Heterologous Proteins Production in Escherichia coli: An Investigation on the Effect of Codon Usage and Expression Host Optimization

Abstract

Objective: The production of heterologous proteins in Escherichia coli is strongly affectedby codon bias. This phenomenon occurs when the codon usage of mRNA coding for theforeign protein differs from that of the bacterium. The ribosome pauses upon encounteringa rare codon and may detach from mRNA, thereby the yield of recombinant protein productionreduces. The aim of this study is to investigate the effect of these codon numbersreductions on the recombinant protein production.Materials and Methods: Since most amino acids are encoded by more than one codon,codons were changed in order to their usage in a special host such as E. coli without anytransformation in amino acids sequence. Silent mutations in 5' codons of human basicfibroblast growth factor cDNA carried out by site-directed mutagenesis and the expressionlevel of the recombinant protein is analyzed by means of sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and Western blot.Results: Expression level in mutant and wild-type genes indicated a considerable difference.In contrast with the remarkable bands of wild-type gene in all the strains particularly in codonplus strain, there were no significant bands related to mutant gene in SDS-PAGE analysis.Conclusion: Because of the same conditions of mutant and wild-type genes during thetranslation and transcription, this significant difference may relate to mRNA efficiency fortranslation. Our results indicate that increased stability of 5' mRNA secondary structuresin E. coli prevents efficient translation initiation. Furthermore, wild-type gene significantbands in codon plus strain support the hypothesis that the possible elimination of translationalpauses that increase translation rate leads to over expression.

Keywords

• Codon
• Usage
• Gene Expression
• hbFGF