CLONING AND EXPRESSION OF THE GENE ENCODING HEPATITIS B VIRUS POLYMERASE ON THE SURFACE OF SACCHAROMYCES CEREVISIAE

Abstract

Although hepatitis B virus (HBV) is a DNA virus but their replication is dependent upon reverse transcriptase encoded by the HBV polymerase gene (pol gene). Molecular genetic studies have suggested that the HBV polymerase (Pol), a multifunctional enzyme of about 94 KDa, plays critical roles during the viral life cycle. These functions include priming activity, polymerase activity and nuclease activity. Despite much effort have been made toward expressing the pol gene, the biochemical dissection of the corresponding protein has lagged behind because of the inability to obtain sufficient amounts of soluble enzyme required for such an analysis. Here we reported our study on the expression of Polan the surface of S. cerevisiae. The pol gene with 2496 bp in length was amplified by PCR, cloned into pGEM-Teasy vector and sequenced. The pol gene was then cut out from pGEM-Teasy and inserted into pYDI vector at .Votl and KpnI sites to generate pYDI-pol expression vector. The recombinant pYDI-pol was transformed into yeast S. cerevisiae by electroporation, To express Pol, the yeast cells carrying pYDI-pol were grown in liquid medium YNB-CNN supplemented with 2% of glucose and were induced by adding 2% of galactose at 20'C with shaking at 220 rpm for 12 - 72 hrs. The expression of Pol was analysed by SDS-PAGE and found to be growth-time dependent with the highest protein accumulation at 60 hrs after induction. This result was confirmed by Western blot using the anti Y5-epitope antibodies. The recombinant HBY polymerase greatly facilitate future analyses on pol-protein structure and function and allow the investigation for new anti-HBY drugs specifically, blocking HBY polymerase.