Polyomavirus Small tAntigen: Overproduction inBacteria, Purification, andUtilization forMonoclonal andPolyclonal Antibody Production

Polyomavirus small tantigen was purified fromgenetically engineered Escherichia coli andusedas the immunogen fortheproduction ofpolyclonal andmonoclonal antibodies. A new series ofplasmids forincreased expression ofpolyomavirus T antigens or a T antigen- -galactosidase fusion protein was constructed by replacing sequencescoding fortheribosome-binding site ofpreviously published plasmids witha chemically synthesized sequencethathasa higher degree ofcomplementarity tothe3'endofthe16SrRNA.Cells expressing thefusion protein fromtheplasmid withthesynthetic sequencecontained 5-to10-fold more fusion protein after a 3-hinduction thandidcontrol cells. Pulse-labeling ofcells bearing thenew plasmids revealed that theTantigens weresynthesized athighlevels after induction: 10%oftotal synthesis forsmall t;15%for Py-1387T middle T,atruncated mutantofmiddle T;andprobably 1to5%formiddle T.Small tandPy-1387T middle T,butnotwild-type middle T,were seenasminorbandsintotal cell protein analyzed on sodium dodecyl sulfate-polyacrylamide gels stained withCoomassie blue. Asimple, rapid procedure forpurification of bacterial small tfromthepellet ofsonicated bacteria yielded 1to2mg ofsmalltperliter ofbacterial culture at80to90%homogeneity. High-titer polyclonal rabbit antisera raised against purified small trecognized all three Tantigens andweresuitable forimmunoaffinity purification ofmiddle T.Mousemonoclonal antibodies raised against bacterial small twere offourclasses, immunoprecipitating