Improved Expression of Aggregation-Prone Tau Proteins Using a Spidroin-Derived Solubility Tag

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The efficient overexpression and purification of recombinant proteins, especially of the intrinsically disordered proteins (IDPs), at high yield and acceptable purity are extremely important for many research applications including pure science and drug discovery processes in pharmaceutical industry. In the case of molecular medical targets, which are often IDPs proteins, developing appropriate strategies for their purification from various sources (E.coli or mammalian cultures) is often a huge challenge. Moreover, the inherent tendency of the intrinsically disordered amyloid-forming proteins to aggregate renders overexpression, purification, and researches challenging. There are many well described approaches and procedures showing the way to obtain sufficient protein preparations. However, the obtained amounts of proteins very often are insufficient to perform key experiments or the proteins are inactive due to changes in structure and loss of function as a result of the purification process. Here, in this manuscript the authors decided to try to significantly improve overexpression and purification of the aggregation-prone Tau proteins using the major ampullate spidroin-derived solubility tag (MaSp-NT*). This unique approach involves using this NT* tag as a fusion tag to enhance significantly the solubility and stability of overexpressed non-transmembrane proteins by forming micelle-like particles within the cytosol of E. coli cells (proposed by Kronqvist et al. 2017). The obtained results certainly confirmed that fusion with the NT* tag significantly increased the solubility and yield of highly hydrophobic and aggregation-prone Tau construct. The authors used a set of appropriate techniques that are used to purify this type of recombinant proteins from E. coli cell extracts and developed purification procedures. Although there are no Western Blot results included in the manuscript, the results obtained seem to be reliable in terms of obtaining relatively pure protein preparations (some with low stability). In my opinion, this is a very important work and the readers of Separations should find the paper worth reading. I do have one serious comment, however. The manuscript only describes the effect of using the new tag to increase the production of the recombinant Tau protein variants. The question remains: does this approach allow obtaining active proteins? To increase the quality and validity of this publication, the authors are encouraged to conduct at least some preliminary experiment or experiments showing the activity of these protein preparations, in terms of e.g. aggregation propensity, recording CD spectra, in order to analyse secondary structure characteristics or changes in the structure. Such results are missing.

Author Response

Thank you for the careful reading and constructive feedback on our manuscript.

 

Suggestions for Authors:

In my opinion, this is a very important work and the readers of Separations should find the paper worth reading. I do have one serious comment, however. The manuscript only describes the effect of using the new tag to increase the production of the recombinant Tau protein variants. The question remains: does this approach allow obtaining active proteins? To increase the quality and validity of this publication, the authors are encouraged to conduct at least some preliminary experiment or experiments showing the activity of these protein preparations, in terms of e.g. aggregation propensity, recording CD spectra, in order to analyse secondary structure characteristics or changes in the structure. Such results are missing.

> We have conducted Solution NMR spectroscopy on purified Tau-MTBR and obtained a well-resolved [¹H,¹⁵N]-HSQC spectrum with near-complete assignments of all protein backbone atoms (Figure 5), testifying to the high purity and good quality of the protein sample. (Kevin)

Reviewer 2 Report

Comments and Suggestions for Authors

Muwonge et al described an improved method for the recombinant production of Tau proteins. They approached this task by employing a solubility enhancing tag MaSp-NT* and by multi-step chromatography including cation exchange and size-exclusion chromatography. They showed that this method leads to improved yield of the Tau proteins and high purity. Overall, I find this work valuable to the community. I have a few minor comments about the presentation of the results and additional control experiments that could be added to make the work even stronger:

 

1.    I’m unsure why the lysis buffer wasn’t supplemented with RNaseA and high salt during the purification of MaSp-Tau-P301L, given the resulting nucleic acid contamination. Would the addition of RNaseA and high salt eliminate the need for a subsequent CIEX step in the case of MaSp-Tau-P301L?

Additionally, can the authors provide evidence that the final product is free of nucleic acid contamination (e.g. the A260/A280 ratio, or an agarose gel stained with SYBR gold)?

2.    To demonstrate the quality of Tau proteins purified with this method, it would be beneficial to perform established biochemical assays with these proteins, for example the Thioflavin T assay (Ferrari and Rüdiger 2018) or CD spectra to assess the secondary structure conformation of the purified tau.

3.    In Figure 2-3, it appears that the green arrow indicates the cleaved tag, contrary to the figure legends which suggest it is the red arrow.

4.    In relevant figures, it would be helpful to indicate the position of DnaK contaminant.

5.    In Supplementary Figure S14, the authors should explain what Aβ means. The authors should also indicate which band corresponds to the expressed Aβ.

6. It's unclear from the figures: can the author comment on what's the percent fraction of the dimer and point to relevant data?

Author Response

> Thank you for the careful reading and constructive feedback on our manuscript.

 

Suggestions for Authors:

Overall, I find this work valuable to the community. I have a few minor comments about the presentation of the results and additional control experiments that could be added to make the work even stronger:

 

1. I’m unsure why the lysis buffer wasn’t supplemented with RNaseA and high salt during the purification of MaSp-Tau-P301L, given the resulting nucleic acid contamination. Would the addition of RNaseA and high salt eliminate the need for a subsequent CIEX step in the case of MaSp-Tau-P301L?

Additionally, can the authors provide evidence that the final product is free of nucleic acid contamination (e.g. the A260/A280 ratio, or an agarose gel stained with SYBR gold)?

 

> Yes, the addition of RNase A and high salt in the lysis buffer during the purification of MaSp-Tau-P301L would counter the nucleic acid contamination and eliminate the need for a subsequent CIEX step. We only encoutered the problem of nucleic acid contamination when we switched from LB to TB growth medium and MaSp-Tau-P301L was the first construct we tested. That is why it required an additional CIEX polishing step, but the other two constructs didn’t because we already added RNase A and high salt in their respective lysis buffers.

> We have included the the A260/A280 ratio in the results section as evidence that the final product was free of nucleic contaminantion. (Kevin)

 

 

2. To demonstrate the quality of Tau proteins purified with this method, it would be beneficial to perform established biochemical assays with these proteins, for example the Thioflavin T assay (Ferrari and Rüdiger 2018) or CD spectra to assess the secondary structure conformation of the purified tau.

 

> We conducted Solution NMR spectroscopy on purified Tau-MTBR and obtained a well-resolved [¹H,¹⁵N]-HSQC spectrum with near-complete assignments of all protein backbone atoms (Figure 5), testifying to the high purity and good quality of the protein sample. (Kevin)

 

 

3. In Figure 2-3, it appears that the green arrow indicates the cleaved tag, contrary to the figure legends which suggest it is the red arrow.

 

> Done (Kevin)

 

 

4. In relevant figures, it would be helpful to indicate the position of DnaK contaminant.

 

> Done (Kevin)

 

 

5. In Supplementary Figure S14, the authors should explain what Aβ means. The authors should also indicate which band corresponds to the expressed Aβ.

 

> Done (Kevin)

 

 

 

6. It's unclear from the figures: can the author comment on what's the percent fraction of the dimer and point to relevant data?

 

> Done (Kevin)

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors of the manuscrpt responded positively to my suggestions. The quality and purity of the obtained key preparation were confirmed using high quality NMR spectroscopy. By performing this experiment, the value of this work was significantly increased.

Author Response

 

Thank you again for your feedback and suggestions to further improve our manuscript.