High cell-density fermentation of Pichia pastoris that express endostatin / 重庆医科大学学报

ABSTRACT

Objective:To develop a high cell-density fermentation process in micro DCU-400 fermentor of Pichia pastoris GS115 containing a recombinant vector pPICZ ?/endostatin.Methods:Cells were grown in the basal salts medium(BSM) at 30℃,pO 2≥30%,pH 5.3(growing) or pH 5.0(inducing),and the feed operation using 50% glycerol was performed when dissolved oxygen(pO 2) increased(after approximately 16-18h).It lasted 3-5hrs.When cell density(OD 600 ) reached 450,the inducement was began.After the transition from glycerol-methanol mixture to methanol,methanol from 0.03ml/min/L to 0.08ml/min/L in 8h was used to induce the endostatin expression.The inducement lasted for approximately 60h.Then centrifugation,the supernatant was collected.The expression was examined using 12% SDS-PAGE and ELISA.Results:In inducing phase,OD 600 reached 580(wet wt 275g/L).The production of endostatin is 27.2mg/L,exceeding that in spinner flask(16.3mg/L).Conclusion:The human endostatin had been successfully expressed in 15L fermentor.