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

Reviewer #2: This study by Pavlidou and colleagues explores the expression of membrane proteins on nanodiscs and the possibility to use then as bait to select ligands to conformational epitopes of membrane proteins. The research for protein membrane ligands is an important field in pharmaceutical research. Since those proteins are integrated into membranes they need a lipidic environment to fold properly. Phage display selection on cell surfaces is much more complex than selection using purified antigens. The cell membrane presents a myriad of proteins, carbohydrates and lipids as potential antigens. Their expression level can differ from cell to cell, which renders the presence of a relevant antigen to a minute amount among all the cell membrane components. The method presented here opens an opportunity to use phage display libraries to bind a membrane protein that is folded in a native-like environment and is presented as a pure antigen embedded into nanodics. The data support the conclusions. However some weak points should be mentioned. I present below some questions.

Among the clones chosen for further analysis they should include clone 30: which shows readout values from nanodiscs treated wells higher than the readout values from controls, and also was represented 7 times among the sequenced clones. This phenomenon of being represented many times is a sign of sequential enrichment of phages displaying specific binding against the chosen target. Dis they analysed clone 30?

The authors based their novelty on the fact that this technic allows membrane proteins to be correctly folded in nanodiscs. Results showed on figure 5 reveal that selected clones bind at equivalent levels to the AB loop either in the linear or cyclic conformation. The same results are observed for loop EF. They should explain better the reasons for this. It is possible that the peptides didn't fold correctly on nanodiscs and were presented in a linear form to the peptide phage display library?

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

Reviewer #2: I don't know

Please explain (optional). Reviewer #2: I am not an expert in statistics. But in this field the results are not necessarily analysed by statistical methods.

They analysed a large number of clones by ELISA (171) and sequenced quite a large number too (48). Nevertheless they do not explain if the ELISA showed on figure 4 have been performed on triplicates.

1. Does the manuscript adhere to standards in this field for data availability?

Authors must follow field-specific standards for data deposition in publicly available resources and should include accession numbers in the manuscript when relevant. The manuscript should explain what steps have been taken to make data available, particularly in cases where the data cannot be publicly deposited.

Reviewer #2: Yes

Please explain (optional). Reviewer #2: The authors do not mention about data deposition in publicly available resources, nevertheless it is not a widespread practice in this field. The phage display data are not always collected in the central data resources such as UniProt and NCBI GenPept. Timur Shtatland and co-workers (2007) created and maintains a database of peptide sequences called PepBank. The database is freely available on http://pepbank.mgh.harvard.edu/.

Another database called MimoDB is an information portal to biopanning results of random libraries. In version 3.2, it has 19 146 peptides. The MimoDB database is freely available at http://immunet.cn/mimodb. MimoDB has been developed and maintained by HLAB.

Shtatland T. et al., PepBank - a database of peptides based on sequence text mining and public peptide data sources. BMC Bioinformatics. 2007; 8: 280).

1. 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 below.

Reviewer #2: Yes

Please explain (optional).

Reviewer #2: I am not a native english epeakers, but the english sounds correct to me.

Some minor points: Line 9, pg 2: "as a model polytopic membrane protein" changes to "as a model of polytopic membrane protein" Line 11, pg 2:"to the membrane protein" replace to: "to membrane proteins".

Line 20, pg2: "metabolic pathways » is written twice in this sentence; "in signaling" replace to "in cell signaling"

Line 23, pg2: "because" can be replaced to "indeed"

Line 8m pg2: "selection of known and novel antibodies replace" replace to "selection of antibodies"

Line 23, pg 6 : (PBS)) replace to (PBS).

Line 9, pg 9: ?bR reconstituted into nanodiscs was used to screen an M13 phage library presenting random dodecapeptides. This sentence is confusing; usually the library is used to screen a target, and not the opposite. It would be more appropriate to say "bR was reconstituted into nanodiscs and used as target to be screened by the M13 phage library presenting random dodecapeptides.

Line 13, pg6: "each selection" means each round of selection? / Screening should be replaced to analysed.

Line 11, pg9 : (Thermo Fisher Scientific, Waltham, MA, USA) clear it, this information is already in material and methods (pg6, line 21).

Line 16, pg9: (representative results are shown in Fig. 4) would be beter explained like this: In total, 171 clones were analysed, figure 4 shows the representative results of 9 of them.

Line 25, pg9: "derived" must be replaced by "deduced"

Line 25, pg9: "deduced" must be replaced by "determined"

Line 13, pg 10: "In addition, the clones 43 and 165 showed increased binding signals", should be replaced to "In addition, the clones 43 and 165 showed also high binding signals" because only the clone 165 showed increased binding signals for loop EF compared to loop AB. In the way that is written it is confusing.

1. Additional Comments to the Author (optional)

Please offer any additional comments here, including concerns about dual publication or research or publication ethics.

Reviewer #2: It is not clear whether all clones analysed come from each round of selection or only from the last one.

Line 14, pg 8: The successful expression of bR was clearly visible by its unique purple color after addition of the cofactor retinal indicating correct folding of the protein [27]. It should be interesting to add a picture showing this purple color, otherwise add "data not shown".

In the figure legends: Figure 4: Line 17, pg 15: Numbers below the bars refer to the number of the tested clones. It is confusing what the numbers below the bars represent. It should be more appropriate to say: Numbers below the bars refer to clone names.They do not explain what WT means.

Figure 5: Numbers below the bars refer to clone names.

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Your name and review will not be published with the manuscript.

Reviewer #1: (No Response)

Reviewer #2: Carmela Dantas-Barbosa