Abstract
BackgroundViral protein expression in Escherichia coli (E. coli) is a powerful tool for structural/functional studies as well as for vaccine and diagnostics development. However, numerous factors such as codon bias, mRNA secondary structure and nucleotides distribution, have been indentified to hamper this heterologous expression.ResultsIn this study, we combined computational and biochemical methods to analyze the influence of these factors on the expression of different segments of hepatitis E virus (HEV) ORF 2 protein and hepatitis B virus surface antigen (HBsAg). Three out of five HEV antigens were expressed while all three HBsAg fragments were not. The computational analysis revealed a significant difference in nucleotide distribution between expressed and non-expressed genes; and all these non-expressing constructs shared similar stable 5′-end mRNA secondary structures that affected the accessibility of both Shine-Dalgarno (SD) sequence and start codon AUG. By modifying the 5′-end of HEV and HBV non-expressed genes, there was a significant increase in the total free energy of the mRNA secondary structures that permitted the exposure of the SD sequence and the start codon, which in turn, led to the successful expression of these genes in E. coli.ConclusionsThis study demonstrates that the mRNA secondary structure near the start codon is the key limiting factor for an efficient expression of HEV ORF2 proteins in E. coli. It describes also a simple and effective strategy for the production of viral proteins of different lengths for immunogenicity/antigenicity comparative studies during vaccine and diagnostics development.
Highlights
Viral protein expression in Escherichia coli (E. coli) is a powerful tool for structural/functional studies as well as for vaccine and diagnostics development
The plasmids were recovered from the clones used for the recombinant protein expression and restriction analysis/ DNA sequencing showed that all the genes were correctly inserted into the designated sites without any shifting or mutations
In this report, we investigated the expressivity of hepatitis E virus (HEV) and hepatitis B virus (HBV) recombinant proteins in E. coli when the respective genes are cloned under the control of the T7 promoter in the pET28a(+) vector
Summary
Viral protein expression in Escherichia coli (E. coli) is a powerful tool for structural/functional studies as well as for vaccine and diagnostics development. Numerous factors such as codon bias, mRNA secondary structure and nucleotides distribution, have been indentified to hamper this heterologous expression. Among the available expression systems, the prokaryotic host cells such as E. coli are usually preferred due to the easy culturing procedures and to the low-cost production of high protein yields. The ribosome-profiling studies revealed that the net translation-elongation rate is generally
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
