Formation of recombinant bifunctional fusion protein: A newer approach to combine the activities of two enzymes in a single protein

Patel Nilpa; Kapadia Chintan; R. Z. Sayyed; Hesham El Enshasy; Hala El Adawi; Alaa Alhazmi; Atiah H. Almalki; Shafiul Haque

doi:10.1371/journal.pone.0265969

Peer Review History

Original SubmissionDecember 11, 2021
26 Jan 2022 Decision Letter - Rahul Datta, Editor PONE-D-21-39132Formation of Recombinant Bifunctional Fusion Protein: A Newer Approach to Combine The Activities of Two Enzymes in A Single ProteinPLOS ONE Dear Dr. Sayyed, 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 Mar 12.2022. 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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Additional Editor Comments: The manuscript reports some interesting results but presents some flaws as indicated by the reviewers. I believe that the presentation can certainly be improved. The article some potential, so I recommend major revision for the article. Authors must reply at the questions raised by reviewers that will largely improve the manuscript. [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: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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: 1) Line 39 - The complete biodegradation of chitin-protein-containing waste requires the action of….. Rephrase as - The complete biodegradation of chitin and protein-present in the waste requires the action of 2) Line 43-44 – alkaline protease – why italics 3) Line 49- the - why italics 4) Cu+2, Na+2, and Ca+2- should be revised as Cu2+, Na2+, and Ca2+ 5) Line 64 – add spacing before [1] 6) Line 71- Phytopathogens – phytopathogens 7) Line 77- [3,10,19,11–18] - [3,10-18]. 8) Line 128 – 129 - use proper symbol 9) Line 134 – add space before bp 10) Line 155 – add space after before μg/ml 11) Use proper symbols of ml consistently as mL g/l as g/L, μg/ml as μg/mL, µM/ml as µM/mL, ml/min as mL/min etc 12) Line 189-replace and with was 13) Line 196 – method of [48]? 14) Line 211 – E.Coli ? 15) Line 229 - Co+ , Zn+ , Ca+ , Mg+ , Na+ , Cu+ , Fe+ - ?? 16) Line (β-Mercaptoethanol, Ethylene diamine tetra acetic acid, Sodium dodecyl sulfate – use consistent lower/upper case 17) Line 242-244- Mention the name of statistical software 18) Line 251 – the? 19) Line 307 – Add space before oC 20) Line 330 5mM – add space before mM 21) Legend of Fig 6 – use proper upper/lower cases 22) Line 345 – ampicillin resistant gene (Fig 6)….delete (Fig 6) 23) Line 354-355 - One can take anywhere between 48-72 h incubates samples to get maximum protein is not clear 24) Line 373-the omp ? 25) Line 383 - S. marcescensis -? 26) Line 386- 1 hour – 1 h 27) Line 397 Add space before [63] 28) Line 403- Ca2+ - ? 29) Line an – why italics? Reviewer #2: Formation of Recombinant Bifunctional Fusion Protein: A Newer Approach to Combine by Patel et al. deals with the fusion of two protein genes to form a single fusion protein having activities of two enzymes. This paper deals with the fusion of two enzymes namely; chitinase and protease to form a single fusion protein having the activity of these two enzymes. The authors have checked the activities of chitinase and protease in a single fusion protein. The paper is well written. Abstract summarizes the objectives, significance and outcome of the study Introduction is relevant Methodologies are scientific and sufficiently described Results are well written Discussion is up to the mark However, I would recommend accepting the paper after minor revision. Comments: Line 43-44 – alkaline protease – why italics Cu+2, Na+2, and Ca+2- should be revised as Cu2+, Na2+, and Ca2+ Line 128 – 129 - use proper symbol Units are not properly mentioned example - mL g/l, μg/ml, µM/ml, ml/min etc Line 196 – cite the name of reference Write proper names of microorganisms Example Line 211 – E.Coli and Line 383 - S. marcescensis Symbols of metal ions are not properly written example - Line 229 Line 242-244- Name of statistical software is missing Discussion part should not have the mention of result - Line 345 – (Fig 6) Line 354-355 – rephrase Line 403- Ca2+ - ? ****** 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. Attachments Attachment Submitted filename: PONE.pdf https://doi.org/10.1371/journal.pone.0265969.r001
Revision 1
6 Feb 2022 Author Response Response to Reviewers' comments Reviewer 1# 1) Line 39 - The complete biodegradation of chitin-protein-containing waste requires the action of….. Rephrase as - The complete biodegradation of chitin and protein-present in the waste requires the action of Response:- Line has been edited 2) Line 43-44 – alkaline protease – why italics Response:- Italics has been removed 3) Line 49- the- why italics Response:-Italics has been removed 4) Cu+2, Na+2, and Ca+2- should be revised as Cu2+, Na2+, and Ca2+ Response:- Corrected as per the suggestions 5) Line 64 – add spacing before [1] Response:- Added 6) Line 71- Phytopathogens – Phytopathogens Response:-The fungus is phytopathogenic thus they tend to cause disease. 7) Line 77- [3,10,19,11–18] - [3,10-18]. Response:- Corrected 8) Line 128 – 129 - use proper symbol Response:- 0C has been written properly 9) Line 134 – add space before bp Response:- Space has been added 10) Line 155 – add space after before μg/ml Response :- Corrected 11) Use proper symbols of ml consistently as mL g/l as g/L, μg/ml as μg/mL, µM/ml as µM/mL, ml/min as mL/min etc Response:- The corrections made in entire MS. 12) Line 189- replace and with was Response:- corrected 13) Line 196 – method of [48]? Response:- It was Aretz 1989 ..corrected 14) Line 211 – E.Coli ? Response:- The protein was expressed in E.Coli thus control with and without induction was taken. 15) Line 229 - Co+ , Zn+ , Ca+ , Mg+ , Na+ , Cu+ , Fe+ - ?? Response :- Corrected as per the suggestions 16) Line (β-Mercaptoethanol, Ethylene diamine tetra acetic acid, Sodium dodecyl sulfate – use consistent lower/upper case Response:- Corrected 17) Line 242-244- Mention the name of statistical software Response:- SPSS was used for this. 18) Line 251 – the? Response:- Corrected 19) Line 307 – Add space before oC Response:- Corrected 20) Line 330 5mM – add space before mM Response:- Corrected 21) Legend of Fig 6 – use proper upper/lower cases Response:- Corrected 22) Line 345 – ampicillin resistant gene (Fig 6)….delete (Fig 6) Response:- figure 6 has been deleted 23) Line 354-355 - One can take anywhere between 48-72 h incubates samples to get maximum protein is not clear Response:- Lower IPTG concentration for longer time give little expression yet soluble protein in the solution thus least chances of protein aggregates or inclusion bodies for mation. 24) Line 373- the omp? Response: - Corrected 25) Line 383 - S. marcescensis -? Response:- corrected S. marcescens 26) Line 386- 1 hour – 1 h Response:- Corrected 27) Line 397 Add space before [63] Response:- Corrected 28) Line 403- Ca2+ - ? Response:- Corrected 29) Line an – why italics? Response:- Corrected Reviewer 2# 1) Line 43-44 – alkaline protease – why italics Response:- Corrected 2) Cu+2, Na+2, and Ca+2- should be revised as Cu2+, Na2+, and Ca2+ Response:- Corrected 3) Line 128 – 129 - use proper symbol Response:- Corrected 4) Units are not properly mentioned example - mL g/l, μg/ml, µM/ml, ml/min etc Response:- Corrected 5) Line 196 – cite the name of reference Response:- Corrected 6) Write proper names of microorganisms Example Line 211 – E.Coli and Line 383 - S. marcescens is Response:- E.coli 7) Symbols of metal ions are not properly written example - Line 229 Response:- Corrected as per Cu2+, Na2+, and Ca2+ 8) Line 242-244- Name of statistical software is missing Response:- SPSS 9) Discussion part should not have the mention of result - Line 345 – (Fig 6) Response :- Line and map has been deleted 10) Line 354-355 – rephrase Response : Rephrased as :- Incubation period between 48-72 h yields maximum protein. The 48 h incubated samples achieved sufficient microbial concentration and low concentration of IPTG for longer time will gives better protein yield. This will lead to proper folded and biologically active protein production with least inclusion bodies formation. 11) Line 403- Ca2+ - ? Response:- Corrected as Ca2+ Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0265969.r002
3 Mar 2022 Decision Letter - Rahul Datta, Editor PONE-D-21-39132R1Formation of Recombinant Bifunctional Fusion Protein: A Newer Approach to Combine The Activities of Two Enzymes in A Single ProteinPLOS ONE Dear Dr. Riyazali Zafarali Sayyed, 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 Apr 17 2022 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: https://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, Rahul Datta 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. Additional Editor Comments (if provided): The error bar in the figure is not explained it seems it correspond to the SD which does not make any sense, that is merely for decorative purpose. I highly recommend using a 95% confidence interval instead of a Standard deviation, in the error bar. https://doi.org/10.1371/journal.pone.0265969.r003
Revision 2
5 Mar 2022 Author Response Response to Editor’s note# The error bar in the figure is not explained it seems it correspond to the SD which does not make any sense, that is merely for decorative purpose. I highly recommend using a 95% confidence interval instead of a Standard deviation, in the error bar. Response: The error bars indicate standard deviation. Corrections have been made in Fig 5 and 6 Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0265969.r004
11 Mar 2022 Decision Letter - Rahul Datta, Editor Formation of Recombinant Bifunctional Fusion Protein: A Newer Approach to Combine The Activities of Two Enzymes in A Single Protein PONE-D-21-39132R2 Dear Dr. Sayyed, 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, Rahul Datta Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0265969.r005
Formally Accepted
23 Mar 2022 Acceptance Letter - Rahul Datta, Editor PONE-D-21-39132R2 Formation of Recombinant Bifunctional Fusion Protein: A Newer Approach to Combine The Activities of Two Enzymes in A Single Protein Dear Dr. Sayyed: 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. Rahul Datta Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0265969.r006

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