PONE-D-20-34837

Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies

PLOS ONE

Dear Dr. Bastian Heim,

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PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: N/A

Reviewer #2: I Don't Know

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The manuscript by Bastian Heim et al. entitled “Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies” describes mainly the crucial steps needed to obtain quickly and easily (GPCR) transmembrane proteins ready to crystallization experiments with the aim to solve their three-dimensional structure by X-ray.

In this study, two GPCR proteins were chosen S1P1 (as a positive control with a known structure) and GPR3 as example of a GPCR with an unknown structure. Both the receptors were cloned and overexpressed in E.Coli cells. From the collected IB pellets, the two receptors were purified by a Ni-IMAC chromatography strategy. The two receptors were also refolded on the column and the refolding process was optimized following the results obtained by a CPM thermal shift assay. CPM is a dye that reacts with free cysteines becoming exposed upon unfolding, and the reaction enhances fluorescence. Several detergent mixtures and micelle forming agents were explored. DDM-CHS mixtures proved to confer the highest thermal stability to the refolded GPCRs compared to other detergent mixtures tested. Stability towards aggregation was further improved by varying the solvent composition and addition of phospholipids to mixed-micelles. By SEC chromatography monomers of GPCR were isolated. The refolded monomers were tested by the ligand AF64394, to evaluate the stability effect and so the binding capability of the purified receptors. The refolded monomers were implied also in a crystallization screen using lipidic cubic phase, obtain four crystals potentially usable for the X-ray diffraction experiments.

The authors conclude that: “The same screening and optimization approach may be used for future overexpression and refolding of other GPCRs, allowing to produce scalable amount of native GPCRs using simple and robust techniques. The methods and optimizations established in this study could serve as a generic technique to express, purify and refold GPCRs from E. coli IBs. Furthermore, these refolded receptors show promise for crystallization in LCP.”

The aim of this work is interesting and the results convincing. The idea reported is remarkable and the paper is well done.

The manuscript in the present form demands a light revision before it can be published, so this reviewer suggests a “minor revision” of the paper.

Major issue:

1) From line 256 to 259, MS/MS analysis and ESI- MS/MS sequencing results were not reported. Please add more details.

Minor issue:

Just a few suggestions to improve the readability of the text helping the reader to better understand the paper.

1) The abstract is not attractive; it should arouse curiosity in the reader.

2) About the materials and methods section, please reorganize in a more appropriate chronological order this section.

3) In line 233 there is a mistake…in Table 1Error! Reference source not found, please remove it.

4) Results, in the 3.1 subsection please add more details about the IB solubilization.

5) Line 442 misspelling of “purifiy”.

Reviewer #2: The manuscript by Bastian Heim et al. entitled “Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies” describes the purification, under denaturing conditions, of two G protein-coupled receptors (GPCR), the sphingosine-1-phosphate receptor S1P1 and the orphan receptor GPR3 in E. coli inclusion bodies and the refolding by detergent exchange while bound to the immobilized metal affinity chromatography column.

The authors conclude that the same screening and optimization approach may be used for future overexpression and refolding of other GPCRs, allowing to produce scalable amount of native GPCRs using simple and robust techniques. They conclude, moreover, that the methods and optimizations established in this study could serve as a generic technique to express, purifiy and refold GPCRs from E. coli IBs.

In my opinion the reading of the work is very tiring, the data are not well organized. I admit this manuscript, but with major revision. I believe that these studies need to be described more succinctly. I would suggest to the authors a complete review.

In particular:

- The introduction is very long.

- All graphics have a low resolution.

- Line 91 to 94. Is one colony sufficient to inoculate 200 ml of medium?

- Line 95. With an optical density of 2 is not already beyond the stationary phase?

- Line 102. Why is the concentration no longer 120ug but 70ug?

- Line 105. Is a single hour of post induction at 25 ° C sufficient to produce the protein?

- Line 110. Even if the solution is on ice maybe seven minutes will heat it too much

- Lines 233 to 234. To check

- Lines 256 to 258. The black spots beyond the monomer obtained with the western, why are they all recognized if the antibody is specific against histidine? The bands with molecular weight lower than the monomer could be proteolytic cuts but the aggregates should not exist in denaturing conditions.

- The figures are not in order.

- The figure 3. The colors of the curves in the legends are not seen.

- In figure 3 the curve of 4 x CMC Fos-Choline 14 was not shown.

- Figure 3. The red curves in Graph A do not look similar. I would suggest to describe better.

- Line 301. How do the authors explain the difference?

- Line 302. DDM at 4 x CMC in the presence of CHS resulted in the highest Tm value for GPR3. better for S1P1 DDM at 2 x CMC in the presence of CHS?

- Line 315 to 318. Did the authors quantify the protein? For S1p1 the band seems more evident but for GPR3 no.

- The discussion paragraph is unconvincing, many unresolved points and many assumptions

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Reviewer #1: No

Reviewer #2: No

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Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies

PONE-D-20-34837R1

Dear Dr. Bastian Heim,

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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Kind regards,

Sabato D'Auria

Academic Editor

PLOS ONE