title: Rational design of translational pausing without altering the amino acid sequence dramatically promotes soluble protein expression: a strategic demonstration.
abstract: The production of many pharmaceutical and industrial proteins in prokaryotic hosts is hindered by the insolubility of industrial expression products resulting from misfolding. Even with a correct primary sequence, an improper translation elongation rate in a heterologous expression system is an important cause of misfolding. In silico analysis revealed that most of the endogenous Escherichia coli genes display translational pausing sites that promote correct folding, and almost 1/5 genes have pausing sites at the 3'-termini of their coding sequence. Therefore, we established a novel strategy to efficiently promote the expression of soluble and active proteins without altering the amino acid sequence or expression conditions. This strategy uses the rational design of translational pausing based on structural information solely through synonymous substitutions, i.e. no change on the amino acids sequence. We demonstrated this strategy on a promising antiviral candidate, Cyanovirin-N (CVN), which could not be efficiently expressed in any previously reported system. By introducing silent mutations, we increased the soluble expression level in E. coli by 2000-fold without altering the CVN protein sequence, and the specific activity was slightly higher for the optimized CVN than for the wild-type variant. This strategy introduces new possibilities for the production of bioactive recombinant proteins.
authors.affiliation: Institute of Biomedicine & National Engineering Research Center of Genetic Medicine, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.affiliation: Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
authors.forename: Jingjie
authors.initials: J
authors.lastname: Jin
authors.affiliation: Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Gu
authors.affiliation: Institute of Biomedicine & National Engineering Research Center of Genetic Medicine, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Wei
authors.affiliation: Institute of Biomedicine & National Engineering Research Center of Genetic Medicine, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Lei
authors.affiliation: Institute of Biomedicine & National Engineering Research Center of Genetic Medicine, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China. Electronic address: xiongsheng@jnu.edu.cn.
authors.forename: Sheng
authors.initials: S
authors.lastname: Xiong
authors.affiliation: Key Laboratory of Functional Protein Research of Guangdong Higher Education Institutes, Institute of Life and Health Engineering, College of Life Science and Technology, Jinan University, Guangzhou 510632, China. Electronic address: zhanggong@jnu.edu.cn.
authors.forename: Gong
authors.initials: G
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism; D014176:Protein Biosynthesis; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins
keywords: Antiviral activity; Cyanovirin-N; Escherichia coli; Protein folding; Synonymous substitutions; Translation pausing