Expression of Secreted Neutrophil Gelatinase-Associated Lipocalin in 293T Cell Using the Inducible Dual-Function System
, Chatikorn Boonkrai 3, Trairak Pisitkun 3, Chiraphat Kloypan 1,2 and Sawitree Nangola 1,2,*Round 1
Reviewer 1 Report
Saoin et al established the Tet-On system to modulate the expression of the secreted NGAL protein in a bicistronic manner and enhanced the secretory machinery by inserting the effective signal peptide HSA-SS or Nat-SS. Although this work successfully expressed the secreted NGAL, the logic for emphasizing the importance of NGAL in introduction is confused. In other words, the introduction did not provide a convinced scientific reason for increase the NGAL expression. As this work focus on protein production, the scaled up should be investigated.
Specific comments:
- Why the author only test two signal peptides, are they representative?
- Page 2 line 78, Different approaches for optimizing the recombinant protein should be listed in the introduction.
- What’s the difference between CHO and other mammalian cell, this point should be clarified in introduction.
- The ruler should be added in figure 2a
- The label in Figure 3 is too small not visible.
- The marker in Figure 4a is not visible.
Author Response
Please see the attachment
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Here's my comments/concerns to the authors
1. WB shows very low protein expression but ELISA shows comparable levels of NGAL. Is this a representation problem or is there more degradation of the HSA-NGAL. Can the authors show WB quantitation similar to ELISA quantitation.
2. Grammar and syntax is very poor throughout the manuscript.
3. Entire discussion needs to be written better. For example, the first paragraph says that accumulation of foreign proteins leads to apoptosis. Later, the references quoted are for different mechanisms than accumulation. There is none quoted for NGAL
4. Related to the example above, and the difference in expression/protein levels, the authors should measure the cellular levels of NGAL via ELISA as well to assess the actual levels of the protein as opposed to just a Western Blot on the cells. This will also reveal if there is over or under-accumulation of protein in the cell
5. The authors should explain the role of TRE3Gs/hPKG
Author Response
Please see the attachment
Author Response File: Author Response.pdf
