A Universal Strategy for the Efficient Expression of Nanobodies in Pichia pastoris
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
This manuscript describes the development and optimization of yeast-based expression platforms for nanobodies. The authors describe the investigation of a number of strategies to improve nanobody production, including the use of a wide variety of promotors, the use of various signal peptides, varied gene dosages, the use of molecular chaperones (with varied promotor strength), and integration of the relevant genes into the yeast genome. In addition, the authors investigated the potential nanobody production yields in high-density fermentation using optimized strains. In this way, the authors were able to generate significant increases in nanobody production using their optimized platforms/conditions. The authors effectively describe the next steps for this research and some of the unanswered questions that are raised by the findings of this research. Given the practical utility and emerging importance of nanobodies, this reviewer believes this work would be of interest to researchers working with nanobodies, or labs interested in optimizing the production of various proteins more generally. This reviewer recommends the publication of this manuscript after the minor issues described below have been addressed.
The authors effectively describe the practical utility of nanobodies and why they are useful, but do not provide sufficient detail as to why improving their production is an urgent need. This reviewer would have liked to see additional discussion on the current state of nanobody production to justify the importance of this work.
Although the authors briefly describe why the chosen host organism is a good candidate for nanobody expression, this reviewer would have liked to see additional discussion on this subject, and discussion as to why this yeast organism is preferable for this function relative to other expression candidates.
The authors describe the important issue of antibiotic gene drift and how it motivated some of their design choices, but do not elaborate on this subject. This should be discussed further in the introduction section.
Following Figure 1, “supplementary Table 1” is included in the main manuscript, which presumably should be included in a supplementary information document and not in the main manuscript.
Author Response
Thanks for acknowledging and providing feedback on this manuscript. Briefly, the current state of nanobody production and discussion regarding the yeast system have been incorporated into the discussion section (Lines 421-439), while some background information has been added to the introduction section (Lines 52-57). Supplementary Table S1 has been relocated to the end of the manuscript, and further modifications are requested for the editor.
Please see the attachment for more information.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for Authors
The authors provided a universal strategy for the efficient expression of nanobadois in Pichia pastoris. Based on careful design of recombinant plasmids and expression component optimization on model proteins, the authors set up a strategy for promoter modification, gene dosage optimization, and high-density fermentation increasing production level of Va, Vb, and Vc. Methods, results are clear and well-documented. However, increased production level of model proteins (1.38-fold, 1.89-fold...etc) during this study seems not highly-efficient values for each steps. The authors should explain how much these values are important in the efficient expression of nanobodies in Pichia pastoris.
Author Response
Thanks for your acknowledging and providing feedback on this manuscript. The pertinent discussion has been integrated into the discussion section (Lines 541-557). In summary, it is recognized that each optimization strategy has had a limited impact on Va production. This can be attributed to two reasons. Firstly, many of the optimization outcomes for high-yielding proteins align with the observations in this study. Secondly, proteins with lower initial yields often demonstrate more significant improvements in yield following optimization. For a more detailed discussion, please refer to Lines 541-557.
Please see the attachment for more information.
Author Response File: Author Response.pdf
