中国生物工程杂志

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 pPICZαA. The linearized recombinant palsmid pPICZαA-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 KD 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<0.01). Our data provides the first evidence that sv-cystatin could not only inhibit papain activity, but also suppress the Matrigel invasion of B16F1 Melanoma cell line.