PONE-D-24-50585A universal method for zinc finger protein purificationPLOS ONE

Dear Dr. Mattei,

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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

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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

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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: Liang et.al. reported a universal method for zinc finger protein purification involving two chromatographic steps. As we know, there are different groups of zinc fingers based on the overall shapes pf the protein backbone in the folded domain. Here authors only investigated C2H2 ZF proteins. Does this method apply to other types of protein, such as C4 or C6 ZF proteins? Moreover, purification was the main focus of the studies and the importance of buffer conditions used in purification process was emphasized in the discussion section. However, there is no detailed information in the main text or figures to show this part. Therefore, the manuscript should be revised accordingly before being accepted for publication.

Major points

1. Lack of information related to the optimization of buffer conditions, such as adding L-arginine before or after purification.

2. Lack additional experiments to validate the method to other types of ZF proteins.

Minor points

1. The figures quality can be improved with higher resolution.

2. Line 55, Remove e.g..

3. Line 66-72, “A general method for the purification of ZF…of probes for non-repetitive sequences.” Should move before Line 55.

4. Line 120-121, Wrong place for sentence “IPTG induction led to a prominent band of the expected molecular weight in whole cell lysates (Fig. 1A, compare lanes with and without IPTG).”

5. Fig 1E, the size of the CXCR4L was slightly not consistent in two SDS-PAGE. Why not just simply show the Marker band in the gel too?

6. Line 170, control experiments are needed in Fig.S2. A non-specific probe should be included.

7. Fig.4. is there non-specific binding of TZAP9-11 in telomeres (shown in green)? Whether this signal (extra green signal) comes from Cy3 signal?

8. Line 257, remove second “pH7.5”.

9. Line 260 and line 263, the purpose of using two different buffer, 100 mM L-Arg-HCl vs 100 mM L-Arg.

10. Why only show TZAP9-11 in Fig. 2B?

11. Information related to yield of each 8 tested proteins can be added to table 1.

Reviewer #2: In this manuscript, the authors reported recombinant preparations of various zinc fingers, by use of SUMO fusion. They confirm their production and characterize these ZFs with cysteine labelling and in situ labelling.

Comments:

-The location of cysteine labelling is not confirmed. The authors should have tested the labelling with the WT ZFs. Mass spectrometry analysis should be included.

-The Kd constants should be compared with the literature values, if available.

-In the introduction, the authors should provide a reasoning why ZFs are difficult to produce. Also, an explanation to why SUMO is superior than others should be included.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Comments on PONE-D-24-50585.doc

A universal method for the purification of C2H2 zinc finger arrays

PONE-D-24-50585R1

Dear Dr. Mattei,

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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Kind regards,

Yu-Hsuan Tsai

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

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: Yes

Reviewer #2: (No Response)

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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: Yes

Reviewer #2: Yes

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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

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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: The authors have addressed all the questions I have asked in previous revision process. The current version of manuscript is satisfied for the publication.

Reviewer #2: My concerns have been addressed on cysteine labelling, Kd values, and difficulties on ZFs production. The article is suitable for publication.

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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.

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

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