Abstract

Staphylokinase (Sak) (EC 3.4.99.22) is a 136-aa protein secreted by the lysogenic phase of Staphylococcus aureus and functions as potential clot dissolving agent for treating blood-clotting disorders. Sak converts the zymogen human plasminogen (hPg) to its active serine protease form, plasmin (hPm) and forms 1:1 complex with hPm, which activates other hPg molecules. In this report, we described high-level expression, purification and characterization of a fully active even glycosylated staphylokinase variant SakfC from S. aureus QT08 in a eukaryotic system Pichia pastoris GS115. The sak gene of 411 bp encoding a mature staphylokinase (136 aa, 15.5 kDa) was amplified from S. aureus QT08 genomic DNA, inserted into the expression vector pPICZαA under the control of the AOX1 promoter using methanol as an inducer and expressed in P. pastorisGS115 with a yield of 19 mg/L culture broth. The recombinant staphylokinase (rSak) was purified to homogeneity using ProBondÔ Ni²⁺-affinity chromatography with a specific activity of 20658 U/mg. The enzyme was expressed as an N-glycosylated protein in P. pastoris with a molecular mass of approximately 24 kDa on SDS-PAGE. rSak showed an optimum temperature of 30 to 37°C and optimum pH 7.5 (phosphate buffer) and pH 8 (Tris-HCl buffer). rSak was stable in a temperature range of 15 to 37°C and pH of 4 to 9. Additives (metal ions and detergents) all inhibited slightly or strongly the rSak activity. For the first time, a fully active staphylokinase from S. aureus was expressed in P. pastoriseven it was glycosylated.

Key words: Staphylococcus aureus QT08, Pichia pastoris, fully active staphylokinase,high-level expression, purification, characterization, glycosylation