Abstract

The staphylokinase (Sak) is emerging as an important thrombolytic agent for the treatment of patients suffering from cardiovascular disease. Hence in this study, wereported the cloning, high-level expression, purification and characterization of the Sak variant SakfC from Staphylococcus aureus QT08 in Escherichia coli Bl21. The sakgene of 489 bp encoding a protein (163 amino acids) with a predicted molecular massof 18.5 kDa and pI 7.28 showed 99.8 to 99.6% identity with corresponding sequences from S. aureus strains deposited in GenBank (AF332619, X00127, EF122253 and M57455). The DNA sequence (411 bp) encoding the mature Sak (15.5 kDa) truncated 27 N-terminal amino acids was expressed in E. coli BL21/pESak under the control of the strong promoter tac in the presence of isopropyl-β-D-1-thiogalactopynoside (IPTG) as inducer. The expression level of rSak was estimated at about 42% of the total cellular proteins by densitometry scanning, which is the highest expression level of rSak expressed in any E. coli system. The recombinant staphylokinase was purified by Ni²⁺-ProBondÔ column to a single homogeneous 16-kDa band on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) with a specific activity of 15175 U/mg protein, a recovery yield of 58% and a purification factor of 2.56. The optimal pH and temperature for the rSak activity was 9 and 37°C,respectively. rSak was stable over a temperature range of 25 to 50°C and at pH range of 7 to 9. Metal ions and detergents also showed an inhibitory effect on rSak, especially Zn²⁺ and Cu²⁺ which completely inhibited the enzymatic activity.

Key words: Staphylococcus aureus QT08, staphylokinase, cloning, high-level expression, purification, characterization.

Abbreviation

Sak, Staphylokinase; IPTG, isopropyl-β-D-1-thiogalactopynoside