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A Pichia Pastoris X-33 With OCH1 Gene Deletion And Its Use In Expression Of Glycoprotein GM-CSF

Posted on:2012-09-21Degree:MasterType:Thesis
Country:ChinaCandidate:D C ZhangFull Text:PDF
GTID:2120330332991259Subject:Biochemistry and Molecular Biology
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Pichia pastoris is a single-celled eukaryote, which has many characteristics of prokaryo- tes, such as fast growth, production proteins at high levels, cultivation of low cost, and is capable of many of the post-translational modifications performed by higher eukaryotic cells including, N-glycosylation, is a widely used expression system for production of recombinant proteins. Glycoproteins produced in P. pastoris with nonhuman high mannose-type N-glycans are different from mammalian complex-type N-glycan structures produced in mammalian cells. Immunogenicity in human drastically reduce protein activity and in vivo half-life ect, thus glycoproteins especially therapeutic glycoproteins can't be produced in P. pastoris. In order to obtain a P. pastoris expression system modifying glycoproteins with lower mannose-type N-glycans and could be used for further N-glycosylation modification. In this research, we deleted the OCH1gene of P. pastoris X-33 playing a key role in high mannose-type N-glycosylation processed by double homologous recombination, obtained an X-33(och1-) strain, and this strain was used for expression of GM-CSF protein.In this research, the left and right arms of URA3 were amplified with PCR from the genome DNA of wild P. pastoris X-33 and fused by fusion PCR. Then, it was transformed into X-33 cells, of which the URA3 gene would be deleted by double homologous recom- bination, and a X-33(ura3-)strain was constructed but it does not contain another selected markers.Firstly, the pYES plasmid containing URA3 marker gene was constructed as S. cerevisiae expression vector pYES2; the left and right arms of OCH1 were amplified with PCR from the genome DNA of wild P. pastoris X-33 and fused by fusion PCR, and it was cloned into pYES vector. As a result, the knockout plasmid of pYES-OCH1 was constructed. Then, it was lined by Mluâ… and transformed into X-33(ura3-)cells, the OCH1 gene of X-33(ura3-)would be deleted and a X-33(och1-)strain was obtained.GM-CSF was identified by SDS-PAGE and western blot. the molecular weight of glycopr- otein GM-CSF expressed by X-33(och1-)is about 25 kDa, and significantly smaller than GM-CSF which is about 35 kDa expressed by X-33. Then, the glycoproteins GM-CSF expressed by X-33(och1-)and X-33 were digested with PNGaseF specificity recognition N-sugar chains, we found the molecular weight of them was smaller than 25 kDa. All these suggested the glycoprotein GM-CSF expressed by X-33(och1-)is lower N-glycosylated and different from hyperglycosylated in X-33. Therefore, X-33(och1-)strain could be used as an expression host for production of glycoproteins lacking the outer-chain hypermannoses, and a host used for further N-glycosylation engineering...
Keywords/Search Tags:N-glycosylation, Pichia pastoris X-33, Gene knockout, Granulocyte-macrophage colony-stimulating factor(GM-CSF)
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