High-level expression, purification and properties of a fully active even glycosylated staphylokinase variant SakφC from Staphylococcus aureus QT08 in Pichia pastoris

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 Sak phi C 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 pPICZ alpha A under the control of the AOX1 promoter using methanol as an inducer and expressed in P. pastoris GS115 with a yield of 19 mg/L culture broth. The recombinant staphylokinase (rSak) was purified to homogeneity using ProBond (TM) 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 degrees 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 degrees 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 staphylokinase from S. aureus was expressed in P. pastoris even it was glycosylated.