Construction Of Pichia Pastoris X33 With A Humanized Polysaccharide Core Components By N-linked Glycosylation

Pichia pastoris expression system has characteristics of prokaryotic expression system which are fast growth, cultivation of low cost, high protein yield and so on, but also has the function of eukaryotic which is post-translational processing modifications of protein(such as N-glycosylation). So it is widely used in the expression of recombinant protein. But the excessive modification of glycoprotein can produce high mannose type N-glycan which is different from complex N-glycan of human and mammalian cells. High mannose type N-glycan can lead to immunogenicity in human which is the major factor why P.pastoris cannot be used in most drug glycoprotein production. So it is necessary to transform the N-glycosylation pathway in P.pastoris to overcome the defect. Base on previous work, our lab have obtained the α-1, 6-mannose glycosidase(OCH1) single mutant P.pastoris X33 with α-1, 6-mannose glycosidase(OCH1) and α-1, 3-glycosidase(ALG3) double mutant P.pastoris X33. However, the growth situation of double mutant strain is not conducive to further transformation. This study is used to get the strain whose growth situation has some improved through mutation breeding. At the same time, introduction of α-1, 2-mannosidase(MNS ?) gene for further removal of mannose, and get genetically modified P.pastoris X33(MNS ?). Then the humanized core glycoprotein is obtained by the expression of reporter glycoprotein GM-CSF.In this research, optimized P.pastoris X33 double mutant was obtained by UV mutagenesis and ARTP(Atmospheric and Room Temperature Plasma) mutagenesis. Yeast suspension would be diluted to 1000?m L-1 in each UV mutagenesis, then mutation was coated onto plates and cultured in the darkness. ARTP mutagenesis process need the yeast suspension was adjusted to OD 6-8, then ten times of gradient dilution for plate culture after mutation. After three rounds of UV mutagenesis and a round of ARTP mutagenesis, a lot of positive mutant strains were obtained. Finally we obtained an improved mutant strain in growing situation after twenty times enrichmen screening of those positive mutagenic strains.Double mutant strain was regarded as the object of this research to express fusion gene of endoplasmic reticulum localization sequence of Saccharomyces cerevisiae MNS ? gene and catalytic sequence of Caenorhabditis elegant MNS ? gene. Then fusion gene was integrated into P.pastoris expression vector p GAPZB, get MNS ? import vector p GAPZB-MNS ?. Recombinant vector was linearized by Avr ?? restriction enzyme, and was imported into mutagenic strain by electric conversion with bleomycin(Zeocin) as the sign of screening. Finally we obtain P.pastoris X33(MNS ?).Results of SDS-PAGE and western blot showed that the molecular weight of GM-CSF expressed by P.pastoris X33(MNS ?) was lower than wild-type strain of P.pastoris X33, P.pastoris X33 single mutant and P.pastoris X33 double mutant. Molecular weight of GM-CSF expressed by four strains drop to below 20 k Da after PNGase F enzyme digestion, which indicating that P.pastoris X33(MNS ?) and wide-type strain had undergone different degree of glycosylation. However, the expression level of P.pastoris X33(MNS ?) was still very low even after 500 times by ultrafiltration.