High-level expression, purification, and enzymatic characterization of truncated human plasminogen (Lys531-Asn791) in the methylotrophic yeast Pichia pastoris

Plasmin is a serine protease that plays a critical role in fibrinolysis, which is a process that prevents blood clots from growing and becoming problematic. Recombinant human microplasminogen (rhμPlg) is a derivative of plasmin that solely consists of the catalytic domain of human plasmin and lacks the five kringle domains found in the native protein. Developing an industrial production method that provides high yields of this protein with high purity, quality, and potency is critical for preclinical research. The human microplasminogen gene was cloned into the pPIC9K vector, and the recombinant plasmid was transformed into Pichia pastoris strain GS115. The concentration of plasmin reached 510.1 mg/L of culture medium. Under fermentation conditions, the yield of rhμPlg was 1.0 g/L. We purified rhμPlg to 96% purity by gel-filtration and cation-exchange chromatography. The specific activity of rhμPlg reached 23.6 U/mg. The K m of substrate hydrolysis by recombinant human microplasmin was comparable to that of human plasmin, while rhμPlm had higher k cat /Km values than plasmin. The high purity and activity of the rhμPlg obtained here will likely prove to be a valuable tool for studies of its application in thrombotic diseases and vitreoretinopathies. Reliable rhμPlg production (for use in therapeutic applications) is feasible using genetically modified P. pastoris as a host strain. The successful expression of rhμPlg in P. pastoris lays a solid foundation for its downstream application.