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.
Peng
Y C
YC
Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
Acheson
N H
NH
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D000952:Antigens, Polyomavirus Transforming / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular / Q000379:methods; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D007438:Introns; D010843:Pichia; D010957:Plasmids; D011120:Polyomavirus / Q000235:genetics; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D000952:Antigens, Polyomavirus Transforming; D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase