Fusion expression of Escherichia coli prlC gene and preparation of PrlC proteinase affinity column

Escherichia coli PrlC is a trypsin-like proteinase regulating the cell cycl e. The Escherichia coli prlC gene has been cloned into the pET28a prokaryot ic expression vector. The recombinant fusion protein was produced mostly in the soluble, active form and the expression level amounted to approximatel y 70% of total protein. The recombinant proteinase was efficiently adsorbed to a resin containing immobilized Ni2+ via its amino terminal fusion hexah istidine tail to give a PrlC proteinase affinity column. The adsorbed fusio n proteinase hydrolyzed 4-methylcoumaryl-7-amide of tert-butoxycarbonyl-L-v alyl-L-prolyl-L-arginine (Boc-Val-Pro-Arg-NH-Mec), the specific substrate f or the trypsin-like proteinase activity of E. coli PrlC.