Expression, purification, and characterization of His-tagged penicillin G acylase from Kluyvera citrophila in Escherichia coli

	Expression, purification, and characterization of His-tagged penicillin G acylase from Kluyvera citrophila in Escherichia coli
	Yong, W; Shi, XL; Yuan, ZY; Pei, Z
刊名	PROTEIN EXPRESSION AND PURIFICATION
	2004
卷号	38 期号:1 页码:24-28
关键词	penicillin G acylase Kluyvera citrophila His-tag fusion protein
通讯作者	Pei, Z (reprint author), Fudan Univ, Sch Pharm, Dept Drug Biosynth, Shanghai 200032, Peoples R China.,pzhou@shmu.edu.cn
英文摘要	The DNA fragment encoding Kluyvera citrophila penicillin G acylase (KcPGA) was amplified and cloned into the vector pET28b to obtain a C-terminus His-tagged fusion expression plasmid. The fusion protein KcPGA was successfully overexpressed in Escherichia coli BL21(DE3). The optimal induction concentration of isopropylthio-p-D-galactoside (IPTG) was found to be 5 M The fusion protein was purified in a single step by Ni-IDA affinity chromatograph to a specific activity of 35.3 U/mg protein with a final yield of 89% representing a 23-fold purification. The data presented here suggest that the purified fusion protein is stable with respect to pH and temperature. The optimal pH and temperature of recombinant KcPGA are 8.5 and 55 degreesC, respectively. The K-m and V-max are 17.6 muM and 23.8 U/mg, respectively. Therefore, the high yield and high specific activity of recombinant KcPGA produced in E coli, together with other kinetic parameters, represent an excellent basis for further development of recombinant KcPGA as an immobilized biocatalyst for industrial applications. (C) 2004 Elsevier Inc. All rights reserved.
学科主题	Biochemistry & Molecular Biology; Biotechnology & Applied Microbiology
类目[WOS]	Biochemical Research Methods ; Biochemistry & Molecular Biology ; Biotechnology & Applied Microbiology
关键词[WOS]	PROTEIN
收录类别	SCI
语种	英语
WOS记录号	WOS:000224778800004
内容类型	期刊论文
版本	出版稿
源URL	[http://202.127.25.143/handle/331003/2007]
专题	上海生化细胞研究所_上海生科院生化细胞研究所
推荐引用方式 GB/T 7714	Yong, W,Shi, XL,Yuan, ZY,et al. Expression, purification, and characterization of His-tagged penicillin G acylase from Kluyvera citrophila in Escherichia coli[J]. PROTEIN EXPRESSION AND PURIFICATION,2004,38(1):24-28.
APA	Yong, W,Shi, XL,Yuan, ZY,&Pei, Z.(2004).Expression, purification, and characterization of His-tagged penicillin G acylase from Kluyvera citrophila in Escherichia coli.PROTEIN EXPRESSION AND PURIFICATION,38(1),24-28.
MLA	Yong, W,et al."Expression, purification, and characterization of His-tagged penicillin G acylase from Kluyvera citrophila in Escherichia coli".PROTEIN EXPRESSION AND PURIFICATION 38.1(2004):24-28.