Activity and Integrating Expression of Human Endostatin Produced by Pichia pastoris

Heterologous expression in Pichia pastoris has many of the advantages of eukaryotic expression, proper folding and disulfide bond formation, glycosylation, and secretion. Contrary to other eukaryotic systems, protein production from P. pastoris occurs in simple minimal defined media making this system attractive for production of expressed proteins for purification. Endostatin is a potent and specific antiangiogenic protein capable of inhibiting the growth of murine and xenotransplanted human tumors. Thus far, however, recombinant endostatin prepared from Escherichia coli has been insoluble after purification and therefore inappropriate for clinical settings. In this study, human endostatin gene was integrated into the chromosome of host pichia pastoris by using the yeast inserted plasmid pPICZaA-endo including human endostatin gene, native Saccharomyces cerevisiae a-factor secretion signal, zeocin resistant gene and the AOX1 promoter and transcription terminator (TT) of pichia pastoris electroporation. The recombinant clones were then selected by the plates containing antibiocin-zeocin (100ug/ml), and finally one highly expressed clone was selected by PCR, SDS-PAGE and Western-blot. The yield of expressed endostatin from P. pastoris depended critically on growth conditions, and attainment of high cell densities by fermentation had been shown to improve protein yields up to 15mg/L estimated. Moreover, the protein was easily purified by using a heparin-agarose column. A strengthened antiangiogenic activity in vivo has been identified by a novel and simple CAM (chorioallantoic membrane) technique.