title: Enhancing the recombinant protein expression of halohydrin dehalogenase HheA in Escherichia coli by applying a codon optimization strategy.
abstract: Halohydrin dehalogenases are attractive biocatalysts in producing a series of important chiral building blocks. Recombinant expression of halohydrin dehalogenase from Arthrobacter sp. AD2 (HheA) in Escherichia coli using T7 promoter-based pGEF(+) system revealed much lower expression level than that of the well-studied halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC). In this study, we changed the codon usage in the 5'-end of hheA gene to improve the expression yield of HheA. Our results showed that the expression of HheA could be largely improved by the replacement of G-rich +2 codon (adjacent to the start codon) with less G-containing codons. The expression of one of the resulting mutants HheA-D1 (replaced +2 codon GTG with CCA) was about 4-fold higher and purified yields about 8-fold greater than that of the wild-type HheA. Moreover, the expression level of the resulting HheA variants correlated well with the minimal folding free energy (ΔG) of the mRNA secondary structure surrounding the 5'-end region of the genes. These findings suggested that the G-rich +2 codon of hheA gene might be the main suppressive factor for limiting the recombinant expression of HheA and that +2 codon optimization strategy could be used as a general tool in modulating recombinant protein production in E. coli.
authors: School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China. lixiatang@uestc.edu.cn
authors: Lixia
authors: L
authors: Tang
authors: Rongxiang
authors: R
authors: Jiang
authors: Kai
authors: K
authors: Zheng
authors: Xuechen
authors: X
authors: Zhu
mesh_terms: D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods; D003062:Codon / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D003062:Codon; D011994:Recombinant Proteins; D006867:Hydrolases; C430848:halohydrin dehalogenase