Expression Of The Recombinant Peptide Of Human Cytomegalovirus In The Pichia Pastoris By High Density Culture

In order to express the recombinant peptide of both gp52 and pp150 Oterminal peptides from human cytomegalovirus (HCMV) in the Pichia pastoris, the recombinant plasmids were transformed into Pichia pastor is by electroporation after linearised respectively by Sac I or Bg1II, the transformants were screened in MD plates without histidine. The Mut+and Muts transformants were identifed according to their different growth characteristic. Some of positive transformants were confirmed by PCR. All Mut+and Muts colones were induced respectively with methanol to secrete interesting peptide in shake-flask cultures for four days. After dialysis and lyophilization, the supernate of cultures were identified by SDS-PAGE and Western blotting to find the genetically engineered Pichia pastoris which expressed interesting peptide. The yield and purity of interesting peptide were analyzed. The culture conditions such as culture time, pH of culture medium and concentration of methanol were optimized and applied to high density culture. The genetically engineered Pichia pastoris was induced with methanol for 48h in the fermenter, and the cell density was mensurated every 12h. The yield and purity of interesting peptides were analyzed by SDS-PAGE.The result of PCR showed that six Mut+ colones and a Muts colone were the positive transformants. After all Mut+ and Muts colones were induced respectively with methanol , A Mut+ colone that expressed the protein which could been detected with anti-HCMV polyclonal antibodies was found. The apparent molecular weight of the recombinant protein was 51KD. The Mut+ colone was chosen as the genetically engineered Pichia pastoris, and the culture conditions were optimized. The interesting protein was expressed best when the genetically engineered Pichiapastoriswas induced with 1. 0%methanol in pH6.0 culture medium for 2-3d. After the genetically engineered Pichia pastoris was induced in the fermenter for 48h, the cell density was 124 (OD600) that was 3 times more than that induced in the shake-flask culture for 10d. The yield of interesting peptide in high density culture was 57. 21mg/L that was 6. 4 times more than that induced in the shake-flask culture for 4d. But the content of interesting peptide in the secreted proteins of supernate was less than that of the shake-flask culture in which the content of interesting peptide was 76.5%. Moreover, the concentration of interesting peptide in high density culture was enhanced along with the increasing cell density.The result indicates that the genetically engineered Pichia pastoris which can express the recombinant peptide of both gp52 and pp150 C-terminal peptides from human cytomegalovirus was sieved successfully and the concentration of interesting peptide was heightened in high density culture.