PONE-D-24-45785Bacterial expression, purification and refolding of exceptionally hydrophobic and essential protein: Surfactant Protein-B (SP-B)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: Partly

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

Reviewer #1: N/A

Reviewer #2: N/A

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Reviewer #1: Yes

Reviewer #2: No

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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: Asrat et al. present a well-written description of a promising recombinant production methodology for surfactant protein-B (SP-B). As is well described by the authors, this protein is critical for life (at least in [some?] organisms with lungs) and the lack of a robust method to produce it either recombinantly or synthetically is a limiting factor for both in-depth studies and for therapeutic application. The authors clearly describe and demonstrate a method to produce SP-B (noting that the final construct lacks a 7-residue segment at its N-terminus, has two Met to Leu substitutions, and one Cys to Ser substitution) as a fusion protein with Staphylococcus nuclease A (SN) and with a C-terminal His6-tag. This fusion protein was demonstrated to be targeted to inclusion bodies upon overexpression in the C43 strain of E. coli, enabling relatively straightforward isolation of the protein. CNBr cleavage was then employed to remove the SN tag, followed by Ni2+-IMAC in order to obtain pure recombinant SP-B. The resulting protein structure was investigated by far-UV CD spectroscopy and its surface activity through LB trough evaluation. As a whole, this is a valuable methodology and should be highly useful. I do however feel that there are several points that it would be beneficial to address prior to publication:

1) In the abstract and again in the discussion, it is noted that C43 cells “overproduce intracellular membranes”. I am not sure that this is a fair generalization to make, as this strain was identified by the Walker group as being generally amenable to toxic/membrane protein production while the citation in question is a quite specific case. Perhaps the SP-B fusion construct also encourages or benefits from intracellular membrane production in C43, but I would prefer to see this noted in a more hypothetical manner given that there is limited evidence in the literature for this as a general phenomenon.

2) The inclusion of a C-terminal His-tag may indeed be perturbing to function, but is not rationalized/discussed until the second-last para. of the Discussion. Are there any literature examples of SP-B-dervied proteins/peptides that retain a His-tag and also retain full surface activity? Beyond the lack of charge argument that is advanced on p. 15 on the basis of the pH being used (I presume pH 7.4 – this could be a little clearer in the methods), is there any potential concern that the aromaticity of the His may come into play with, for example, snorkeling type behaviour that is exhibited with aromatic residues? Although it goes beyond the scope of the present study, would a potentially good strategy be to include an additional Met before the His tag to allow its cleavage and then perhaps an N-terminal His-tag for the SN tag in order to perform reverse purification? Overall, it may not be a concern that there is a C-terminal His6-tag, but I would prefer to see this feature of the recombinant SP-B construct be discussed more broadly through the manuscript (especially making this clear in the results section).

3) Given the importance of S-S bonds in SP-B, I was a bit surprised that there was no discussion of oxidation during refolding until the final para. of the Discussion. It seems that this is being anticipated to take place in concert with refolding on the Ni2+ column? What conditions are being used in that case, and isn’t the pH applied sub-optimal to favour disulfide formation? (Or, is this mirroring the conditions used to refold Mini-B with concomitant disulfide formation?) While it is noted that the bonded state is difficult to assess, would it be feasible to use either near-UV CD (especially comparing the appropriate region of the spectrum for the purified protein without reducing agent vs. following reduction) or comparing SDS-PAGE under non-reducing and reducing conditions to go beyond the inference that the near-full-length SP-B should behave similarly to Mini-B?

4) Relating to (3), what is known about the necessity or lack thereof of the S-S induced dimerization of SP-B? (i.e., is there concern about the Cys to Ser substitution that was incorporated in terms of functional applicability of this recombinant protein?)

5) When carrying out far-UV CD, it’s not fully clear how the protein concentration was determined, nor why the particular methodology chosen was used to infer helical content. Given that there are, for example, deconvolution datasets specific to membrane proteins (recognizing that SP-B isn’t an integral membrane protein), how different might those results be? How does the percentage of helicity inferred from evaluating normalized CD spectra compare to the expectations based on secondary structure predictions shown in the schematic figures? And could the dramatic differences in helical percentage in different environments be an artifact of the helical estimation methodology used rather than a true representation of differences in helicity in these environments? If the disulfide bonds are indeed formed (point 3) it seems hard to rationalize such extreme changes in helical content.

6) Based on the speculation that bacterial proteases are leading to products of the approximate MW of the SP-B, are there any insights from the sequence of the fusion protein as to which protease(s) might be doing so and what the cleavage product would be?

7) The figure caption for Fig. 4 (B) is a little unclear (i.e., what is the “different” gel that is referred to? How was the monoclonal antibody detected?)

8) It’s unclear what’s meant by “only the near 8.5 kDa band material binds to the monoclonal anti-SP-B mouse antibody”.

9) The authors consistently refer to the protein as showing “some” surface activity. This seems an odd choice of wording and perhaps could be either made more specific in nature or just referred to as being surface active?

Some additional minor comments:

10) It would be good to provide reference(s) for the statement that the anomalous migration by SDS-PAGE is expected (p. 9)

11) Abstract and again on p. 7 – italicize Staphylococcus?

12) Abstract – specify that C43 is an Escherichia coli strain. (See also comment about whether it is suitable to generalize statement finding of intracellular membrane production.)

13) p. 7, para. 1 – correct “presented in this work presents”; spell out Escherichia on first use [here]

14) Fig. 2 caption – capitalize “Amino” at start of (B)

15) p. 13, first para. of discussion – probably more suitable to refer to this as “first report” rather than “first publication”?; second sentence – remove comma after permit?

16) p. 14, 2nd para. – not fully clear how an MD simulation (presumably MD, rather than “molecular simulation”?) “might” show cholesterol binding sites; the entire sentence is worded rather speculatively.

17) Methods are fairly well described, but some additional references for sources and precedents for key protocols would be beneficial to include

18) Section 5.7 – include “spectroscopy” in title? Rate of data collection and pitch? Was buffer/blank correction employed? (“Baseline correction” is mentioned, but this has a different meaning and it’s not fully clear how this would be carried out in CD?)

19) Section 5.8 – Specify measurement being carried out in the section title rather than instrument; the wt% per mg/mL of lipids in a v/v solution is very hard to follow - perhaps this could be described in a different way?; I believe that units for water should be MOhm x cm (not just MOhm)?; It would also be good to clarify what was done with the three measurements that were carried out for each sample (i.e., are these cyclic responses that are considered individually or were these averaged, or some other treatment?)

Reviewer #2: The manuscript describes to my knowledge the first recombinant production and purification of (almost) full-length human SP-B. Results are valuable for basic research on this protein and potentially for other, including therapeutic, applications.

The manuscript has some flaws (1) in the methods section, which is incomplete, and (2) in the results & discussion, where some conclusions are not supported by the results shown and therefore seem too speculative.

Methods:

• Expression (p17): Culture volume? Type of culture (e.g. shake flask)? Wet cell mass would also be helpful to know.

• First stage purification (p18): Is there a centrifugation step missing before adding the IMAC resin? How much IMAC resin was added? Apparently, after batch adsorption, the resin is transferred to a column (what type?), this information is missing. What is the "loading buffer"?

• Dialysis (p18): Transferring a hydrophobic protein into distilled water by dialysis should result in aggregation: was aggregation assessed at that stage, or was turbidity observed? What is the protein yield of the dialysis step? I would expect considerable loss, as precipitated protein might stick to the dialysis tubing. How was protein concentration measured anyway?

• P18, bottom: "pellet" should read "lyophilisate".

• 2nd stage purification (p19). Calling two different buffers "loading buffer" and "second loading buffer" is maybe not the best idea. Please assign unique names to solvents.

• Lipid preparation (p19) "After each cycle of freezing, the lipids were thawed at 50°C to break the large multilamellar vesicles into smaller vesicles". Provide a reference confirming that this method leads to smaller vesicles. To my knowledge, freeze-thaw cycles lead to vesicle fusion, i.e. the opposite of the desired effect.

Results not supported by data

• Folding state: as you call your procedure "refolding", one would assume a native state of the protein. The CD data vary widely in alpha helix content, depending on the environment. Caption of Fig. 5 suggests the protein is near-native in DPC/SDS. However, SDS induces alpha helix in almost all proteins, therefore, this conclusion cannot be taken. I suggest recording a CD spectrum in SDS only as a control.

• In the methods section, numerous lipid/detergent mixtures are given, but for only three of those results are presented. Why? It would be interesting to see how the protein behaves (alpha helix content) in the other mixtures.

• Disulphide bond formation (p15): no evidence of correct disulphide formation in results. You refer to results from related peptide without explaining how disulphide formation was determined there.

• Function (p13): If I understood the procedure correctly, the protein is transferred back to organic solvent before being added to the lipid film. If that is the case, prior "refolding" would be irrelevant. I am aware that this is how SP-B from lung tissue has been tested before. To me it seems that this peptide, due to its small size and overall structure is assumed to "refold" from any starting conformation if it is transferred into a suitable lipid/detergent environment.

• Role of CHAPS (p14): Highly speculative. If true, will e.g. CHAPSO or CHS work as well (specific interaction with steroid structure)? This could be tested easily. In the purification, CHAPS is used below its CMC (~5 mM), meaning that it doesn't form micelles. Also, 6M urea further reduces hydrophobic interactions. Have you tested to solubilize in urea without CHAPS, or, alternatively, in urea with other detergents below their CMC? This could be the reason why the His tag is available in CHAPS, as binding of the detergent to the protein will be weak anyway.

• Oligomer formation (p14): highly speculative, and could be easily tested, e.g. by analytical SEC.

Miscellaneous:

• Resolution of figures is very poor. Hard to read e.g. MWs on PAGE:

• Headers: 2.6: "Surface activity", 5.8: Surface activity measurement

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Reviewer #1: No

Reviewer #2: No

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Bacterial expression, purification and folding of exceptionally hydrophobic and essential protein: Surfactant Protein-B (SP-B)

PONE-D-24-45785R1

Dear Dr. Booth,

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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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: N/A

Reviewer #2: (No Response)

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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: (No Response)

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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: (No Response)

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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: All concerns and comments following from the initial submission have been comprehensively and very conscientiously addressed by the authors. I feel this manuscript is now suitable for publication.

Reviewer #2: (No Response)

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Reviewer #1: No

Reviewer #2: No

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