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	Heterologous expression, purification, and characterization of cytochrome P450sca-2 and mutants with improved solubility in Escherichia coli
	Li, Pan ; Guan, Hong ; Li, Jin ; Lin, Zhanglin
	2010-10-12 ; 2010-10-12
关键词	Cytochrome P450sca-2 Recombinant expression E. coli Solubility Directed evolution GFP TISSUE-SELECTIVE INHIBITOR COENZYME-A REDUCTASE HMG-COA REDUCTASE STREPTOMYCES-CARBOPHILUS PRAVASTATIN SODIUM DIRECTED EVOLUTION ML-236B COMPACTIN SWISS-MODEL MICROBIAL HYDROXYLATION PROTEIN SOLUBILITY Biochemical Research Methods Biochemistry & Molecular Biology Biotechnology & Applied Microbiology
中文摘要	Pravastatin, an important cholesterol lowering drug, is currently produced by hydroxylation of mevastatin (ML-236B) with Streptomyces carbophilus, in which the enzyme P450sca-2 plays a key role. Little information on the recombinant expression of this enzyme is available. As it is of industrial interest to develop an alternative simplified enzymatic process for pravastatin, as a first step, further study on the heterologous expression of this enzyme is warranted. We report here, for the first time, the purification, and characterization of P450sca-2 expressed in Escherichia coli. A synthetic gene encoding P450sca-2 was designed to suit the standard codon usage of E. coli. Expression of P450sca-2 in E. coli under optimized conditions yielded about 100 nmol purified active P450sca-2 per liter. Directed evolution was further carried out to improve the soluble expression level. In the absence of a facile and sensitive assay, green fluorescent protein (GFP) was used as a reporter to enable high-throughput screening. After three rounds of evolution by error-prone PCR and DNA shuffling, six almost totally soluble mutants were obtained, with the soluble expression levels dramatically improved by about 30-fold. For six most frequently occurring mutations, the corresponding single mutants were created to dissect the effects of these mutations. A single mutation, P159A, was found to be responsible for most of the enhanced solubility observed in the six mutants, and the corresponding single mutant also retained the hydroxylation activity. Our study provides a foundation for future work on improving functional expression of P450sca-2 in E. coli. (C) 2008 Elsevier Inc. All rights reserved.
语种	英语 ; 英语
出版者	ACADEMIC PRESS INC ELSEVIER SCIENCE ; SAN DIEGO ; 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA
内容类型	期刊论文
源URL	[http://hdl.handle.net/123456789/79615]
专题	清华大学
推荐引用方式 GB/T 7714	Li, Pan,Guan, Hong,Li, Jin,et al. Heterologous expression, purification, and characterization of cytochrome P450sca-2 and mutants with improved solubility in Escherichia coli[J],2010, 2010.
APA	Li, Pan,Guan, Hong,Li, Jin,&Lin, Zhanglin.(2010).Heterologous expression, purification, and characterization of cytochrome P450sca-2 and mutants with improved solubility in Escherichia coli..
MLA	Li, Pan,et al."Heterologous expression, purification, and characterization of cytochrome P450sca-2 and mutants with improved solubility in Escherichia coli".(2010).

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