Continuous Secretion of Human Epidermal Growth Factor Based on Escherichia coli Biofilm
and Hanjie Ying 1Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors report construction and initial characterisation of a modified E.coli strain containing 5 copies of a human epidermal growth factor (hEGF) expression cassette, integrated into each of 5 defined loci of the chromosome. Overexpression of each of four genes involved in cellulose formation, from expression plasmids, increased biofilm formation and hEGF production during biofilm-immobilized continuous fermentation. This is a nicely performed study and the results contribute towards improving E.coli expression systems for recombinant proteins.
Major comments
Figure 2a: please give details in the figure legend stating what material was used for the Standard (7th lane from the left), what volume of the 5 mg/L was loaded into the lane, and an explanation for why the standard migrates differently from the presumed hEGF protein in the lanes labelled C1 through C5.
Minor comments
Methods, table 1: Please change the “sources” fields for E.Coli BL21(DE3), pET30a-PelB-hEGF , pET28a, and pBbE1a, to show where those materials were originally obtained (before being stored in the author’s lab). If a plasmid or strain was created de-novo within the author’s lab please provide either method of creation or a citation where those details have been published previously.
Results line 231: Typo “Mm” should read “mM”
Results line 280: Typo “well plates” should read “96 well plates”
Results line 282-283: Possible typo, what is a “pore plate”? Please define
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsComments to the authors
The authors present an interesting methodological strategy study aimed at the production of epidermal growth factor considering the regeneration effects of this factor (already documented in the literature with tissue regeneration properties), demonstrating that under the expression of selection of bacteria producing E. coli biofilms, higher concentrations of the factor can be obtained by applying these conditions, and where the formation of biofilms was analyzed by strategies such as crystal violet staining and confocal microscopy, supporting the activity of biofilm formation as sustainable for the production of epidermal growth factor. My suggestions to the authors are minor changes related to the presentation of the manuscript, which I refer to below:
Material and methods section
It would be important for them to include the specifications of brands of reagents and equipment used in their methodology such as Luria Bertoni, isopropyl-D-thiogalactopyranoside (IPTG, kanamycin among others mentioned from lines #100-198, which are not described.)
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsContinuous secretion of human epidermal growth factor based in Escherichia coli biofilm.
Zhang et al.
EGF is an important biotech product with a range of uses. Optimization of its production in simple, cost effective and easily scalable systems is a major challenge and of great interest. In this manuscript Zhang are co-workers build on previous work from the same group (reference 15) to develop a continuous secretion system for human EGF based on an E.coli biofilm. The increase in EGF yields they report is significant.
However, there are two major issues which prevent a full evaluation of the manuscript at this time. Specifically, these are:
1) Nowhere in this manuscript is the exact construct used detailed nor is it detailed how any hEGF made is secreted to the media after being folded in the periplasm.
2) Of more importance nowhere in the manuscript (nor in publication 15) is evidence given that EGF is produced.
Regarding point 2 the authors state they analyze production by two methods i) SDS-PAGE; ii) HPLC.
i) SDS-PAGE. This method does not properly validate EGF production. In publication 15 Figure 5A there is a clear host protein that runs at the same position as the EGF control. In this manuscript in figure 1 the control lysate contains virtually no proteins i.e. apparently EGF integration into the genome both greatly inhibits secretion of most other host proteins and prevents cell lysis (or there is a loading error) and the standard (unknown source) is barely visible. Both of these are in contrast to publication 15.
ii) While the basics of the HPLC method are detailed in the methods section, no details of what the samples loaded are given (secretome?) and no results are shown. At a minimum, traces showing co-migration of EGF produced in this system with a standard and the lack of a corresponding peak in controls sample not producing EGF are required, along with details of whether the quantification in figure 3, 7 and 8 and table 3 were determined by densitometric analysis of SDS-PAGE or HPLC.
In addition to these minimal requirements, the authors should consider other validation methods that the protein observed is EGF and whether it contains native disulfide bonds. MS is one possible method.
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
