[Insertional mutagenesis of protease 2A of the poliomyelitis virus and its substrate, simultaneously expressed in Escherichia coli cells].
In order to clarify the structural features of protein substrates which determine their sensitivity towards poliovirus 2A protease, a high-efficiency bacterial expression system for cDNA of the poliovirus genome fragment has been developed. The expressed protein encompasses the C-end half of the VP1 capsid protein. 2A protease, and a large portion of the 2B protein. Virus-specific products were found in the insoluble fraction of bacterial cell lysates which were mainly represented by two proteins. These proteins appeared to be produced via the cleavage of the expressed protein by 2A protease at the Tyr-Gly site located between the VP1 protein and the 2A protease proper. The accumulation of two viral proteins can be prevented by a four amino acid insertion into the 2A gene locus in close vicinity of the putative catalytic His residue. The determinants of specificity toward the 2A action are located within the region flanking the cleavable Tyr-Gly dipeptide from its N-side.
Slobodskaia
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Gorbalenia
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D017955:Poliovirus / Q000235:genetics*; D015183:Restriction Mapping; D013379:Substrate Specificity; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D004274:DNA, Recombinant; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus
Insertsionnyĭ mutagenez proteazy 2A virusa poliomielita i ee substrata, odnovremenno ékspressiruemykh v klekakh Escherichia coli.