Purification of recombinant bacterial collagens containing structural perturbations

Sonal Gahlawat; Vikas Nanda; David I. Shreiber

doi:10.1371/journal.pone.0285864

Peer Review History

Original SubmissionMarch 7, 2023
23 Mar 2023 Decision Letter - Yong Wang, Editor PONE-D-23-06762Purification of recombinant bacterial collagens containing structural perturbationsPLOS ONE Dear Dr. Shreiber, 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 submit your revised manuscript by May 07 2023 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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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. Additional Editor Comments (if provided): Comments from Reviewer#1: This manuscript by Gahlawat et al described a simple laboratory effort to purify a collagen like protein (CLP) having Gly-to-X mutations that was first expressed as a fusion protein. They provided evidence showing protease TEV is better suited for the removal of the His-tagged V-domain than trypsin because of the folding problem related to the mutation. Yet, there is no data and no mention on the folding of the peptides. Did the digestion act on monomer during purification or on folded trimer? Can the triple helix form without the removal of V-domain? If not, how can they claim the varied sensitivity of the fusion protein to proteases is related to the ‘partially folded structure’? Other major problems: Figure 1 is missing. The optimization experiments are notoriously difficult to reproduce. The authors did not mention the reproducibility of the results. Overall, the work felt like an incomplete effort and did not have enough reproducible results to meet the requirement of a research article. Comments from Reviewer#2: The authors present an alternative method for purifying recombinant bacterial collagens (Collagen-like proteins, CLP) containing structural perturbations or more specifically the mutation Gly to Arg. The motivation for developing the purification method is that the widely used purification method, which uses trypsin digestion to remove the affinity tag, also degrade the structural altered CLP. Instead of trypsin, TEV protease and TEV protease cleavage is used for removal of the affinity tag. In total the authors make four constructs with TEV or Trypsin cleavage sites combined with native CLP or CLP containing the Gly-Arg mutations. By blabla and enzymatic digestion assay, they demonstrate that CLPs containing Gly→Arg mutations are readily digested by trypsin while digestion with TEV protease only cleaved the affinity tag. The manuscript would benefit from: • Specify the rationale for choosing to substitute Glycine with Arginine • Indicate the number of the amino acid substitution or specify which glycine was substituted in the integrin binding site. • Reference for the contribution of the mutation Gly to Arg change the structure of CLP • Reference or argument that Gly to X would have same effect as Gly to Arg • Figure 1: o Panel C: Missing figure text. o Panel A: � Exchange “Protease cleavage sites” with “TEV or Trypsin cleavage site”. � Specify which glycine was mutated in the integrin binding site • Line 79: forgo --> forgot [Note: HTML markup is below. Please do not edit.] 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: Yes Reviewer #2: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 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 ****** 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 ****** 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 authors present an alternative method for purifying recombinant bacterial collagens (Collagen-like proteins, CLP) containing structural perturbations or more specifically the mutation Gly to Arg. The motivation for developing the purification method is that the widely used purification method, which uses trypsin digestion to remove the affinity tag, also degrade the structural altered CLP. Instead of trypsin, TEV protease and TEV protease cleavage is used for removal of the affinity tag. In total the authors make four constructs with TEV or Trypsin cleavage sites combined with native CLP or CLP containing the Gly-Arg mutations. By blabla and enzymatic digestion assay, they demonstrate that CLPs containing Gly→Arg mutations are readily digested by trypsin while digestion with TEV protease only cleaved the affinity tag. The manuscript would benefit from: • Specify the rationale for choosing to substitute Glycine with Arginine • Indicate the number of the amino acid substitution or specify which glycine was substituted in the integrin binding site. • Reference for the contribution of the mutation Gly to Arg change the structure of CLP • Reference or argument that Gly to X would have same effect as Gly to Arg • Figure 1: o Panel C: Missing figure text. o Panel A: � Exchange “Protease cleavage sites” with “TEV or Trypsin cleavage site”. � Specify which glycine was mutated in the integrin binding site • Line 79: forgo forgot Reviewer #2: This manuscript by Gahlawat et al described a simple laboratory effort to purify a collagen like protein (CLP) having Gly-to-X mutations that was first expressed as a fusion protein. They provided evidence showing protease TEV is better suited for the removal of the His-tagged V-domain than trypsin because of the folding problem related to the mutation. Yet, there is no data and no mention on the folding of the peptides. Did the digestion act on monomer during purification or on folded trimer? Can the triple helix form without the removal of V-domain? If not, how can they claim the varied sensitivity of the fusion protein to proteases is related to the ‘partially folded structure’? Other major problems: Figure 1 is missing. The optimization experiments are notoriously difficult to reproduce. The authors did not mention the reproducibility of the results. Overall, the work felt like an incomplete effort and did not have enough reproducible results to meet the requirement of a research article. ****** 6. 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. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. <quillbot-extension-portal></quillbot-extension-portal> https://doi.org/10.1371/journal.pone.0285864.r001
Revision 1
26 Apr 2023 Author Response PONE-D-23-06762 Purification of recombinant bacterial collagens containing structural perturbations We would like to thank the reviewers for their effort and time in reviewing the manuscript. We sincerely appreciate their valuable and insightful comments, which helped us in improving the quality of the manuscript. We have addressed the comments specific to each reviewer below. We have uploaded this in a formatted version with the added figure to the revised documents, labeled "Response to Reviewers". Reviewer 1 1. Specify the rationale for choosing to substitute Glycine with Arginine. Thank you for the suggestion. We have included the rationale for choosing Gly→Arg mutation in lines 83-87. Lines 83-87: Structurally distorted triple helices were generated through the introduction of Gly→Arg mutations within the integrin-binding site of CL domains. The Gly→Arg mutation would provide differential folding effects and has been associated with collagen disorders, including Osteogenesis Imperfecta and Ehlers-Danlos Syndrome and its subtypes (https://eds.gene.le.ac.uk/). 2. Indicate the number of the amino acid substitution or specify which glycine was substituted in the integrin binding site. As listed in lines 120-122, Gly at position #201 and #444 were mutated to Arg residues. Within the integrin-binding site, GFPGER, the second Gly residue (highlighted in bold) was mutated to Arg amino acid, which has further explained in lines 176-178 and Fig 2 legend. Lines 176-178: The VCL2(G→R) mutant protein includes a single Gly→Arg mutation (GFPRER) near the middle of the CL domain within the IBS sequence to have minimum impact on the folding and assembly of the triple helix Fig 2B: Version of the VCL2 construct with Gly→Arg within the IBS, where the second Gly residue of GFPGER was mutated to Arg (highlighted by “★”). 3. Reference for the contribution of the mutation Gly to Arg change the structure of CLP. Reference 22 (line 238) supports the impact of Gly→Arg mutation on the triple helix structure. Additional references have been added in support of the argument (28-30). 4. Reference or argument that Gly to X would have same effect as Gly to Arg. We have added text in lines 252-256 justifying this claim. Lines 252-256: Replacing the trypsin cleavage site with TEV offers a reliable method to isolate CL domains containing structural perturbations irrespective of the type of substituted amino acid, the location of the mutation, and the surrounding local sequence, all of which play a decisive role in determining the conformation and stability of the triple helix. 5. Figure 1: a. Panel C: Missing figure text. It is unclear which figure text is missing. The submitted Fig 2C contains a table, which describes the design of protein constructs, including the presence or absence of Gly substitution and the type of protease cleavage site (trypsin vs. TEV). b. Panel A: i. Exchange “Protease cleavage sites” with “TEV or Trypsin cleavage site”. ii. Specify which glycine was mutated in the integrin binding site i. We have exchanged the “protease cleavage sites” with “TEV or Trypsin cleavage site” in Fig 2A. ii. We have updated the Fig 2B legend to highlight which Gly residue was mutated to Arg within the integrin-binding site. Fig 2B: Version of the VCL2 construct with Gly→Arg within the IBS, where the second Gly residue of GFPGER was mutated to Arg (highlighted by “★”). 6. Line 79: forgo forgot There is no spelling error in line 79. Because of Gly→X mutations on the triple helix structure, it is challenging to remove an affinity tag and isolate collagen-like domains containing such mutations using trypsin enzyme. For that reason, authors intentionally do not cleave the affinity tag to prevent protein degradation. Reviewer 2 1. This manuscript by Gahlawat et al described a simple laboratory effort to purify a collagen like protein (CLP) having Gly-to-X mutations that was first expressed as a fusion protein. They provided evidence showing protease TEV is better suited for the removal of the His-tagged V-domain than trypsin because of the folding problem related to the mutation. a. Yet, there is no data and no mention on the folding of the peptides. We have added circular dichroism (CD) spectra in Supplementary Information (Fig S1) for the different CLPs demonstrating that the constructs properly fold into a triple helix as evidenced by a minimum and a maximum peak around 198-200 nm and 220-225 nm, respectively. b. Did the digestion act on monomer during purification or on folded trimer? The trypsin digestion assays were performed on the folded trimers and the digestion profile was visualized using SDS-PAGE. Due to the inclusion of denaturing chemicals and reducing agents, only the monomers will be seen. c. Can the triple helix form without the removal of V-domain? If not, how can they claim the varied sensitivity of the fusion protein to proteases is related to the ‘partially folded structure’? Yes, the bacterial collagen triple helix can form without the removal of V-domain. 2. Figure 1 is missing. Fig 1 is attached. 3. The optimization experiments are notoriously difficult to reproduce. The authors did not mention the reproducibility of the results We agree with the reviewer that optimization experiments are challenging. Using the reagents and chemicals listed in the Materials and Methods section of the manuscript, we obtained similar yields and purity of proteins in multiple batches, thus offering reproducible results. Since multiple factors can impact protein expression and purification, which ultimately affects protein yield and purity, it is always recommended to tailor the expression and purification of each protein construct by screening different culturing conditions. We emphasize that optimization is not the main theme of Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0285864.r002
3 May 2023 Decision Letter - Yong Wang, Editor Purification of recombinant bacterial collagens containing structural perturbations PONE-D-23-06762R1 Dear Dr. Shreiber, 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. 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. Kind regards, Yong Wang Academic Editor PLOS ONE Additional Editor Comments (optional): 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 ******** 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: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A ****** 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: 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 ****** 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: All comments have been addressed satisfactorily. I have no additional questions. The use of TEV proteases for purification of CLP has been demonstrated satisfactorily. ****** 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. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No ******** <quillbot-extension-portal></quillbot-extension-portal> https://doi.org/10.1371/journal.pone.0285864.r003
Formally Accepted
9 May 2023 Acceptance Letter - Yong Wang, Editor PONE-D-23-06762R1 Purification of recombinant bacterial collagens containing structural perturbations Dear Dr. Shreiber: 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Yong Wang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0285864.r004

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