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rdf:type |
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lifeskim:mentions |
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pubmed:issue |
3
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pubmed:dateCreated |
1986-12-29
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pubmed:databankReference |
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pubmed:abstractText |
Polyomavirus small t antigen was purified from genetically engineered Escherichia coli and used as the immunogen for the production of polyclonal and monoclonal antibodies. A new series of plasmids for increased expression of polyomavirus T antigens or a T antigen-beta-galactosidase fusion protein was constructed by replacing sequences coding for the ribosome-binding site of previously published plasmids with a chemically synthesized sequence that has a higher degree of complementarity to the 3' end of the 16S rRNA. Cells expressing the fusion protein from the plasmid with the synthetic sequence contained 5- to 10-fold more fusion protein after a 3-h induction than did control cells. Pulse-labeling of cells bearing the new plasmids revealed that the T antigens were synthesized at high levels after induction: 10% of total synthesis for small t; 15% for Py-1387T middle T, a truncated mutant of middle T; and probably 1 to 5% for middle T. Small t and Py-1387T middle T, but not wild-type middle T, were seen as minor bands in total cell protein analyzed on sodium dodecyl sulfate-polyacrylamide gels stained with Coomassie blue. A simple, rapid procedure for purification of bacterial small t from the pellet of sonicated bacteria yielded 1 to 2 mg of small t per liter of bacterial culture at 80 to 90% homogeneity. High-titer polyclonal rabbit antisera raised against purified small t recognized all three T antigens and were suitable for immunoaffinity purification of middle T. Mouse monoclonal antibodies raised against bacterial small t were of four classes, immunoprecipitating either all three polyomavirus T antigens, small t and middle T only, primarily small t, or middle T and large T in preference to small t. One of the latter monoclonal antibodies also immunoprecipitated large T but not small t of simian virus 40, suggesting that the site recognized by this antibody may be functionally important. None of the monoclonal antibodies yielded an immunoprecipitate active in phosphorylating middle T in vitro.
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pubmed:grant |
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pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-149110,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-168683,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-186787,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-203944,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-229972,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-229973,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-229974,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-2424008,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-2981329,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-2987699,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-2987799,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-3012562,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-3016331,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-3088563,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-4887520,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-5432063,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-558524,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6091900,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6095063,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6098822,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6158095,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6169844,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6175960,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6179082,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6204863,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6254066,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6260373,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6262529,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6287461,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6290466,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6294132,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6294529,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6304524,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6308618,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6312293,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6313968,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6325177,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6325896,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6327264,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-6984190,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-803646,
http://linkedlifedata.com/resource/pubmed/commentcorrection/3023660-942051
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Dec
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pubmed:issn |
0022-538X
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:volume |
60
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
1075-84
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pubmed:dateRevised |
2009-11-19
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pubmed:meshHeading |
pubmed-meshheading:3023660-Antibodies, Monoclonal,
pubmed-meshheading:3023660-Antigens, Viral, Tumor,
pubmed-meshheading:3023660-Binding Sites,
pubmed-meshheading:3023660-Chemical Precipitation,
pubmed-meshheading:3023660-Cloning, Molecular,
pubmed-meshheading:3023660-Escherichia coli,
pubmed-meshheading:3023660-Polyomavirus,
pubmed-meshheading:3023660-Ribosomes,
pubmed-meshheading:3023660-Viral Fusion Proteins
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pubmed:year |
1986
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pubmed:articleTitle |
Polyomavirus small t antigen: overproduction in bacteria, purification, and utilization for monoclonal and polyclonal antibody production.
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pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't
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