Cloning, expression, purification, and characterization of arginine kinase from Locusta migratoria manilensis.
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 Km, Kd, 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 kcat 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.
Wu
Qing-Yun
QY
College of Life Science, Shandong Agricultural University, Shandong Taian 271018, People's Republic of China.
Li
Feng
F
Zhu
Wen-Jing
WJ
Wang
Xiao-Yun
XY
D000818:Animals; D001122:Arginine Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies / Q000378:metabolism; D007700:Kinetics; D048188:Locusta migratoria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008974:Mollusca / Q000201:enzymology; D017510:Protein Folding; D020673:Protein Renaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013050:Spectrometry, Fluorescence; D013696:Temperature
D018076:DNA, Complementary; D011994:Recombinant Proteins; D001122:Arginine Kinase