1887

Abstract

The poliovirus protease 2A has been produced to high levels in using the inducible system that utilizes T7 RNA polymerase. The protease coding sequences that contained an additional AUG to start translation were cloned in pET vectors. Synthesis of 2A was induced by IPTG or IPTG plus rifampicin, the levels of the protein made being higher when IPTG alone was used. The expression of the protein is not toxic for cells and can be readily visualized by Coomassie blue staining of total bacterial protein extracts separated in polyacrylamide gels. Centrifugation of the broken bacterial cells sediments more than 95% of the 2A synthesized at a 95% purity level after sarkosyl treatment. Antibodies raised against 2A in recognize a 16K protein in poliovirus-infected cells. In addition, 2A shows activity in trans as measured by the cleavage of p220 in HeLa cell extracts and by cleavage of a poliovirus protein substrate that contains the junction between the P1 and P2 polypeptides.

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/content/journal/jgv/10.1099/0022-1317-74-12-2645
1993-12-01
2019-11-22
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http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-74-12-2645
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