Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review provides an overview of various additivities to enhance the expression levels of soluble recombinant proteins in E. coli. The topic is interesting. However, there are several issues needed to be addressed.

1 Ln 200- Ln 215, I would suggest rewriting this part to a more concise manner.

2 Ln 252- Ln 254, the relevant references should be provided.

3 Figure 2, it is weak to show the typical scheme and examples. The details of the additives and relevant references should be provided in this figure.

4 Ln 333, 'convulsions and the future perspectives', this section is written very vaguely. The current limitation, future perspectives and development should be added.

 

Comments on the Quality of English Language

Minor editing of English is required.

Author Response

We extend our sincere gratitude to the reviewer for their insightful comments and valuable feedback on our manuscript. In this response, we address each of the reviewer's comments and revisions, aiming to strengthen the overall quality of our work. We appreciate the opportunity to improve our manuscript and contribute to the advancement of scientific knowledge in this field.

 

Comment 1:

Ln 200- Ln 215, I would suggest rewriting this part to a more concise manner.

Response 1:

Thank you for your comment. We have revised the section of the text as you suggested, focusing on condensing expressions and simplifying sentence structure. The updated version is provided below:

“Numerous studies provide compelling evidence for the efficacy of osmotic shock in increasing the expression levels of poorly soluble enzymes in E. coli. In Oganesyan's work, six out of nine proteins showed improved expression in LB medium with the addition of 0.5 M NaCl together with 1 mM betaine [52]. The addition of 660 mM sorbitol and 2.5 mM betaine resulted in a 2.4-fold increase in the yield of dimethylallyl pyrophosphate:5'-AMP dimethylallyltransferase at 37 °C, whereas the addition of 1000 mM sorbitol with betaine at 25 °C resulted in a 6.5-fold increase [53]. 500 mM sorbitol without betaine increased the soluble forms of three out of eight enzymes by approximately 1.5 to 2 times [54]. Co-expression with chaperones and supplementation with 0.5 M sorbitol increased yields of transforming growth factor beta 3 [55]. Further enhancement was achieved with 1 M trehalose, while other osmolytes like ethylene glycol, arginine hydrochloride, and sucrose did not increase soluble protein yield.”

 

Comment 2:

Ln 252- Ln 254, the relevant references should be provided.

Response 2:

We have deleted sentence “This may be due to the lower efficiency of ethanol or its lesser popularity as an additive” because we did not find any articles that compared the effectiveness of ethanol and sorbitol supplements.

 

Comment 3:

Figure 2, it is weak to show the typical scheme and examples. The details of the additives and relevant references should be provided in this figure.

Response 3:

Comments on the scheme are provided within the text of the chapter. We believe that providing extensive recommendations on the scheme might make it more challenging for the reader to comprehend. If we have misunderstood the intent of your comment, we would appreciate the opportunity to clarify further.

 

Comment 4:

Ln 333, 'convulsions and the future perspectives', this section is written very vaguely. The current limitation, future perspectives and development should be added.

Response 4:

In response to your suggestion, we have revised the conclusion. We hope that these additions improve the overall comprehensibility of the article. If you have any further suggestions or specific areas you would like us to focus on, please feel free to let us know.

Reviewed conclusion:

“Enhancing the expression level of soluble recombinant proteins is an important task for molecular biology and biotechnology. Introducing additives in culture medium is a technically straightforward method for optimizing the expression levels of recom-binant proteins. Expression systems based on E. coli are among the most popular for obtaining recombinant proteins. This review provides a comprehensive overview of the use of additives to enhance the expression levels of recombinant proteins in E. coli in soluble form.

The most popular and researched approaches include changing the inducer con-centration and adding osmolytes. Based on studies [52] and [54], approximately half of the cases show increased expression levels of recombinant proteins in soluble form under osmotic shock conditions. This effectiveness is comparable to the efficiency of co-expression with chaperones [91].

The number of studies focusing on the impact of ethanol on the expression levels of recombinant proteins is significantly lower compared to osmolytes. It would be inter-esting to see research comparing the effectiveness of adding various osmolytes, ethanol, and other additives on the expression of a wide range of different proteins. Most studies focus on the impact of a single additive. It is unclear how the combination of additives will affect the expression levels of recombinant proteins.

Temperature is an important factor in optimizing the expression of recombinant proteins, and lowering it appears to increase the effectiveness of osmolyte and ethanol additives [53, 74, 75]. Predicting the impact of any additive in advance is not feasible; therefore, it is most practical to test additives that most commonly lead to increased expression levels of soluble enzyme forms.

It can be assumed that as the experimental knowledge base expands and our un-derstanding of E. coli metabolism and regulation grows, researchers will continue to discover new additives that lead to increased yields of soluble enzymes. The rapidly advancing field of metabolic engineering may also aid in creating E. coli strains with metabolic pathways adapted for the highly efficient expression of recombinant proteins, including those requiring specific additives.”

Reviewer 2 Report

Comments and Suggestions for Authors

This review paper was organized well so that it can be accepted after minor revision.

In line 182, the title of section 4 is the same as section 3. It should be fixed. 

This paper deals with several recent progress to increase the production of more sobuble proteins in cytoplasm of E. coli. This is a problem in the field of biotechnology, but still has not been solved clearly. I found this manuscript deals with many methodologies to solve the problem and summarized it in a organized way. Only minor concerns about this manuscript are

 

In page 5, Line 182

The title of this section must be something else, not Glucose, Lactose and Glycerol. Please fix it.

 

page 7, Line 287-292

While the title of the paper suggests a focus on additives for soluble recombinant protein expression, the content in lines 287-292 primarily discusses about improved activity of proteins by the addition of cofactors, which may not be directly linked with solubility. However, to comprehensively address the topic of protein overexpression, it woukd be beneficial to include a discussion on how additives have been observed to influence the solubility of proteins in previous research. Please modify this explanation in accordance to the overal theme of the manuscript.

  page 7 , Line 294

The phrase "expression levels" in the statement "Not all vitamin additives or their precursors can always increase expression levels" might also not be directly related to the topic. It seems pertinent to choose a term that encompasses both the aspects of protein expression and enzymatic activity. A more inclusive phrasing might better reflect the varied impacts of additives on both the expression and functional activity of proteins in these contexts.

 

Author Response

We would like to express our gratitude to the reviewer for their thoughtful critique and constructive feedback on our manuscript. In this response, we have carefully considered each of the reviewer's remarks and have made revisions accordingly to improve the overall quality and impact of our article.

 

Comment 1:

In page 5, Line 182

The title of this section must be something else, not Glucose, Lactose and Glycerol. Please fix it.

Response 1:

Section renamed to “Osmolytes and Osmoprotectants”

 

Comment 2:

page 7, Line 287-292

While the title of the paper suggests a focus on additives for soluble recombinant protein expression, the content in lines 287-292 primarily discusses about improved activity of proteins by the addition of cofactors, which may not be directly linked with solubility. However, to comprehensively address the topic of protein overexpression, it woukd be beneficial to include a discussion on how additives have been observed to influence the solubility of proteins in previous research. Please modify this explanation in accordance to the overal theme of the manuscript.

Response 2:

Indeed, when using pyridoxine or pyridoxal phosphate, the increase in active protein yield appears to be mainly associated with the enhancement of enzyme specific activity. We have revised the text to the following:

“The addition of cofactors can increase the yield of activity not only by increasing solubility, but also by increasing the specific enzymatic activity. The addition of 0.02 mM pyridoxal phosphate (pyridoxine-5-phosphate) increased the yield of active glutamate decarboxylase by 2-2.5 times and simultaneously double increased in glutamate decarboxylase specific activity [87]. Pyridoxine can be taken up by E. coli cells and used for the synthesis of pyridoxal phosphate [88]. The addition of pyridoxine at concentrations above 0.05 mM allows for an almost 1.8-fold increase in the yield of active glutamate decarboxylase and 1.5-fold increase in the specific activity of enzyme. [89]. The addition of pyridoxine also resulted in a 2.8-fold increase in stability of glutamate decarboxylase.”

 

Comment 3:

page 7 , Line 294

The phrase "expression levels" in the statement "Not all vitamin additives or their precursors can always increase expression levels" might also not be directly related to the topic. It seems pertinent to choose a term that encompasses both the aspects of protein expression and enzymatic activity. A more inclusive phrasing might better reflect the varied impacts of additives on both the expression and functional activity of proteins in these contexts.

Response 3:

Thank you for your feedback regarding the phrase "expression levels" in our statement. We have considered your suggestion and revised the sentence accordingly. Here is the updated version: "Not always vitamin additives or their precursors can increase the yield of the active soluble form of the protein." We believe this modification better reflects the varied impacts of additives on both protein expression and functional activity. If you have any further suggestions or concerns, please let us know.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Accepted in present form.