Construction Of HRP-Transgenic Pichia Pastoris,and Preparation Of The Recombinant HRP

Horseradish peroxidase is a heme-containing glycoprotein usually used in medical diagnostics and coupled enzyme assays.Due to wide substrate specificity and high catalytic activity and stability,HRP is often employed in bio-and immunosensors,chemiluminescent,fluorescent,and electrochemical detection systems,as well as in DNA and protein microchips with colorimetric detection.In this study we aim to subclone and express HRP gene constitutively and inductively in yeast Pichia pastoris.In this research,HRP gene was expressed in yeast P.pastoris as follow:(1)HRP gene was amplified by PCR reaction under appropriate conditions and right designed primers(P1.P2.P3).(2)The amplified fragment of HRP was integrated into prokaryotic and eukaryotic expression vectors,which were constructed into the open reading frame of the constitutive and inducible expression vectors p GAPZ?A and p PIC9K-His.The expression vectors p GAPZ?A-HRP and p PIC9K-His-HRP were successfully constructed.(3)The expression vectors p GAPZ?A-HRP and p PIC9K-His-HRP were transformed into chemically competent E.coli DH5? cells,and the transformants were verified by colony PCR.(4)The expression vectors p GAPZ?A-HRP and p PIC9K-His-HRP were finally transformed into yeast P.pastoris GS115 host cells by electroporation method in order to obtain the recombinant strains.The obtained recombinant strains were verified by colony PCR.And the integration of expression vectors into the genome of the host cells was further verified by screening the multicopy strain using high concentration of Zeocin for those strains integrated in P.pastoris through p GAPZ?A vector and Geneticin for those integrated into P.pastoris through p PIC9K-His vector(5)By inducing the recombinant cells(10),and by analyzing them in pellet and supernatant form using SDS-PAGE method,the bands were found to be 64 k Da and approximately 63 k Da which were consistent with the expected molecular weight(65k Da&63k Da).This Indicated that exogenous protein can be secreted into the extracellular medium,and intracellularly by breaking the cell wall the intracellular protein can be obtained.