Research Of The Glycosylation Modification In Pichia Pastoris Expression System

With the increasing demand for therapeutic protein,yeast expression system has gradually attracted people’s attention.The rapid development of genetic engineering make it possible to humanized glycosylation modification in yeast. Our laboratory obtained a non-hyperglycosylation mutant GJK01 (ura3-,OCH I-) by use of twice homologous recombination in order to knockout Pichia pastoris alpha-1,6-mannose transferase (OCHIp) gene,then block its high mannose pathway.The ER positioning signal of Saccharomyces cerevisiae a-1,2-mannanase was mixed together with MDSI from Arabidopsis thaliana and was transformed to Pichia pastoris. A glycoengineering yeast who could secret Man5GlcNAc2 N-glycan was obtained. The goal of yeast humanized glycosylation modification was achieved and the glycoengineering yeast will be used to produce therapeutic glycoproteins. But the yeast expression system still has some flaws.N-glycan sites on recombinant glycoproteins produced in yeast can be underglycosylated and O-glycan sites can be overglycosylated,which hampered its application.The Lishimania major N-glycosyltransferase gene STT3D was used to solve the first flaw.The gene was transformed to yeast strain 4-32 through the selecting marker URA3 gene and expressed under the control of an inducible alcohol oxidase I promoter.SDS-PAGE,Western Blot and peptide-N-asparigine amidase F was used to analyze the N-glycosylation degree of anti HER2 antibody expressed in 4-32-STT3D.The result of SDS-PAGE showed that anti HER2 antibody expressed by 4-32-HL were two bands:the upper band with the molecular weight 55×103 was glycosylated protein, which was the same with commercial anti HER2 antibody,while the lower band with the molecular weight 50×103. And all the bands became 50×103 after the digestion of PNGase F.These bands were proved to be antibody components by Western Blot. Meanwhile, anti HER2 antibody expressed in engineering yeast 4-32-HL-STT3D had only one band of 55×103 without non-glycosylated 50×103. The granulocyte-macrophage colony-stimulating factor was used as a reporter protein to verify the function of STT3D.The result of SDS-PAGE showed that GM-CSF expressed in ordinary yeast 4-32-GM-CSF had two components:22×103 and 20×103, while only one component with the molecular of 22×103 in 4-32-GM-CSF-STT3D had, and all the GM-CSF became 18×103 after the digestion of PNGase F. As the GM-CSF has two N-glycosylated sites,Asn27 and Asn37, Respectively, we concluded that the 22x103 band was formed because both sites were N-glycosylated and the 20x103 was formed because only one site was N-glycosylatedAs the STT3D gene was expressed under the control of an inducible alcohol oxidase I promoter, we detected the growth rate of glycoengineering yeast(+STT3D) in induce and non-induce with methanol compaired with yeast( — STT3D), respectively. Statistical analysis showed that STT3D had no effect on the growth rate of glycoengineering yeast without induction,as p=0.032>0.01;while great effect was observed when STT3D was induced to express,as p<0.0001.For the problem of overglycosylated modification in glycoengineering yeast,the conventional method was to knockout its O-glycosyltransferase gene in yeast.Considering that O-glycosyltransferase genes were necessary for yeast grew, a inducible manner was taken to repress their expression.Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associate system 9 attracted our attention owing to its targeted deletion function.Dcas9, a variety form of Cas9,was chosed because of its ability to bind to target sites and inactivation of endonuclease activity.The dcas9 was under the control of D6 promoter. In order to make the dcas9 protein translocate into nucleus;nuclear localization signal ORF was added to the 3’ terminal of dcas9 gene and both 5’terminal and 3’terminal. Pmt-gRNA consisted of pmt1-gRNA、pmt2-gRNA and pmt4-gRNA in series and anchored to the upstream of the start codon of pmtl,pmt2 and pmt4.Two repressor vectors named pGE-D6-dcas9-pmt-gRNA and pGE-D6-NLS-dcas9-pmt-gRNA were constructed.Each of the repressor vectors was translocated into JC307 yeast strain through the selecting marker URA3,respectively.Then a protein granulocyte colony stimulating factor was choosed as the reporter protein who had only O-glycosylation sites.MALDI-TOF-MS was used to detect the effect of repression vector.The result showed that G-CSF expressed in yeast strain with one NLS had two peaks:one 9801.9,another19614, the same with the native strain,who also had two peaks, respectively 9803.2 and 19612.9.This result told us that the repressor expression pGE-D6-dcas9-pmt-gRNA had no effect on repressing yeast overglycosylation.But G-CSF expressed in yeast strain with two NLS had four peaks, 9645.4,9806.9,19296.3,and 19620.7,respectively.Among them,19620.7 minus 19296.3 was 324.4.As we know,the molecular weight of a mannose residue is 160,it meant that the reporter protein G-CSF was kept from overglycosylation modification by pGE-D6-NLS-dcas9-pmt-gRNA.As a result,the repression vector pGE-D6-NLS-dcas9-pmt-gRNA played a role in preventing the overglycosylation modification of reporter protein in yeast.