mRNA secondary structure at start AUG codon is a key limiting factor for human protein expression in Escherichia coli.

Codon usage and thermodynamic optimization of the 5'-end of mRNA have been applied to improve the efficiency of human protein production in Escherichia coli. However, high level expression of human protein in E. coli is still a challenge that virtually depends upon each individual target genes. Using human interleukin 10 (huIL-10) and interferon alpha (huIFN-alpha) coding sequences, we systematically analyzed the influence of several major factors on expression of human protein in E. coli. The results from huIL-10 and reinforced by huIFN-alpha showed that exposing AUG initiator codon from base-paired structure within mRNA itself significantly improved the translation of target protein, which resulted in a 10-fold higher protein expression than the wild-type genes. It was also noted that translation process was not affected by the retained short-range stem-loop structure at Shine-Dalgarno (SD) sequences. On the other hand, codon-optimized constructs of huIL-10 showed unimproved levels of protein expression, on the contrary, led to a remarkable RNA degradation. Our study demonstrates that exposure of AUG initiator codon from long-range intra-strand secondary structure at 5'-end of mRNA may be used as a general strategy for human protein production in E. coli.