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

Objective: The production of heterologous proteins in Escherichia coli is strongly affected by codon bias. This phenomenon occurs when the codon usage of mRNA coding for the foreign protein differs from that of the bacterium. The ribosome pauses upon encountering a rare codon and may detach from mRNA, thereby the yield of recombinant protein pro- duction reduces. The aim of this study is to investigate the effect of these codon numbers reductions 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 any transformation in amino acids sequence. Silent mutations in 5ʹ codons of human basic fibroblast growth factor cDNA carried out by site-directed mutagenesis and the expression level of the recombinant protein is analyzed by means of sodium dodecyl sulfate polyacry- lamide gel 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 codon plus 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 the translation and transcription, this significant difference may relate to mRNA efficiency for translation. Our results indicate that increased stability of 5ʹ mRNA secondary structures in E. coli prevents efficient translation initiation. Furthermore, wild-type gene significant bands in codon plus strain support the hypothesis that the possible elimination of transla- tional pauses that increase translation rate leads to over expression.