High level expression in Escherichia coli cells and purification of poliovirus protein 2Apro.
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.
Martínez-Abarca
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F
Centro de Biología Molecular Severo Ochod (CSIC-UAM), Universidad Autónoma de Madrid, Canto Blanco, Spain.
Alonso
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MA
Carrasco
L
L
D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D039562:Eukaryotic Initiation Factor-4F; D008969:Molecular Sequence Data; D010448:Peptide Initiation Factors / Q000378:metabolism; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D013379:Substrate Specificity; D014764:Viral Proteins
D039562:Eukaryotic Initiation Factor-4F; D010448:Peptide Initiation Factors; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus