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

ABSTRACT: 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 (rh?Plg) 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 rh?Plg was 1.0 g/L. We purified rh?Plg to 96% purity by gel-filtration and cation-exchange chromatography. The specific activity of rh?Plg reached 23.6 U/mg. The K m of substrate hydrolysis by recombinant human microplasmin was comparable to that of human plasmin, while rh?Plm had higher k cat /Km values than plasmin. The high purity and activity of the rh?Plg obtained here will likely prove to be a valuable tool for studies of its application in thrombotic diseases and vitreoretinopathies.Reliable rh?Plg production (for use in therapeutic applications) is feasible using genetically modified P. pastoris as a host strain. The successful expression of rh?Plg in P. pastoris lays a solid foundation for its downstream application.

SUBMITTER: Liu R

PROVIDER: S-EPMC4460660 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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

Liu Rongzeng R Zhao Bing B Zhang Yanling Y Gu Junxiang J Yu Mingrong M Song Houyan H Yu Min M Mo Wei W

BMC biotechnology 20150609

<h4>Background</h4>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 ...[more]

PMID: 26054637

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Project description:BackgroundPichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.ResultsA fully compartmentalized metabolic model of P. pastoris (iPP668), composed of 1,361 reactions and 1,177 metabolites, was reconstructed based on its genome annotation and biochemical information. The constraints-based flux analysis was then used to predict achievable growth rate which is consistent with the cellular phenotype of P. pastoris observed during chemostat experiments. Subsequent in silico analysis further explored the effect of various carbon sources on cell growth, revealing sorbitol as a promising candidate for culturing recombinant P. pastoris strains producing heterologous proteins. Interestingly, methanol consumption yields a high regeneration rate of reducing equivalents which is substantial for the synthesis of valuable pharmaceutical precursors. Hence, as a case study, we examined the applicability of P. pastoris system to whole-cell biotransformation and also identified relevant metabolic engineering targets that have been experimentally verified.ConclusionThe genome-scale metabolic model characterizes the cellular physiology of P. pastoris, thus allowing us to gain valuable insights into the metabolism of methylotrophic yeast and devise possible strategies for strain improvement through in silico simulations. This computational approach, combined with synthetic biology techniques, potentially forms a basis for rational analysis and design of P. pastoris metabolic network to enhance humanized glycoprotein production.

Dataset Information

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

Publications

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

Similar Datasets

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