title: Constitutive expression of human angiostatin in Pichia pastoris using the GAP promoter.
abstract: The GAP gene promoter was amplified from P. pastoris GS115 and used to replace the AOX1 promoter (P(AOX1)) on pPIC9K resulting in plasmid pGAP9K. The recombinant expression vector pGAP9K-AS was constructed by inserting the angiostatin gene(AS) into pGAP9K. pGAP9K-AS was then transformed into P. pastoris GS115. The multi-copy integration transformant P. pastoris GS115 (pGAP9K-AS) was used to investigate the constitutive expression of angiostatin in P. pastoris. The expression of angiostatin reached its peak after 4 d of culture in P. pastoris GS115 (pGAP9K-AS) while the angiostatin expressed in P. pastoris GS115 (pPIC9K-AS) after 4 d of induction or 5 d of culture is only 70% of that expressed by P. pastoris GS115 (pGAP9K-AS). The AS expression in inducible system reached the peak after 6 d of induction but the expressed AS was only 86% of that from constitutive system. The results of anti-angiogenic and antitumor activity assay showed that AS expressed from both constitutive and inducible system inhibited the CAM angiogenesis and suppressed B16 melanoma in C57BL/6J mouse and that the tumor inhibition rates reached 90.63% and 90.54%, respectively. The above data indicates that the constitutive promoter P(GAP) can served as an effective alternative to the inductive promoter P(AOX1) to express AS and other proteins in P. pastoris.
authors.affiliation: The Key Laboratory of Gene Engineering of Ministry of Education, Department of Biochemistry, Zhongshan University, Guangzhou 510275, China.
authors.forename: Ai-Lian
authors.initials: AL
authors.lastname: Zhang
authors.forename: Jin-Xian
authors.initials: JX
authors.lastname: Luo
authors.forename: Tian-Yuan
authors.initials: TY
authors.lastname: Zhang
authors.forename: Shou-Cai
authors.initials: SC
authors.lastname: Chen
authors.forename: Wen-Jun
authors.initials: WJ
authors.lastname: Guan
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D043168:Angiostatins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002642:Chick Embryo; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D006801:Humans; D008297:Male; D008546:Melanoma, Experimental / Q000188:drug therapy; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000494:pharmacology; D017422:Sequence Analysis, DNA
chemical_list: D020533:Angiogenesis Inhibitors; D011994:Recombinant Proteins; D043168:Angiostatins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases