title: Expression of the non-glycosylated kringle domain of tissue type plasminogen activator in Pichia and its anti-endothelial cell activity.
abstract: The two-kringle domain of tissue-type plasminogen activator (TK1-2) has been identified as a potent angiogenesis inhibitor by suppressing endothelial cell proliferation, in vivo angiogenesis, and in vivo tumor growth. Escherichia coli-derived, non-glycosylated TK1-2 more potently inhibits in vivo tumor growth, whereas Pichia expression system is more efficient for producing TK1-2 as a soluble form, albeit accompanying N-glycosylation. Therefore, in order to avoid immune reactivity and improve in vivo efficacy, we expressed the non-glycosylated form of TK1-2 in Pichia pastoris and evaluated its activity in vitro. When TK1-2 was mutated at either Asn(117) or Asn(184) by replacing with Gln, the mutated proteins produced the glycosylated form in Pichia, of which sugar moiety could be deleted by endoglycosidase H treatment. When both sites were replaced by Gln, the resulting mutant produced a non-glycosylated protein, NQ-TK1-2. Secreted NQ-TK1-2 was purified from the culture broth by sequential ion exchange chromatography using SP-sepharose, Q-spin, and UNO-S1 column. The purified NQ-TK1-2 migrated as a single protein band of approximately 20 kDa in SDS-PAGE and its mass spectrum showed one major peak of 19,950.71 Da, which is smaller than those of two glycosylated forms of wild type TK1-2. Functionally, the purified NQ-TK1-2 inhibited endothelial cell proliferation and migration stimulated by bFGF and VEGF, respectively. Therefore, the results suggest that non-glycosylated TK1-2 useful for the treatment of cancer can be efficiently produced in Pichia, with retaining its activity.
authors.affiliation: Cancer Research Institute, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea.
authors.forename: Sang-Bae
authors.initials: SB
authors.lastname: Lee
authors.forename: Ho-Kyun
authors.initials: HK
authors.lastname: Oh
authors.forename: Hyun-Kyung
authors.initials: HK
authors.lastname: Kim
authors.forename: Young Ae
authors.initials: YA
authors.lastname: Joe
mesh_terms: D020533:Angiogenesis Inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D018082:Kringles / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000235:genetics*; D013329:Structure-Activity Relationship; D013937:Thymidine Kinase / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology*; D042442:Vascular Endothelial Growth Factors / Q000494:pharmacology
chemical_list: D020533:Angiogenesis Inhibitors; D042442:Vascular Endothelial Growth Factors; D016222:Fibroblast Growth Factor 2; C105339:thymidine kinase 2; D013937:Thymidine Kinase; C092717:thymidine kinase 1; D010959:Tissue Plasminogen Activator