Expression of Recombinant Snake Venom Cystatin in Yeast Pichia pastoris and Its Effects on B16F1 Melanoma Invasion in vitro
- VernacularTitle:蛇毒半胱氨酸蛋白酶抑制剂的酵母表达及其对B16细胞体外侵袭的抑制作用
- Author:
Rong WAN
;
Jun SONG
;
Hai-Ying ZHENG
;
Xiao-Yan ZHANG
;
Xu LIN
;
Jian-Yin LIN
;
- Publication Type:Journal Article
- Keywords:
sv-cystatin Pichia pastoris Secreted expression B16F1 cell Invade
- From:
China Biotechnology
2006;0(07):-
- CountryChina
- Language:Chinese
-
Abstract:
To investigate the biological role of snake venom cystatin(sv-cystatin) in tumor progression, the cDNA of sv-cystatin amplified by PCR from pUC18-cystatin plasmid was cloned into methanol-inducible expression vector pPICZ?A. The linearized recombinant plasmid pPICZ?A-cystatin was transfered into Pichia pastoris, strain GS115 by electrophoration. Transfermants with phenotype Mut+ selected were identified by PCR analysis and induced in 1.0% methanol. The reombinant sv-cystatin protein was examined by SDS-PAGE, Western blot analysis. The molecular mass of expression product was about 14 kDa and approximately 16 mg/L of recombinant sv-cystatin was produced from one of GS115-cystatin transformants. The chromatography purified protein could reduce the activity of papain. The ability of B16F1 cells treated with recombinant sv-cystatin to invade the reconstituted basement membrane decreased significantly (P
