Cloning, expression, purification, and characterization of arginine kinase from Locusta migratoria manilensis.

Cloning, expression, purification, and characterization of arginine kinase from Locusta migratoria manilensis. Research Abstract Details 

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Cloning, expression, purification, and characterization of arginine kinase from Locusta migratoria manilensis. Abstract Text:

Qing-Yun Wu,Feng Li,Wen-Jing Zhu,Xiao-Yun Wang,Qing-Yun Wu,Feng Li,Wen-Jing Zhu,Xiao-Yun Wang,Qing-Yun Wu,Feng Li,Wen-Jing Zhu,Xiao-Yun Wang,

Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. The gene encoding Locusta migratoria manilensis AK was cloned and expressed in Escherichia coli by two prokaryotic expression plasmids, pET-30a and pET-28a. The recombinant protein was expressed as inclusion bodies using pET-30a. After denaturation, the recombinant AK was successfully renatured and confirmed to be enzymatically active. Addition of Tween-20 and SDS to the dilution system led to higher renaturation efficiency. Using another expression plasmid, pET-28a, and changing the expression conditions resulted in a soluble and functional form of AK, which was purified by an improved method using Sephadex G-75 chromotography to a final yield of 358 mg L(-1) of LB medium. Some parameters for the renatured and soluble forms of AK, including K(m), K(d), specific activity, electrophoretic mobility and isoelectric focusing, were identical with those of AK obtained directly from L. migratoria manilensis leg muscle. Comparison of kinetic constants with those of AKs from other sources indicated that L. migratoria manilensis AKs have the highest k(cat) and stronger synergistic substrate binding. The first report of a concise purification method enables the enzyme to be prepared in large quantities. This research should enable further detailed investigations of the enzymatic mechanism by site directed mutagenesis techniques.

Cloning, expression, purification, and characterization of arginine kinase from Locusta migratoria manilensis. Publishing Authors By Initials

QY Wu,F Li,WJ Zhu,XY Wang,QY Wu,F Li,WJ Zhu,XY Wang,QY Wu,F Li,WJ Zhu,XY Wang,

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Cloning, expression, purification, and characterization of arginine kinase from Locusta migratoria manilensis. Journal Published:

PUBLICATION TYPE: Journal Article

Journal: Comparative biochemistry and physiology. Part B, B

VOLUME: 148

Page Numbers: 355-62

Journal Abbreviation: Comp. Biochem. Physiol. B, Bio

ISSN: 1096-4959

DAY: 14

MONTH: 07

YEAR: 2007

Cloning, expression, purification, and characterization of arginine kinase from Locusta migratoria manilensis. Information

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LANGUAGE: eng

NlmUniqueID: 9516061

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Grant and Affiliation Information for Cloning, expression, purification, and characterization of arginine kinase from Locusta migratoria manilensis.

AFFILIATION: College of Life Science, Shandong Agricultural University, Shandong Taian 271018, People's Republic of China.

Country: England

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MEDLINETA: Comp Biochem Physiol B Biochem

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