PMID:22112566
| Citation |
Tang, L, Jiang, R, Zheng, K and Zhu, X (2011) Enhancing the recombinant protein expression of halohydrin dehalogenase HheA in Escherichia coli by applying a codon optimization strategy. Enzyme Microb. Technol. 49:395-401 |
|---|---|
| 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. |
| Links |
PubMed Online version:10.1016/j.enzmictec.2011.06.021 |
| Keywords |
Arthrobacter/enzymology; Arthrobacter/genetics; Base Sequence; Biotechnology/methods; Codon/chemistry; Codon/genetics; Escherichia coli/enzymology; Escherichia coli/genetics; Gene Expression Regulation, Bacterial; Hydrolases/genetics; Hydrolases/metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Nucleic Acid Conformation; Recombinant Proteins/genetics; Recombinant Proteins/metabolism |
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