Journal
ENZYME AND MICROBIAL TECHNOLOGY
Volume 49, Issue 4, Pages 395-401Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.enzmictec.2011.06.021
Keywords
Halohydrin dehalogenase; Recombinant expression; +2 Codon optimization; pGEF(+) expression vector; Purification
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Funding
- National Natural Science Foundation of China [20872014]
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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 colt 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 (Delta G) 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. (C) 2011 Elsevier Inc. All rights reserved.
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