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Expression of vascular basement membrane derived multifunctional peptide in Pichia pastoris and identification of its bioactivity
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Abstract:
To express vascular basement membrane-derived multiple peptide (VBMDMP) in Pichia pastoris and to identify its bioactivity. Methods: PCR technique was used to obtain the target fragment GST-VBMDMP, which was then cloned into pPIC9K vector. The resultant vector pPIC9K-GST-VBMDMP was transfected into Pichia pastoris cells with spheroplast and the positive recons was identified. The His + Muts transformant was shake-cultured in MGY at 30 ℃. The culture supernatant (1 ml) was collected for preservation at -70 ℃ every 24 hours while maintaining the concentration of methanol at 0.5% in the culture medium. SDS-PAGE analysis was used to detect the protein expression after 8 days and then animal and cell experiments were performed with GST-VBMDMP. Results: SDS-PAGE analysis found that protein express increased during 1-7 days and decreased after 8 days in MGY medium; GST-VBMDMP fused protein was obtained by Glutathione Sepharose 4B and it inhibited the artery endothelial cell tube structure formation in C57BL／6 mouse; GST-VBMDMP（ 2, 6, 10 mg/kg）also significantly inhibited the primary cancer Lewis mouse （tumor inhibitor rates being 96.6%, 82.1%, and 61.2%, respectively）and metastatic lung tumors（tumor inhibitor rates being 96.8 %, 87.9 %, and 75.3%, respectively）（P<0.01）. Conclusion: VBMDMP can inhibit mouse artery endothelial cell tube structure formation and Lewis mouse primary, metastatic lung tumors.
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