Expression And Activity Assay Of T-reesei α-1, 2-mannosidase In Pichia Yeast

Pichia expression system is one of the most frequently used expression systems for producing foreign proteins.This study is the beginning of the yeast N-glycosylation engineering project in our lab. We planned to introduce a T.reesei ?-l,2-mannosidase and localize it into the yeast endoplasmic reticulum(ER)to reduced the length of the mannose residues on the reporter glycoprotein.Total RNA of T.reesei was extracted with Trizol reagents and RT-PCR was used to amplify the cDNA sequence of mds. The 944th base pair of G in mds gene was changed into A by site directed mutation,thus removing the Xhol restriction enzyme site . The ER-retaining tag HDEL was added to the mds gene and cloned into the expression vector of pPIC9,resulting in pPIC9-a-M. Mds gene with S.cerevisise a-mating factor signal sequence after removing BamHI site was cut from plasmid pPIC9-a-M and cloned into same enzyme digested pAO815,generating plasmid pAO815-α-M. Human p-IFN having only one N-glycosylation site on its 80th amino-acid was used as a reporter gene.The P-IFN was amplified by PCR from the plasmid pUC19-B and subcloned into the plasmid pAO815-a-A,resulting in pAO815-a-B. Plasmid pAO815-a-B-M was constructed by inserting digested P-IFN gene with S.cerevisise a-mating factor signal sequence into plasmid pAO815-a-M,which can express P-IFN and MnsI.After transformation of Pichia pastoris GS115 with plasmid pAO815-α-B or pAO815-α-B-M respectively.two transformants expressing only p-IFN or p-IFN and MnsI were screened by induction in shake flask and SDS-PAGE analysis of the supernatant.The strain only expressing p-IFN was named PPMB1. The other was named PPMGl.The specific activity of p-IFN expressed in PPMB1 was 3.6 xl07 u/ml , while 1.7 xl05 u/ml in PPMG1. Both recombinant P-IFN were recovered from SDS-PAGE and analyzed by MALDI-TOF-MS.The results showed the molecular weight of P-IFNexpressed in PPMG1 is different from that expressed in PPMB1, indicating mannose residues on the reporter protein were changed, thus showing -l,2-mannosidase activity in Pichia pastoris.