Abstract: There are still difficulties in regulating the expression and solubility of heterologous proteins in E. coli. In this study, a protein expression optimization method that combines overlapping gene structure and 5′-end sequence optimization of mRNA was developed. Utilizing the 5′-end tag of the pET-28a plasmid vector expression box, and the target gene introduced into TG before the start codon formed an overlapping gene type expression structure of TGATG. Then, by designing degenerate primers, synonymous codon mutations of 14 consecutive codons at the 5′-end of a heterologous gene mRNA were performed to change the secondary structure of mRNA and realize the screening of overlapping gene structure strength. This method can affect the expression of heterologous protein without changing the plasmid composition and external conditions, and regulate the solubility of the protein by affecting the rate of the translation process. Several examples of heterologous gene expression optimization in this study have proved the role of overlapping gene structures in regulating the soluble expression of proteins. It provided a simple optimization strategy for the regulation of the expression and solubility of heterologous proteins in E. coli.

Key words: E. coli, heterologous expression, synonymous codon, mRNA structure, overlapping genes