title: Expression, purification, and biological characterization of the amino-terminal fragment of urokinase in Pichia pastoris.
abstract: Urokinase (uPA) and its receptor (uPAR) play an important role in tumor growth and metastasis. Targeting the excessive activation of this system as well as the proliferation of the tumor vascular endothelial cell would be expected to prevent tumor neovasculature and halt the tumor development. In this regard, the amino-terminal fragment (ATF) of urokinase has been confirmed as effective to inhibit the proliferation, migration, and invasiveness of cancer cells via interrupting the interaction of uPA and uPAR. Previous studies indicated that ATF expressed in Escherichia coli was mainly contained in inclusion bodies and also lacked posttranslational modifications. In this study, the biologically active and soluble ATF was cloned and expressed in Pichia pastoris. The recombinant protein was purified to be homogenous and confirmed to be biologically active. The yield of the active ATF was about 30 mg/l of the P. pastoris culture medium. The recombinant ATF (rATF) could efficiently inhibit angiogenesis, endothelial cell migration, and tumor cell invasion in vitro. Furthermore, it could inhibit in vivo xenograft tumor growth and prolong the survival of tumor-bearing mice significantly by competing with uPA for binding to cell surfaces. Therefore, P. pastoris is a highly efficient and cost-effective expression system for large-scale production of biologically active rATFs for potential therapeutic application.
authors.affiliation: The State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093 Jiangsu, P.R. China.
authors.forename: Jianping
authors.initials: J
authors.lastname: Li
authors.forename: Yuli
authors.initials: Y
authors.lastname: Lin
authors.forename: Hongqin
authors.initials: H
authors.lastname: Zhuang
authors.forename: Zi-Chun
authors.initials: ZC
authors.lastname: Hua
mesh_terms: D020816:Amino Acid Motifs; D020533:Angiogenesis Inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*; D000818:Animals; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply / Q000187:drug effects; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009389:Neovascularization, Pathologic / Q000188:drug therapy; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology*
chemical_list: D020533:Angiogenesis Inhibitors; D011994:Recombinant Proteins; D014568:Urokinase-Type Plasminogen Activator