Extracellular Production of the Taiwan-Native Norovirus P Domain Overexpressed in Pichia pastoris
Kristina Hedfalk
Round 1
Reviewer 1 Report
Extracellular production of the Taiwan-native norovirus P domain overexpressed in Pichia pastoris.
The manuscript by Chien et al presents the overproduction of the Taiwan-native norovirus P domain (N o V P) in the methylotrophic yeast P. pastoris. Four different construct designs were evaluated, compared and the protein of interest purified from a scaled-up growth in fermenter and further characterized with respect to size, shape and binding.
The manuscript is clearly structured, and the figures support well the written information. The main strength with the manuscript is the evaluation of the extracellular production of N o V P in P. pastoris and the high yield achieved. There is, however, some room for improvement when it comes to descriptive information on the procedures used and for the evaluation, see comments below.
In principle, the conclusion is well supported but could be improved based on the comments below.
Major concerns:
1. Section 2.2. The description of the construct design needs more information so that the reader understand how it is done. For example, is the plasmid linearized before transformation into P. pastoris? For the constructs containing HAC1 in addition to N o V P, are the genes placed in sequence and produced from the same promoter? A figure showing the expression cassettes and final protein products would be most helpful.
2. Fig 1C and 3A. The whole gel should be shown for proper conclusions to be drawn.
3. Fig, 1E and F. There is indeed a difference in the amount observed for Oph and aph (1F). Could this explain the difference seen in 1E?
4. Fig. 3B and Fig. 4A. The bands and peaks of higher molecular weight should be reflected on.
5. Table 3. The yield in the supernatant is extracted after 116 hours of growth, while the eluted fraction is from 96 hours of methanol induction. It is not fully clear that this is the same growth parameters, please clarify this, also in the method section. Furthermore, the volumes for the various fractions should be properly stated in the method section, maybe also in the table, so that the evaluation of the total protein could be understood. Also, total protein should be given as an amount, not concentration. Finally, the way to calculate purity should be properly described.
6. Section 3.4 and Discussion. Why is the sample taken at 96 hours of methanol induction and not 116 hours, as stated maximum in figure 2C? Or did you start the MeOH induction after 20 hours? Please clarify this in the method section and in the figure. This comment is also valid for comment 5 above.
7. The first paragraph of the Discussion section is a bit repetitive, please correct this.
Minor concerns:
1. Pichia pastoris should be written in italic, please check throughout the text and correct.
2. Section 2.7. Would be good to have some information on the purification procedure, like ‘In brief…’.
3. Figure legends. The first sentence should be in bold, the rest not. Please check all figure legends and correct.
4. Figure 1A. An independent samples Tukey test is referred to, but the outcome is not stated. Please clarify.
5. Figure 1D. Aliquotes for Western Blot, please clarify whether the same volume was taken in all cases.
6. Figure legends. Standard deviation is referred to in Figure legend 1A, later SE is mentioned. Do you mean standard error, as in SE, or standard deviation, as in SD? Please check through out the text and clarify.
7. Figure legend 2. Please clarify which construct is produced.
8. Figure 5. Orange and yellow lines are referred to but not shown like that. Please correct.
Author Response
Please see the upload file.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Chen, Yu and Huang, highlight the suitability of Pichia pastoris for the extracellular production of norovirus P domain. They provide strong evidence that P domain secretion is improved by altering the secretion signal to include Ost1 and show further improvement through the co-expression of the transcriptional regulator Hac1. The authors go on to show the suitability of these expression clones for fermentation and the purification of highly pure P domain through ion exchange chromatography. The TEM analysis shows small particles and potentially dimers of the P domain before showing that these particles/dimers interact with saliva HGBA. Overall, I would like to commend the authors on a well-executed and well-written manuscript and would like to recommend this paper for publication once the minor comments below are addressed.
Figure 4A – The kDa sizes noted on the graph aren’t clear for the reader, it may be better if the authors used lines to point to each peak to increase clarity.
Figure 4B – Would it be possible for the authors to add some quantification regarding the number of particles which appear as dimers or as P-particles either through measuring sizes of the particles or through 2D classification? This would help give an overall picture of the particles produced by fermentation.
Figure 5 – The figure legend describes the lines on the graph as different colours than those represented. Additionally, perhaps the authors could consider providing the concentrations of Type B saliva used in this assay to aid with reproducibility rather than the dilutions currently stated on the graph.
Discussion – I would like to see the authors offer some context on the suitability of these P-dimers/particles as potential vaccines and what sort of responses we would expect to see? Are they of a size which would lead to uptake and drainage towards lymph nodes and therefore germinal centres? Would this provide protection and does this region induce neutralising and cross-neutralising antibodies? What are the advantages of using P-dimers only above using full length VP1 including the S domain and whether or not any consideration should be given to VP2 in the context of norovirus vaccine development?
Author Response
Please see the upload file.
Author Response File: Author Response.pdf
