Open AccessHigh level expression in Escherichia coli cells and purification of poliovirus protein 2Apro
Author(s) -
Francisco MartínezAbarca,
Miguel A. Alonso,
Luís Carrasco
Publication year - 1993
Publication title -
journal of general virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.55
H-Index - 167
eISSN - 1465-2099
pISSN - 0022-1317
DOI - 10.1099/0022-1317-74-12-2645
Subject(s) - biology , escherichia coli , poliovirus , microbiology and biotechnology , t7 rna polymerase , lac operon , affinity chromatography , rna , protease , biochemistry , virology , virus , gene , enzyme , bacteriophage
The poliovirus protease 2Apro has been produced to high levels in Escherichia coli 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 2Apro 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 E. coli 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 2Apro synthesized at a 95% purity level after sarkosyl treatment. Antibodies raised against 2Apro in E. coli recognize a 16K protein in poliovirus-infected cells. In addition, 2Apro 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.