir, Favipiravir, Ribavirin, Galidesivir, and EIDD-2801, have been shown to efficiently inhibit SARS-CoV-2 replication in cell-based assays (14–17) but their efficiency in humans remains to be assessed, rendering the search for new inhibitors still of interest.”

- Reference 17 does not have relevance to this statement.

ii. Page 3 – “RdRp is composed of 3 viral non-structural proteins (nsp) named nsp7, nsp8 and nsp12. The core component nsp12 hosts the catalytic polymerase activity (18) which is greatly enhanced by the two accessory subunits nsp7 and nsp8 (8,19).”

- Please cite Kirchdoerfer and Ward, 2019 and Reference 18 (Subissi et al., 2014) since these studies were the first to demonstrate the importance of nsp7 and nsp8 for nsp12 activity. Remove references 8 and 19 since they are not relevant here.

iii. Page 3 – “Recently, RdRp has been a subject of intensive structural biology efforts, yielding high resolutions cryo-EM structures of the RdRp apo form (20,21), bound with RNA (22,23), and with inhibitors (23,24).”

- Reference 21 is misplaced. The structure presented in this reference contains RNA. Remove reference 24 as it does not pertain to the sentence. Also add “bound to other factors” (cite Chen et al. 2020).

iv. Page 3 – “The production of sufficient amounts of heterologous RdRp with a native structure and full biological activity is a prerequisite for the discovery, optimization and comprehensive evaluation of new drugs directed against SARS-CoV-2, including in High Throughput Screening (HTS) assays involving very large chemical libraries (25).”

- Reference 25 is about the structure PolD complexes and does not have any relevance to HTS.

v. Page 3 – “The main strategies employed so far for the overexpression of recombinant RdRp consists in expressing and purifying the 3 subunits separately before assembling the complex in vitro (19,22).”

- References 19 through 22 all purify nsp7, nsp8, and nsp12 separately to and reconstitute the RdRp in vitro. Please add these citations to this statement. Also Cite Chen et al. 2020 as well as they also purified separately and reconstituted.

vi. Page 3 – “Moreover, while nsp7 and nsp8 express readily in Escherichia coli, nsp12 shows limited solubility in bacterial expression systems and is often produced in insect cells (20,22,24).”

- Reference 24 does not belong here. Cite Chen et al. 2020 as well since they use E. coli to express these proteins.

vii. Page 9 – “Recent structural studies have revealed that this N-terminal region of nsp8 is flexible and gets ordered when the RNA duplex exits from the enzyme’s active site (22).”

- Please cite Reference 21 (Hillen et al. 2020) instead of Reference 22 as Hillen et al. made the first structural observations that the N-terminus of nsp8 contacts the upsteam RNA.

viii. Page 9 – “Yet, this region is not required for RNA polymerase activity but improves its processivity by perpetuating the interactions with the RNA backbone (22).”

- Cite Reference 18 (Subissi et al., 2014) not Reference 22 since Subissi et al. showed that nsp7 and nsp8 are important for polymerase processivity.

ix. Page 14 – “(C) Two orthogonal views of the cryo-EM structure of the SARS-CoV-2 RdRp (PDB code: 6YYT) (22).”

- The reference cited for PDB 6YYT is wrong. Please correct to Reference 21 (Hillen et al. 2020).

3. Authors claim to have a yield of “~100 μg pure RdRp complex” (Page 9) after size exclusion chromatography. However, the sample is not “pure” as it contains a significant proteolyzed product (nsp8-CTD) that is clearly visible after size exclusion chromatography (Figure 2D).

4. Authors claim that “the complex [purified RdRp] has an extended shape, consistent with the RdRp structure” (Page 9) and references Figure 1C and cites References 21 and 22. However, these structures are extended due to the presence of a duplexed RNA scaffold. Analytical ultracentrifugation experiments were performed on the “purified RdRp” (Page 9) and thus lack RNA. Can authors address this discrepancy? In addition, the expected size of an intact RdRp is predicted to be 163 kDa but the authors report 145 kDa (Figure 3A). Why is the purified complex smaller by 18 kDa?

5. Authors perform negative-stain EM to claim that the sample is “homogeneous and not aggregated” (Page 9), however the image presented in Figure 3B is uninformative since the image is low-resolution and the particle population are not shown (no 2D classes). Authors should show that the particles observed by negative-stain EM are in fact intact RdRp particles by reconstructing a 3D volume from the particles. Additionally, the negative stain EM sample was prepared at 0.05 mg/mL (which is magnitudes lower than the concentrations used in cryo-EM and even some biochemical assays) so it is unsurprising that the sample is “not aggregated” (Page 9).

6. The activity of the purified RdRp (Figure 3C) is much weaker compared to reconstituted RdRp (combining nsp12 with nsp7/8, see primer extension assay in Figure 1 of Hillen et al. 2020). Even after 60 mins of incubation, the purified RdRp does not extend all the 20mer primer RNA. Despite this, the authors claim the purified RdRp is “functionally active” (Page 10).

Minor comments

1. Page 2, Abstract – “Characterization of the purified recombinant SARS-Cov-2 shows that it forms a tetramer with the expected stoichiometry.”

- Insert “RdRp” between “SARS-CoV-2” and “shows”.

2. Page 4 – “Here, we describe an alternative strategy to produce the SARS-CoV-2 RdRp directly in E. coli, using a single polycistronic construct.”

- This construct is NOT polycistronic as nsp12 and nsp7/8 are expressed on two separate T7 promoters.

3. Page 4 – “The open reading frames (ORF) of the nsp7, nsp8 and nsp12 genes from SARS-CoV-2virus were synthesized commercially by geneArt (Thermo Fisher)”

- Are these genes made using viral codons or were they codon optimized for expression in E. coli?

4. Page 5. Change “chimio-competent” to “chemocomponent”

5. Page 5. Change “1,4 kPa” to “1.4 kPa”

6. Page 6. Change “histrap fractions” to “HisTrap fractions”

7. Page 6. Change “heparin hiTrap HP” to “HiTrap Heparin HP”

8. Nsp 12 is a cysteine-rich protein (29 cysteines in primary protein sequence), however buffers used for purification and biochemistry do not contain any reducing agents (BME, DTT, TCEP, etc.). Could the authors justify using oxidizing conditions (no reducing agents present) for their purification and biochemistry?

9. The RNA duplex was prepared in water and annealed by only “heating for 2 min at 70 °C” (Page 7) followed by slow cooling to room temperature. Could the authors justify annealing the RNA in water (absence of salt or buffer) and using a lower denaturation temperature?

10. Page 8 – “A polycistronic plasmid was employed to co-express the nsp7, nsp8 and nsp12 subunits of the SARS-CoV-2 RdRp in E. coli.”

- Incorrect usage of the word polycistronic. Nsp12 and nsp7/8 are made from two separate T7 promoters.

Reviewer #2: The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes the current COVID-19 pandemic. The RNA dependent RNA polymerase (RdRP), consisting of nsp7, nsp8, and nsp12, is the key enzyme of the SARS-CoV-2 and a very good antiviral target. Currently, they have been multiple studies about the function and structural studies of the RdRP. The existing strategies used for the overproduction of RdRp involve the expression and purification the 3 subunits separately before assembling the complex in vitro. This paper describes an alternative strategy of using a polycistronic construct to produce the SARS-CoV-2 RdRp in E. coli.

Overall, it is a good paper, but the following questions need to be addressed.

1. Provide the plasmid sequence.

2. Page 12:” The complex was eluted using a 25 mL linear gradient of imidazole (Buffer B: 50 mM Tris-HCl at pH 8, 500 mM NaCl, 500 mM imidazole).” From Fig. 2A, it should be 50ml.

3. The authors should provide the 2D classification of Negative stain images, and the defocus at -3 µm is a little high. And the NS-EM image Fig. 3B shows it has some aggregation.

4. The authors should provide the data of Matrix-Assisted Laser Desorption/Ionization (MALDI-TOF/TOF) analysis.

5. The image of the RNA Primer extension assays (Fig. 3C) should be clearly labeled, especially the control at 0 min and protein only and RNA only. Besides, more assays designed is better.

6. There has another band in the first lane of Fig. 3C, what is that?

7. Pg. 10, “it forms a tetramer with the expected stoichiometry.” This seems vague, perhaps state what the “expected stoichiometry” is.

8. There seems to be a typo on the first line of page 14 (the “and” should be before the MgCl2, not after).

9. They perhaps need to expand on the functions of NSP7 and NSP8 cofactors in the complex instead of just NSP12.

10. This is probably not as important, but they specifically mention that they incubated the cells with agitation (180rpm, pg 11) during the induction step but not in the previous step(s) when they were culturing the cells.

11. Also, a plasmid map would be beneficial in visualizing the final vector.

Reviewer #3: Summary:

This work by Madru et al needs major revision if it is to be published. The authors claim that they have constructed a polycistronic expression system is false as the design utilizes two separate promoters that are regulated independently. The methods used to both express and purify the RdRp complex require further development as the purification has numerous steps that could be optimized.

The authors incorrectly reference other papers throughout the manuscript which indicates a lack of attention to detail or knowledge about the field.

Plos one paper review:

• This is not actually polycistronic – its a duet expression vector with two separate T7 promoters. See methods.

There is evidence in support of Remdesivir's use in humans (it is incorrect to say that their efficiency in humans remains to be tested) as there have been several clinical trials conducted.

References starting at reference 19 are off by one reference.

“Recently, RdRp has been a subject of intensive structural biology efforts, yielding high resolutions cryo-EM structures of the RdRp apo form(20,21), bound with RNA (22,23), and with inhibitors (23,24)”

Gao et al is numbered reference 19 in the references section. Please change this line to:

“Recently, RdRp has been a subject of intensive structural biology efforts, yielding high resolutions cryo-EM structures of the RdRp apo form(19,20), bound with RNA (21,22), and with inhibitors (22,23)”

In ref 19, the authors co-expressed nsp7 and nsp8 which has been done in other recent manuscripts Chen, Malone et al, 2020. The statement “The main strategies employed so far for the overexpression of recombinant RdRp consists in expressing and purifying the 3 subunits separately before assembling the complex in vitro” is incorrect.

“Moreover, while nsp7 and nsp8 express readily in Escherichia coli, nsp12 shows limited solubility in bacterial expression systems and is often produced in insect cells (20,22,24).” Wang et al use insect cell expression, this statement is likely made to reference Peng and Hillen et al.

“These approaches lengthen the protein expression and purification steps” would make better sense than “These approaches multiply the protein expression and purification steps”

Methods:

Authors need to state if ORFs were codon optimized, and how.

The design is not polycistronic. Polycistronic implies that the three genes are transcribed as a single transcript that has separate RBS sites (there are two T7 promoters so two mRNA transcripts in this design).

Given use of codon plus BL21 cells, I am assuming that the ORFs of nsp12/7/8 are not codon optimized. Please confirm as mentioned in previous point if these cells are necessary.

Protein expression:

Please elaborate what is LBKC. Was this supposed to say ‘LB’?

Is 1 mM IPTG correct for overnight expression?

‘conserved at -80’ – Use of ‘kept’ or ‘stored’ would be better.

Purification:

Would doing the heparin step pre the nickel purification step be better? The authors state that there was nucleic acid contamination of the Nickel elution. Do the authors lose protein in the flow-through for the nickel step due to poorer binding of the nucleic acid bound RdRp complex to the Ni-NTA?

Please rephrase description of the protein purification. The use of Nickel buffer A, Nickel Buffer B, Heparin buffer A, Heparin buffer B, SD200 buffer as descriptors for the purification buffers maybe more reader friendly. The authors should also specify reducing agents that were utilized in each of these buffers.

Can the authors comment on the need to use the Q column post the use of a heparin column? Have the authors compared running the heparin column over a 20CV gradient and selecting only those fractions containing nsp12?

“The purification was finally polished using..” - Please re-phrase.

The authors should specify if they add glycerol prior to flash-freezing.

RNA primer extension assays:

Statement reads:

“The primer extension assay was performed with 500 nM of purified RdRp complex in the presence of 500 μM NTPs and 100 nM RNA duplex, in a reaction buffer containing 20 mM Na-Hepes pH 8, 50 mM NaCl, 3 mM MgCl2 and.”

Please remove the ‘and.’ or complete the sentence.

Results and discussion:

“The UV spectra of the eluted fractions showed a large DNA contamination with A260/A280 ratio of 1.4 from this stage” – Insert ‘an’ after ‘with - Replace ‘from’ with ‘at’

“Finally, the purification was polished using a size-exclusion chromatography showing one single peak” – Remove ‘polished’ with ‘completed” and ‘showing’ with ‘which showed’

“Recent structural studies have revealed that this N terminal region of nsp8 is flexible and gets ordered when the RNA duplex exits from the enzyme’s active site (22).” – Rephrase “gets ordered”. Please cite Hillen et al for this statement.

“Yet, this region is not required for RNA polymerase activity but improves its processivity by perpetuating the interactions with the RNA backbone (22)” -Please rephrase. This region is required for polymerase activity as determined by the phenotypic reduced viral replication in the presence of nsp8 K58 mutations as shown in subissi et al, 2014. The use of ‘improves’ implies a non-lethal phenotype which is not the case.

“An overall yield of ~100 µg pure RdRp complex was obtained from 1 g of E. coli pellet after the final size exclusion chromatography.” – It is custom to give the yield as mg / L of culture if specifying at all.

Biophysical and functional characterization…

“In addition, the purified RdRp complex was applied onto glowdischarged carbon coated EM grids, and stained with a 2% uranyl formate aqueous solution. Images were recorded with a defocus of -3�m, at the instrument magnification of 49000” –The underlined above is redundant since stated in the methods.

Figures:

Fig 1

The reader would require a higher resolution image first and foremost.

The authors use fig 1 B (i.e pdb 6YYT from Hillen et al which is cited incorrectly as wang et al ) as a discussion piece for why their AUC frictional coefficient was 1.5. The authors are purifying the RdRp complex in the absence of RNA. They have correctly stated that the N-terminus of nsp8 is ordered in the presence of upstream RNA. In its absence, the N terminus is disordered and the resultant comment that the authors make regarding an “extended shape” in relation to the AUC data needs to be further developed.

The structure is also quite pixelated.

Fig 2

The reader would require a higher resolution image first and foremost.

In relation to the Nickel purification step, the authors should comment on the need to include 500 mM imidazole in nickel buffer B. It is more common when using a nickel column to do step wise washes of 20-50 mM imidazole prior to elution with 250 mM imidazole.

Fig 3

The reader would require a higher resolution image first and foremost.

Not sure what information is being provided by the negative stain image. The AUC shows the absence of aggregation on its own.

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Reviewer #3: No

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PLoS One. 2021 Apr 29;16(4):e0250610. doi: 10.1371/journal.pone.0250610.r002

Author response to Decision Letter 0

11 Mar 2021

REVIEWER #1:

The manuscript by Madru et al. describe a new strategy to purify SARS-CoV-2 RdRp (nsp7/82/12) in E. coli and provide biochemical characterization to demonstrate the quality of the purified, recombinantly expressed RdRp. This study outlines a fast purification scheme for the RdRp which may have applications in general RdRp research and RdRp inhibitor drug discovery. The study uses a series of chromatographic steps to purify the RdRp followed by analytical centrifugation, negative-stain electron microscopy (EM), and primer extension assays to test the quality of the RdRp.

I do feel that a pipeline that would lead to a high yield of the holo-RdRp ((nsp7/82/12) expressed in bacteria would greatly facilitate the efforts many researches trying to screen for inhibitors, so the significance is high and I appreciate's this group's intent. However, the data here do not support that the authors do indeed get a higher yield of protein that the other protocols using bacterial expression data. Indeed, 100 µg from 1.5 L seems rather low and the methods used to purify are not that different from already published methods.

We understand the reviewer’s concerns. Therefore, while preparing this revised version of the manuscript, we have designed a new plasmid named pRSFDuet-1(14his-nsp8/nsp7)(nsp12) and a new strategy to purifiy RdRP. With this new strategy, we are able to produce a full-length complex by fusing the 14His tag to the N-terminus of nsp8. With this new construct, the purification yield has been increased passing 5-fold 0.2 to 1 mg of complex per liter of culture, and no proteolysis is observed. The new purification protocol has been improved and simplified. Consistently, the biophysical and functional characterization of this newly purified complex has been improved. In the revised manuscript, we have shown that active RdRP can be obtained from one single affinity chromatography step, giving to this plasmid far-reaching potential in high throughput screening methods.

We sincerely apologize for our mistakes in the numbering of the references. We wanted this new construct to be available as quickly as possible, to help the scientific community that is fighting against covid-19 and did not carefully enough check the accuracy of the references.

Major critiques

1. The purification described in the paper is largely similar to that described in:

Chen J, Malone B, Llewellyn E, et al. Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex [published online ahead of print, 2020 Jul 28]. Cell. 2020;S0092-8674(20)30941-7. doi:10.1016/j.cell.2020.07.033

Even though the purification protocol is similar, our method allows the RdRp production from one single plasmid. The table below describes the recombinant expression system used to date for RdRp production in the literature. To our knowledge, our approach is unique and never published so far. This offers specific advantages that are now better described in the current version of the manuscript. With our novel construct, we are also able to obtain pure and active RdRP following one single Ni-NTA purification step.

NSP12 NSP8 NSP7

Yin et al. https://science.sciencemag.org/content/368/6498/1499

inssect cells

E.coli

Wang et al. https://doi.org/10.1016/j.cell.2020.05.034

E.coli E.coli E.coli

Hillen et al.

Kokic et al. https://www.nature.com/articles/s41586-020-2368-8

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804290/ inssect cells E.coli E.coli

Gao et al.

Yan et al.

Yan et al. https://science.sciencemag.org/content/368/6492/779

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7675986/

https://www.sciencedirect.com/science/article/pii/S0092867420315336?via%3Dihub#fig3 E.coli E.coli

Peng et al. https://doi.org/10.1016/j.celrep.2020.107774

inssect cells E.coli E.coli

nsp7L8 fusion in E.coli

Gordon et al.

Tchesnokov et al. https://www.jbc.org/content/295/20/6785

https://www.jbc.org/content/early/2020/09/23/jbc.AC120.015720

inssect cells (polyprotein with nsp5)

Shannon et al. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7499305/

E.coli E.coli E.coli

nsp7L8 fusion in E.coli

Chen et al. 10.1016/j.cell.2020.07.033

E.coli E.coli

Naydenova et al. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7896311/ inssect cells (polyprotein with nsp5)

E.coli

Chien et al.

Jockusch et al. 10.1021/acs.jproteome.0c00392

10.1016/j.antiviral.2020.104857

inssect cells E.coli E.coli

Li et al. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883726/ E.coli E.coli

Dangerfield et al. https://www.sciencedirect.com/science/article/pii/S2589004220310464

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7859716/ E. coli + chaperones

E. coli

2. Authors do not reference the appropriate papers and citations are misplaced throughout the manuscript. The following are a few examples out of many where citations are lacking, incorrect, or misplaced:

i. Page 3 - “So far, Remdesivir, Favipiravir, Ribavirin, Galidesivir, and EIDD-2801, have been shown to efficiently inhibit SARS-CoV-2 replication in cell-based assays (14–17) but their efficiency in humans remains to be assessed, rendering the search for new inhibitors still of interest.”

- Reference 17 does not have relevance to this statement.

This sentence has been removed.

ii. Page 3 – “RdRp is composed of 3 viral non-structural proteins (nsp) named nsp7, nsp8 and nsp12. The core component nsp12 hosts the catalytic polymerase activity (18) which is greatly enhanced by the two accessory subunits nsp7 and nsp8 (8,19).”Please cite Kirchdoerfer and Ward, 2019 and Reference 18 (Subissi et al., 2014) since these studies were the first to demonstrate the importance of nsp7 and nsp8 for nsp12 activity. Remove references 8 and 19 since they are not relevant here.

Corrected and added.

iii. Page 3 – “Recently, RdRp has been a subject of intensive structural biology efforts, yielding high resolutions cryo-EM structures of the RdRp apo form (20,21), bound with RNA (22,23), and with inhibitors (23,24).”

- Reference 21 is misplaced. The structure presented in this reference contains RNA. Remove reference 24 as it does not pertain to the sentence. Also add “bound to other factors” (cite Chen et al. 2020).

Corrected and added.

iv. Page 3 – “The production of sufficient amounts of heterologous RdRp with a native structure and full biological activity is a prerequisite for the discovery, optimization and comprehensive evaluation of new drugs directed against SARS-CoV-2, including in High Throughput Screening (HTS) assays involving very large chemical libraries (25).”Reference 25 is about the structure PolD complexes and does not have any relevance to HTS.

Removed.

v. Page 3 – “The main strategies employed so far for the overexpression of recombinant RdRp consists in expressing and purifying the 3 subunits separately before assembling the complex in vitro (19,22).”

- References 19 through 22 all purify nsp7, nsp8, and nsp12 separately to and reconstitute the RdRp in vitro.

Please add these citations to this statement. Also Cite Chen et al. 2020 as well as they also purified separately and reconstituted.

Corrected and added.

vi. Page 3 – “Moreover, while nsp7 and nsp8 express readily in Escherichia coli, nsp12 shows limited solubility in bacterial expression systems and is often produced in insect cells (20,22,24).”

- Reference 24 does not belong here. Cite Chen et al. 2020 as well since they use E. coli to express these proteins.

Corrected and added.

vii. Page 9 – “Recent structural studies have revealed that this N-terminal region of nsp8 is flexible and gets ordered when the RNA duplex exits from the enzyme’s active site (22).”

- Please cite Reference 21 (Hillen et al. 2020) instead of Reference 22 as Hillen et al. made the first structural observations that the N-terminus of nsp8 contacts the upsteam RNA.

Corrected.

viii. Page 9 – “Yet, this region is not required for RNA polymerase activity but improves its processivity by perpetuating the interactions with the RNA backbone (22).”

- Cite Reference 18 (Subissi et al., 2014) not Reference 22 since Subissi et al. showed that nsp7 and nsp8 are important for polymerase processivity.

This sentence has been removed.

ix. Page 14 – “(C) Two orthogonal views of the cryo-EM structure of the SARS-CoV-2 RdRp (PDB code: 6YYT) (22).”

- The reference cited for PDB 6YYT is wrong. Please correct to Reference 21 (Hillen et al. 2020).

This figure has been removed.

3. Authors claim to have a yield of “~100 μg pure RdRp complex” (Page 9) after size exclusion chromatography. However, the sample is not “pure” as it contains a significant proteolyzed product (nsp8-CTD) that is clearly visible after size exclusion chromatography (Figure 2D).

This is not true anymore with our new construct that is no longer proteolyzed and from which we can obtain higher yields. The new plasmid construct yields full-length native complex.

4. Authors claim that “the complex [purified RdRp] has an extended shape, consistent with the RdRp structure” (Page 9) and references Figure 1C and cites References 21 and 22. However, these structures are extended due to the presence of a duplexed RNA scaffold. Analytical ultracentrifugation experiments were performed on the “purified RdRp” (Page 9) and thus lack RNA. Can authors address this discrepancy? In addition, the expected size of an intact RdRp is predicted to be 163 kDa but the authors report 145 kDa (Figure 3A). Why is the purified complex smaller by 18 kDa?

First of all, we aimed here to demonstrate the homogeneity of the purified complex and not to accurately measure the molecular weight (MW). However, the apparent MW of 145kDa was indeed lower than the theoretical value (111 + 22 + 22 + 9 = 164 kDa with 14His tag). This discrepancy could be explained by two main reasons: (i) the pRSFDuet-1(14his-nsp12)(nsp7/nsp8) yielded a partially proteolyzed complex, lacking the ~10 kDa nsp8 N-terminal region, (ii) the weak contribution of the ~4.5 kDa 14His TEV-cleavable tag to the sedimentation.

Our new construct, pRSFDuet-1(14his-nsp8/nsp7)(nsp12), described in the current manuscript version allows the purification of the whole complex, without proteolysis. The new AUC sedimentation profile exhibits one main peak, corresponding to a 160 kDa complex much closer to the theoretical value (106.5 + 26.5 + 26.5 + 9 = 168.5 with two 14-His tag). We attribute this ~5% error to the low contribution of the two 14His flexible tags to the sedimentation.

In addition, the sentence “the complex [purified RdRp] has an extended shape, consistent with the RdRp structure” has been replaced by “the frictional ratio of 1.55 suggests that the heterotetramer is slightly elongated”.

5. Authors perform negative-stain EM to claim that the sample is “homogeneous and not aggregated” (Page 9), however the image presented in Figure 3B is uninformative since the image is low-resolution and the particle population are not shown (no 2D classes). Authors should show that the particles observed by negative-stain EM are in fact intact RdRp particles by reconstructing a 3D volume from the particles. Additionally, the negative stain EM sample was prepared at 0.05 mg/mL (which is magnitudes lower than the concentrations used in cryo-EM and even some biochemical assays) so it is unsurprising that the sample is “not aggregated” (Page 9).

Following the reviewer’s #1 comment, and as suggested by the reviewer #3, we have decided to remove the negative-stain EM image from the manuscript. Indeed, the present study aims to propose a production method and not a structural study. The current protocol yields a pure complex, biochemically and biophysically characterized through a 3-step quality control showing (i) full-length subunits on SDS-PAGE analysis, (ii) absence of aggregates in AUC assay (iii) polymerase activity on primer extension assays. In our opinion, reconstructing an EM 3D model using our purified RdRP is beyond the scope of this paper.

6. The activity of the purified RdRp (Figure 3C) is much weaker compared to reconstituted RdRp (combining nsp12 with nsp7/8, see primer extension assay in Figure 1 of Hillen et al. 2020). Even after 60 mins of incubation, the purified RdRp does not extend all the 20mer primer RNA. Despite this, the authors claim the purified RdRp is “functionally active” (Page 10).

The functional assay has been repeated more than 3 times with the newly purified RdRp, and have shown better results (see new Fig 4B and 4C). The full-length product is now detected after only 30 seconds at 37˚C, with a strong signal that stabilizes after 5 min.

Minor comments

1. Page 2, Abstract – “Characterization of the purified recombinant SARS-Cov-2 shows that it forms a tetramer with the expected stoichiometry.”

- Insert “RdRp” between “SARS-CoV-2” and “shows”.

Corrected.

2. Page 4 – “Here, we describe an alternative strategy to produce the SARS-CoV-2 RdRp directly in E. coli, using a single polycistronic construct.”

- This construct is NOT polycistronic as nsp12 and nsp7/8 are expressed on two separate T7 promoters.

We agree with the reviewer #1. The word polycistronic has been removed from the manuscript.

3. Page 4 – “The open reading frames (ORF) of the nsp7, nsp8 and nsp12 genes from SARS-CoV-2virus were synthesized commercially by geneArt (Thermo Fisher)”

- Are these genes made using viral codons or were they codon optimized for expression in E. coli?

The ORFs of nsp7, nsp8 and nsp12 were optimized for expression in E. coli. it is now specified in the materiel and method section.

4. Page 5. Change “chimio-competent” to “chemocomponent”

Done.

5. Page 5. Change “1,4 kPa” to “1.4 kPa”

Corrected.

6. Page 6. Change “histrap fractions” to “HisTrap fractions”

Corrected.

7. Page 6. Change “heparin hiTrap HP” to “HiTrap Heparin HP”

Corrected.

8. Nsp12 is a cysteine-rich protein (29 cysteines in primary protein sequence), however buffers used for purification and biochemistry do not contain any reducing agents (BME, DTT, TCEP, etc.). Could the authors justify using oxidizing conditions (no reducing agents present) for their purification and biochemistry? `

Considering reviewer #1’s comment, we have repeated the purification in presence of 5 mM DTT in all buffers. Results were identical.

9. The RNA duplex was prepared in water and annealed by only “heating for 2 min at 70 °C” (Page 7) followed by slow cooling to room temperature. Could the authors justify annealing the RNA in water (absence of salt or buffer) and using a lower denaturation temperature?

We have calculated the melting temperature of the RNA primer which is 58.5˚C. Heating for 2 min at 70˚C should be therefore good enough to denature and re-hybridize the RNA duplex. In addition, we perform RNA duplex denaturation and renaturation in water to avoid hydrolysis promoted by magnesium ions (See AbouHaidar MG, Ivanov IG. Non-Enzymatic RNA Hydrolysis Promoted by the Combined Catalytic Activity of Buffers and Magnesium Ions. Zeitschrift für Naturforschung C. 1999 Aug 1;54(7–8):542–8.).

10. Page 8 – “A polycistronic plasmid was employed to co-express the nsp7, nsp8 and nsp12 subunits of the SARS-CoV-2 RdRp in E. coli.”

- Incorrect usage of the word polycistronic. Nsp12 and nsp7/8 are made from two separate T7 promoters.

We agree with the reviewer #1. The word polycistronic has been removed from the manuscript.

REVIEWER #2:

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes the current COVID-19 pandemic. The RNA dependent RNA polymerase (RdRP), consisting of nsp7, nsp8, and nsp12, is the key enzyme of the SARS-CoV-2 and a very good antiviral target. Currently, they have been multiple studies about the function and structural studies of the RdRP. The existing strategies used for the overproduction of RdRp involve the expression and purification the 3 subunits separately before assembling the complex in vitro. This paper describes an alternative strategy of using a polycistronic construct to produce the SARS-CoV-2 RdRp in E. coli.

Overall, it is a good paper, but the following questions need to be addressed.

1. Provide the plasmid sequence.

We have added the full annotated plasmid sequence in the supplementary Figure 2. It is also available online on the Addgene website.

2. Page 12:” The complex was eluted using a 25 mL linear gradient of imidazole (Buffer B: 50 mM Tris-HCl at pH 8, 500 mM NaCl, 500 mM imidazole).” From Fig. 2A, it should be 50ml.

Corrected.

3. The authors should provide the 2D classification of Negative stain images, and the defocus at -3 µm is a little high. And the NS-EM image Fig. 3B shows it has some aggregation.

As explained to the reviewer #1 (5th point), we have decided to remove the negative staining EM Figure in the revised manuscript.

4. The authors should provide the data of Matrix-Assisted Laser Desorption/Ionization (MALDI-TOF/TOF) analysis.

We agree with the reviewer #2. The identification by mass spectrometry of the additional band should have been shown in the first version of the manuscript. However, this problem is obsolete now with our new construct. Indeed, this fourth band is no longer present with the new protocol and the new construct, we think there is no need to put it in the current version.

5. The image of the RNA Primer extension assays (Fig. 3C) should be clearly labeled, especially the control at 0 min and protein only and RNA only. Besides, more assays designed is better.

Functional assays have been re-designed, optimized, and clearly labeled (See the new Fig 4).

6. There has another band in the first lane of Fig. 3C, what is that?

This Figure has been replaced.

7. Pg. 10, “it forms a tetramer with the expected stoichiometry.” This seems vague, perhaps state what the “expected stoichiometry” is.

This sentence has been corrected as follows: “Characterization of the purified recombinant SARS-CoV-2 RdRp shows that it forms a complex with the expected (nsp7)(nsp8)2(nsp12) stoichiometry”.

8. There seems to be a typo on the first line of page 14 (the “and” should be before the MgCl2, not after).

Corrected.

9. They perhaps need to expand on the functions of NSP7 and NSP8 cofactors in the complex instead of just NSP12.

The introduction has been modified and the functions of nsp7 and nsp8 are now better described: “These structures showed the nsp12 core bound to a heterodimer nsp7-nsp8 and an additional nsp8 at a different binding site. The two nsp8 copies expose long N-terminal alpha helices that slide along the exiting RNA to prevent premature dissociation.”.

10. This is probably not as important, but they specifically mention that they incubated the cells with agitation (180rpm, pg 11) during the induction step but not in the previous step(s) when they were culturing the cells.

Agitation conditions are now specified along the protocol.

11. Also, a plasmid map would be beneficial in visualizing the final vector.

We have added the full annotated plasmid map in the supplementary Figure 1.

REVIEWER #3:

This work by Madru et al needs major revision if it is to be published. The authors claim that they have constructed a polycistronic expression system is false as the design utilizes two separate promoters that are regulated independently. The methods used to both express and purify the RdRp complex require further development as the purification has numerous steps that could be optimized. The authors incorrectly reference other papers throughout the manuscript which indicates a lack of attention to detail or knowledge about the field.

We understand the reviewer’s concerns. Therefore, while preparing this revised version of the manuscript, we have designed a new plasmid named pRSFDuet-1(14his-nsp8/nsp7)(nsp12) and a new strategy to purifiy RdRP. With this new strategy, we are able to produce a full-length complex by fusing the 14His tag to the N-terminus of nsp8. With this new construct, the purification yield has been increased 5-fold from 0.2 to 1 mg of complex per liter of culture, and no proteolysis is observed. The new purification protocol has been improved and simplified. Consistently, the biophysical and functional characterization of this newly purified complex has been improved. In the revised manuscript, we have shown that active RdRP can be obtained from one single affinity chromatography step, giving to this plasmid far-reaching potential in high throughput screening methods. We sincerely apologize for our mistakes in the numbering of the references. We wanted this new construct to be available as quickly as possible to help the scientific community that is fighting against covid-19 and did not carefully enough check the accuracy of the references.

1) This is not actually polycistronic – its a duet expression vector with two separate T7 promoters. See methods.

We agree with the reviewer #3. The word polycistronic has been removed from the manuscript.

2) There is evidence in support of Remdesivir's use in humans (it is incorrect to say that their efficiency in humans remains to be tested) as there have been several clinical trials conducted.

Corrected in the new introduction.

3) References starting at reference 19 are off by one reference.

Corrected.

4) “Recently, RdRp has been a subject of intensive structural biology efforts, yielding high resolutions cryo-EM structures of the RdRp apo form (20,21), bound with RNA (22,23), and with inhibitors (23,24)”

Gao et al is numbered reference 19 in the references section. Please change this line to:

“Recently, RdRp has been a subject of intensive structural biology efforts, yielding high resolutions cryo-EM structures of the RdRp apo form (19,20), bound with RNA (21,22), and with inhibitors (22,23)”

Corrected.

5) In ref 19, the authors co-expressed nsp7 and nsp8 which has been done in other recent manuscripts Chen, Malone et al, 2020. The statement “The main strategies employed so far for the overexpression of recombinant RdRp consists in expressing and purifying the 3 subunits separately before assembling the complex in vitro” is incorrect.

This sentence has been modified as follows: “The main strategies employed so far for the overexpression of recombinant RdRp consists in expressing and purifying the catalytic nsp12 subunit and the accessory nsp7-nsp8 subunits separately before assembling the complex in vitro”.

6) “Moreover, while nsp7 and nsp8 express readily in Escherichia coli, nsp12 shows limited solubility in bacterial expression systems and is often produced in insect cells (20,22,24).” Wang et al use insect cell expression, this statement is likely made to reference Peng and Hillen et al.

Corrected.

7) “These approaches lengthen the protein expression and purification steps” would make better sense than “These approaches multiply the protein expression and purification steps”

Corrected.

Methods:

8) Authors need to state if ORFs were codon optimized, and how.

Given use of codon plus BL21 cells, I am assuming that the ORFs of nsp12/7/8 are not codon optimized. Please confirm as mentioned in previous point if these cells are necessary.

The ORFs of nsp7, nsp8 and nsp12 were optimized for expression in E. coli. it is now specified in the materiel and method section.

9) The design is not polycistronic. Polycistronic implies that the three genes are transcribed as a single transcript that has separate RBS sites (there are two T7 promoters so two mRNA transcripts in this design).

We agree with the reviewer #3. The word polycistronic has been removed from the manuscript.

Protein expression:

10) Please elaborate what is LBKC. Was this supposed to say ‘LB’?

This sentence has been corrected as follows “was grown overnight in Lysogeny Broth medium

supplemented with 100 μg/mL kanamycin (LBK).”

11) Is 1 mM IPTG correct for overnight expression?

The induction parameters have been optimized (see new Fig 2), including inducer concentration, post-induction time and post-induction temperature.

12) ‘conserved at -80’ – Use of ‘kept’ or ‘stored’ would be better.

Corrected.

Purification:

13) Would doing the heparin step pre the nickel purification step be better? The authors state that there was nucleic acid contamination of the Nickel elution. Do the authors lose protein in the flow-through for the nickel step due to poorer binding of the nucleic acid bound RdRp complex to the Ni-NTA?

There is no longer DNA contamination using the pRSFDuet-1(14his-nsp8/nsp7)(nsp12).

14) Please rephrase description of the protein purification. The use of Nickel buffer A, Nickel Buffer B, Heparin buffer A, Heparin buffer B, SD200 buffer as descriptors for the purification buffers maybe more reader friendly. The authors should also specify reducing agents that were utilized in each of these buffers.

Corrected.

15) Can the authors comment on the need to use the Q column post the use of a heparin column? Have the authors compared running the heparin column over a 20CV gradient and selecting only those fractions containing nsp12?

The new protocol includes only one step of anion exchange chromatography.

16) “The purification was finally polished using..” - Please re-phrase.

Corrected: “The purification final step involves..”

17) The authors should specify if they add glycerol prior to flash-freezing.

The new Figure 4C shows the influence of freezing condition on RdRp activity.

RNA primer extension assays:

18) “The primer extension assay was performed with 500 nM of purified RdRp complex in the presence of 500 μM NTPs and 100 nM RNA duplex, in a reaction buffer containing 20 mM Na-Hepes pH 8, 50 mM NaCl, 3 mM MgCl2 and.”Please remove the ‘and.’ or complete the sentence.

Corrected.

Results and discussion:

19) “The UV spectra of the eluted fractions showed a large DNA contamination with A260/A280 ratio of 1.4 from this stage” – Insert ‘an’ after ‘with - Replace ‘from’ with ‘at’

This sentence has been removed.

19) “Finally, the purification was polished using a size-exclusion chromatography showing one single peak” – Remove ‘polished’ with ‘completed” and ‘showing’ with ‘which showed’

Corrected.

20) “Recent structural studies have revealed that this N terminal region of nsp8 is flexible and gets ordered when the RNA duplex exits from the enzyme’s active site (22).” – Rephrase “gets ordered”. Please cite Hillen et al for this statement.

This sentence has been corrected as follows: “This flexible region becomes ordered when the RNA duplex exits from the enzyme’s active site.”

21) “Yet, this region is not required for RNA polymerase activity but improves its processivity by perpetuating the interactions with the RNA backbone (22)” -Please rephrase. This region is required for polymerase activity as determined by the phenotypic reduced viral replication in the presence of nsp8 K58 mutations as shown in subissi et al, 2014. The use of ‘improves’ implies a non-lethal phenotype which is not the case.

This sentence has been removed from the manuscript.

22) “An overall yield of ~100 µg pure RdRp complex was obtained from 1 g of E. coli pellet after the final size exclusion chromatography.” – It is custom to give the yield as mg / L of culture if specifying at all.

The yield is now given in mg of protein / L of culture

23) Biophysical and functional characterization…

24) “In addition, the purified RdRp complex was applied onto glowdischarged carbon coated EM grids, and stained with a 2% uranyl formate aqueous solution. Images were recorded with a defocus of -3�m, at the instrument magnification of 49000” –The underlined above is redundant since stated in the methods.

These data have been removed.

Figures:

25) Fig 1

The reader would require a higher resolution image first and foremost.

The authors use fig 1 B (i.e pdb 6YYT from Hillen et al which is cited incorrectly as wang et al ) as a discussion piece for why their AUC frictional coefficient was 1.5. The authors are purifying the RdRp complex in the absence of RNA. They have correctly stated that the N-terminus of nsp8 is ordered in the presence of upstream RNA. In its absence, the N terminus is disordered and the resultant comment that the authors make regarding an “extended shape” in relation to the AUC data needs to be further developed.

The structure is also quite pixelated.

As explained to the reviewer #1 (4th point), the AUC analysis has been improved.

26) Fig 2

The reader would require a higher resolution image first and foremost.

In relation to the Nickel purification step, the authors should comment on the need to include 500 mM imidazole in nickel buffer B. It is more common when using a nickel column to do step wise washes of 20-50 mM imidazole prior to elution with 250 mM imidazole.

We agree with the reviewer #3. However, as the RdRp produced from the new pRSFDuet-1(14his-nsp8/nsp7)(nsp12) contains two different 14His tags, it is much strongly bound to the Nickel resin. We have thus decided to keep the same imidazole range for the elution step.

We followed the advice of Reviewer #3 by including a 5% Histrap Buffer B wash step which greatly enhance the chromatogram profile.

27) Fig 3

The reader would require a higher resolution image first and foremost.

Not sure what information is being provided by the negative stain image. The AUC shows the absence of aggregation on its own.

As explained to the reviewer #1 (5th point), we have decided to remove the negative stain EM from the manuscript.

Attachment

Submitted filename: REVIEWERS_comments_060321.docx

Click here for additional data file. (45.5KB, docx)

Decision Letter 1

Filippo Prischi

25 Mar 2021

PONE-D-20-24456R1

Fast and efficient purification of SARS-CoV-2 RNA dependent RNA polymerase complex expressed in Escherichia coli

PLOS ONE

Dear Dr. Sauguet,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Reviewers agree that the paper has improved and all comments raised have been properly answered. One reviewer has suggested some very minor editorial changes (see attached document). If you could respond to these 5 comments, I'll then be happy to accept the paper for publication.

==============================

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Attachment

Submitted filename: Madru et al..pdf

Click here for additional data file. (109.1KB, pdf)
PLoS One. 2021 Apr 29;16(4):e0250610. doi: 10.1371/journal.pone.0250610.r004

Author response to Decision Letter 1

6 Apr 2021

-----------------------------------------------------------------------------------------------------------

The revised manuscript by Madru et al. is greatly improved. The significance of obtaining active RdRp rapidly would facilitate high-throughput screens, and the relative ease of obtaining active protein with one step is commendable. I have a few comments that need addressing, but I do recommend acceptance with some revision. Because the manuscript does not have page or line numbers, I have copied and pasted where my comments are addressing:

1.”The main strategies employed so far for the overexpression of recombinant RdRp

consists in expressing and purifying the catalytic nsp12 subunit and the accessory nsp7-nsp8 subunits separately before assembling the complex in vitro (11,22,24,28–30).”

-The authors, in their response, cited Dangerfield's 2021 work which also employs a co- expression system, so this claim is false. This paper should be cited within this manuscript. This submitted manuscript's advantage is that the preps can be performed in one day and yield active enzymes. I appreciate the authors did initiate these studies before Dangerfield's paper was published. Therefore, they could state while their manuscript was in review, another group published a co-expression system but one that uses at least 4 for five steps.

This paper is now cited in our manuscript : “While this manuscript was under review, a method for producing tag-free SARS-CoV-2 RdRp in E. coli has been reported by Dangefield et al. (32).Their approach requires many more steps than traditional purification of tagged proteins, but yields 7 mg of active enzyme per liter of culture.”

2. “Our fast, single-day purification protocol results in a stable and active complex that can be used in most protein biochemistry laboratories for drug screening, as well as for routine functional and structural studies.”

-This approach of a one-day prep shows the protein is active, but they did not show the prep to be suitable for structural studies. Please remove that claim.

This sentence has been modified as follows: “Our fast, single-day purification protocol results in a stable and active complex that can be used in most protein biochemistry laboratories for drug screening as well as for functional studies.”

3. “Depending on the applications, the N-terminal 14his-tag fused to nsp8 can be easily

removed following TEV-protease cleavage.”

-This statement should be modified to state that they have not tested if the tag is removable and the effect on the prep. If the authors did remove the tag, they should show the resulting gels and biochemical activity.

This sentence has been modified as follows: “Depending on the applications, the N-terminal 14his-tag fused to nsp8 can be removed following TEV-protease cleavage. We recommend to do so after the HiTrapQ step. The RdRp could thus be incubated overnight with TEV-protease at 4˚C in the presence of 1mM DTT before the final size exclusion chromatography. However, since the 14his tag is cleavable, we did not verify RdRp activity after cleavage in the present study.”

4. The plasmid is not yet deposited on Addgene- do the authors plan to do so when the paper is published? If so, change “Our construction has been made available to the entire

scientific community through the Addgene plasmid repository (Addgene ID: 165451) upon publication of this manuscript.”

The plasmid is indeed not yet available and still held for publication. The sentence has been modified as suggested by the reviewer.

5. In the conclusion:

“The resulting sample is pure, homogeneous, properly active, and therefore suitable for drug screening and extensive site directed mutagenesis, as well as for functional and structural studies.”

-Reword as pure and homogeneous means the same in this context. Properly active is redundant; change to active. Remove extensive. To state that the resulting sample (from the single-day prep) is suitable for structural studies has not been shown. They could say that the one-day prep is ideal for biochemical studies, and while the three-step prep SHOULD be suitable for structural studies BUT that remains to be shown. Otherwise, the conclusion can be construed as misleading.

The conclusion has been modified as follows: “Motivated by E. coli’s broad accessibility, ease of culture, rapid growth rates and proven scalability, we developed an efficient expression and purification system for the SARS-CoV-2 RdRp complex in this bacterial host. Active RdRp can be immobilized and isolated from bacterial lysate in one step by nickel affinity purification, facilitating high-throughput screens and biochemical studies. Furthermore, the three-step purification protocol yields an intact and correctly assembled (nsp7)(nsp8)2(nsp12) complex that should be suitable for other purpose as structural studies. Our construction has been made available to the entire scientific community through the Addgene plasmid repository (Addgene ID: 165451) upon publication of this manuscript.”

Attachment

Submitted filename: R2RPONE-D-20-24456.docx

Click here for additional data file. (44.5KB, docx)

Decision Letter 2

Filippo Prischi

12 Apr 2021

Fast and efficient purification of SARS-CoV-2 RNA dependent RNA polymerase complex expressed in Escherichia coli

PONE-D-20-24456R2

Dear Dr. Sauguet,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Filippo Prischi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Filippo Prischi

15 Apr 2021

PONE-D-20-24456R2

Fast and efficient purification of SARS-CoV-2 RNA dependent RNA polymerase complex expressed in Escherichia coli

Dear Dr. Sauguet:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Dr. Filippo Prischi

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 File

    (PDF)

    Click here for additional data file. (598.8KB, pdf)
    Attachment

    Submitted filename: REVIEWERS_comments_060321.docx

    Click here for additional data file. (45.5KB, docx)
    Attachment

    Submitted filename: Madru et al..pdf

    Click here for additional data file. (109.1KB, pdf)
    Attachment

    Submitted filename: R2RPONE-D-20-24456.docx

    Click here for additional data file. (44.5KB, docx)

    Data Availability Statement

    All relevant data are within the paper and its Supporting Information files.

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