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.

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

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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Please submit your revised manuscript by Oct 29 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

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Filippo Prischi

Academic Editor

PLOS ONE

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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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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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 #1: No

Reviewer #2: No

Reviewer #3: No

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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.

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

Please submit your revised manuscript by May 09 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Filippo Prischi

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. 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 Response)

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. 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: please see attached. I submitted as attachment to color code responses and my comments. It's easier as I had to copy and paste text from manuscript as line numbers were not included in the manuscript.

Reviewer #2: I want to thank the authors for addressing my initial comments. Following the revision to the article, I do not have more questions now.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Madru et al..pdf

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.

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Kind regards,

Filippo Prischi

Academic Editor

PLOS ONE

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