Production of active polyomavirus large T antigen in yeast Pichia pastoris.

The coding region of polyomavirus large T antigen was engineered into the genome of the methylotrophic yeast Pichia pastoris by use of the vector pHIL-D2. Expression of large T antigen was induced by methanol under the control of the strong alcohol oxidase (AOX1) promoter. Large T antigen was purified by immunoaffinity chromatography. We showed that yeast-derived large T antigen bound specifically to a DNA fragment that contains the polyomavirus replication origin, protected the four known major binding sites in the origin against DNase I digestion, and could unwind the strands of an origin-containing DNA fragment in an ATP-dependent manner. This system therefore provides a convenient and inexpensive source of biologically active polyomavirus large T antigen for in vitro studies.