Abstract
Cytochrome P450 (CYP) 2C9 is of major importance in drug metabolism. However, the low yield of recombinant CYP2C9 protein in E. coli strains prevents its extensive use in the study of in vitro drug metabolism. In the present study, Taguchi design and desirability function were first used to investigate the effect of medium components (glycerol, δ-ALA, IPTG, ampicillin, chloramphenicol, inoculum density, peptone, thiamine, trace elements, NH4Cl, and MgSO4) on recombinant human CYP2C9 production by E. coli DH5α. An L12 (211) orthogonal array was used to design the experiments to screen out the most influential factors. The CYP concentration and the specific content of CYP were considered as two product quality variables. A desirability function was applied to combine these two qualities as a single objective function. Optimization via central composite design (CCD) was then undertaken to yield the best performance. The confirmation experiments indicated that the expression performance under the optimized conditions was better than those obtained under other conditions. A compromise between conflicting goals, such as achievement of good yield of recombinant CYP2C9 and facility of the following purification, was found by means of the desirability function D. This is the first report that combined Taguchi design and CCD, and performed experiments in a multiresponse framework to optimize the production of human CYP in a recombinant E. coli strain.
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Acknowledgement
We thank Dr. E.F. Johnson (Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA) for kindly providing expression plasmid pCW2C9dH. This work is supported by 973 Program (2003CCA03400) of the Ministry of Science and Technology of China.
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Hao, D.C., Zhu, P.H., Yang, S.L. et al. Optimization of Recombinant Cytochrome P450 2C9 Protein Production in Escherichia coli DH5α by Statistically-Based Experimental Design . World J Microbiol Biotechnol 22, 1169–1176 (2006). https://doi.org/10.1007/s11274-006-9158-9
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DOI: https://doi.org/10.1007/s11274-006-9158-9