Staphylokinase (SAK), a nonglycosylated single-chain protein secreted by certain strain of Staphylococcus aureus, functions as profibrinolytic agent by virtue of its ability to convert human plasminogen (hPg) to its active serine protease form, plasmin (hPm). Secretion of SAK was designed in Pichia pastoris that is methylotrophic yeast, capable of metabolizing methanol as its sole carbon source. The Pichia expression vector, pPIC9K has AOX1 promoter and a pre-pro peptide signal sequence of mating factor. AOX1 promoter was tightly regulated and highly induced by methanol. The SAK gene was introduced into pPIC9K vector. The resulting expression vector, pPIC-SAK, was transformed into the P. pastoris GS115 by electroporation. Secretion of recombinant SAK was confirmed by skim-milk human plasminogen overlay test. Mixture of glycosylated and nonglycosylated SAK was secreted into the culture medium at the level of about 300mg/liter. Glycosylated SAK occupies about 80% in the total SAK secretion amount. Both of glycosylated and nonglycosylated SAKs were separated through CM-chromatography and ConA affinity chromatography. Nonglycosylated SAK shows plasminogen activation activity, while glycosylated SAK did not have the activity. However, removal of glycans in glycosylated SAK recovered the SAK activity. As result, inactive glycosylated recombinant SAK that consists of major part in secreted SAK was easily purified by ConA affinity chromatography and subsequently converted to biologically active SAK by N-glycosidase treatment.