title: High-level expression, purification, and enzymatic characterization of truncated human plasminogen (Lys531-Asn791) in the methylotrophic yeast Pichia pastoris.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. liur12@fudan.edu.cn.|Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. liur12@fudan.edu.cn.
authors.forename: Rongzeng
authors.initials: R
authors.lastname: Liu
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. zhaobing828@163.com.|Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. zhaobing828@163.com.
authors.forename: Bing
authors.initials: B
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. zhangyl@shmu.edu.cn.|Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. zhangyl@shmu.edu.cn.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. morris_gu@icloud.com.|Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. morris_gu@icloud.com.
authors.forename: Junxiang
authors.initials: J
authors.lastname: Gu
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. charis_yu@icloud.com.|Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. charis_yu@icloud.com.
authors.forename: Mingrong
authors.initials: M
authors.lastname: Yu
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. hysong@shmu.edu.cn.|Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. hysong@shmu.edu.cn.|Collaborative Innovation Center for Biotherapy, Sichuan University, Huaxi Campus: No.17 People's South Road, Chengdu, 610041, China. hysong@shmu.edu.cn.
authors.forename: Houyan
authors.initials: H
authors.lastname: Song
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. minyu@shmu.edu.cn.|Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. minyu@shmu.edu.cn.
authors.forename: Min
authors.initials: M
authors.lastname: Yu
authors.affiliation: Key Laboratory of Metabolism and Molecular Medicine, Ministry of Education, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. weimo1025@shmu.edu.cn.|Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Fudan University, 138 Yixueyan Rd, Shanghai, 200032, China. weimo1025@shmu.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Mo
mesh_terms: D019149:Bioreactors / Q000382:microbiology; D003001:Cloning, Molecular; D005285:Fermentation; D005343:Fibrinolytic Agents / Q000494:pharmacology; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010958:Plasminogen / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology
chemical_list: D005343:Fibrinolytic Agents; D010446:Peptide Fragments; C111879:microplasminogen protein, human; D010958:Plasminogen