ABSTRACT
Carbon source plays an important role in the constitutive expression of foreign proteins in Pichia pastoris. In present study, glucose , glycerol , methanol and oil acid, was used respectively as the only carbon source to constitutively express hAS in Pichia pastoris GS115 (pGAP9K-AS)in shaking flask. The result shows that oleic acid is the best (163 mg/L) compared with glycerol (83mg/L), glucose (76 mg/L)and methanol (57 mg/L). Since oleic acid is insoluble in water, glycerol was used as the carbon source in the high-density cell culture of GS115 (pGAP9K-AS) in a 30 liter bioreactor and 169 mg/L of angiostatin was obtained after 48h of culture. The expressed angiostatin is immunologically active as shown by Western blotting. The recombinant hAS inhibits bFGF induced CAM angiogenesis and suppresses the growth of B16 melanoma in C57BL/6J mice. The tumor inhibition rate is 90% after 12 days of treatment. Statistics analysis revealed that the tumor volume difference of mice between the hAS group and PBS group is prominent (P < 0.01).
Subject(s)
Animals , Humans , Mice , Angiogenesis Inhibitors , Genetics , Therapeutic Uses , Angiostatins , Genetics , Therapeutic Uses , Bioreactors , Microbiology , Culture Media , Pharmacology , Fermentation , Glycerol , Pharmacology , Melanoma, Experimental , Drug Therapy , Mice, Inbred C57BL , Pichia , Genetics , Metabolism , Recombinant Proteins , Genetics , Therapeutic UsesABSTRACT
Human tumstatin(hTumstatin)cDNA was amplified from recombinant plasmid pET-3c-tum, cloned in frame with the signal sequence in yeast vector pPICZalphaA and transformed into Pichia pastoris GS115 by electroporation. The expression of hTumstatin in GS115(pPICZalpha-tum)was then induced by methanol and secreted into the culture medium, with a yield of 25mg/L as shown by SDS-PAGE and Western blotting. The expressed hTumstatin was purified to more than 85% purity using a simple one-step SP-Sepharose cation exchange chromatography. The MTT and chick chorioallantoic membrane assay showed that the yeast produced hTumstatin could inhibit the proliferation of human umbilical vein endothelial cells and the neovascularization induced by bFGF. Hoechst 33258 fluorescent staining also demonstrated the apoptotic change in endothelial cellular nuclear morphology.
Subject(s)
Humans , Angiogenesis Inhibitors , Metabolism , Autoantigens , Genetics , Metabolism , Cell Proliferation , Cells, Cultured , Collagen Type IV , Genetics , Metabolism , DNA, Complementary , Genetics , Electroporation , Endothelial Cells , Cell Biology , Pichia , Genetics , Metabolism , Recombinant Proteins , Genetics , Metabolism , Umbilical Cord , Cell BiologyABSTRACT
Kringle 1-3 domain is a recently found angiogenesis inhibitor with anti-angiogenesis and anti-tumor activity. The kringle 1-3 gene was amplified by PCR technique using angiostatin gene as template. After DNA sequencing, the PCR product was cloned into pPIC9K resulting in recombinant plasmid pPIC9K13 which was then transformed into Pichia pastoris GS115. The high copy integration transformants screened by PCR and G418 methods were cultivated in flasks. The K1-3 was expressed and secreted to the medium and has immunogenic activity as shown by SDS-PAGE and Western blotting. High cell density culture was carried out in 30-liter and 80-liter bioreactor, the biomass reaches 300 OD after methanol induction, and the expressed product is 200 mg/L. The fermentation supernatant was purified by Streamline SP and Phenyl Sepharose Chromatography, the product appears as a single band on SDS-PAGE, with a purity of 95%-96%. The purified product has anti-angiogenesis and anti-tumor activity.