Expression α-1, 2-mannosidase In Mutant Type Pichia Pastoris

Yeast is a widely used host for recombinant protein expression.However,glycoproteins derived from yeast contain N-glycan of high mannose type and were usually hyperglycosylated. This study is indispensable to the yeast N-glycosylation engineering project.We planned to introduction theα-1,2-mannosidase(MDSI)into the yeast genome,which catalyzes an essential proceeding of N-glycan structures from Man₈GlcNAc₂ to Man₅GlcNAc₂.α-1,2-mannosidase come from various eukaryotes,which belongs to typical typeⅡmembrane glycosidase,and cleavesα-1,2-linked mannose specially.Only localized suitable position,α-1,2-mannosidase can catalyze the substrate glycan in the cell.So three leaders were screened, they were MnsI from Golgi of S.cerevisiae,Sec12 from ER of S.cerevisiae and Sec12 from ER of P.pastoris.MDSI genes of two sources,respectively from Homo sapiens and Arabidopsis thaliana were chose in this study,and the DNA encoding itself location signal peptide or stem region peptide was cleaved four kinds of MDSI gene fragment:Hmdsi(△63),Hmdsi(△185),ATmdsi(△48),ATmdsi(△80)were obtained.After composition the three leaders,twelve different MDSI gene expression vector were acquired.And were used to transform a mutation P.pastoris GJK01.Finally the N-glycan of a model protein HSA/GM-CSF was analysis by fluorescence-assisted carbohydrate electricity(FACE).The results suggested that the combination of MnsI and ATmdsi(△80)can cleave N-glycan from Man₈GlcNAc₂ to Man₅GlcNAc₂.