Expression of horseshoe crab arginine kinase in Escherichia coli and site-directed mutations of the reactive cysteine peptide.
Arginine kinase (AK) from the horseshoe crab Limulus polyphemus was expressed in Escherichia coli. The bulk of expressed protein resided in insoluble inclusion bodies. However, approximately 3 mg enzyme protein/l culture was present as active soluble AK. The AK-containing expression vector construct was subjected to site-directed mutagenesis via a polymerase chain reaction-based megaprimer protocol. The AK reactive cysteine peptide was engineered so that it was identical to the corresponding peptide sequence of creatine kinase, another member of the guanidino kinase enzyme family. The resulting expressed protein had a considerably reduced specific activity but was still specific for arginine/arginine phosphate. No catalytic activity was observed with other guanidine substrates (creatine, glycocyamine, taurocyamine, lombricine). The reactive cysteine peptide, characteristic of all guanidino kinases, very likely plays a minimal role in determining guanidine specificity.
Strong
S J
SJ
Department of Biological Science, Florida State University, Tallahassee 32306-3050, USA.
Ellington
W R
WR
D000595:Amino Acid Sequence; D000818:Animals; D001122:Arginine Kinase / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003402:Creatine Kinase / Q000737:chemistry; D003545:Cysteine / Q000032:analysis*; D004926:Escherichia coli; D006146:Guanidines; D006737:Horseshoe Crabs; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis
D006146:Guanidines; D010455:Peptides; D011994:Recombinant Proteins; D003402:Creatine Kinase; D001122:Arginine Kinase; D003545:Cysteine