Cloning and inducible synthesis of poliovirus nonstructural proteins.
The poliovirus nonstructural protein-encoding genes have been cloned and expressed in Escherichia coli using the inducible system described by Studier and Moffat [J. Mol. Biol. 189 (1986) 113-130] and Studier [J. Mol. Biol. 219 (1991) 37-44]. The two genes encoding the poliovirus proteases, 2Apro and 3Cpro, were cloned together with their flanking regions in order to test the ability of the polyprotein precursors synthesized to cause proteolytic cleavage and generate mature forms. Both proteases were synthesized and showed activity upon induction in this system. Previously, it had not been possible to produce the three poliovirus nonstructural proteins, 2B, 2C and 3A, and some of their precursors, 2C3AB, 2C3A and 3AB, at high levels in E. coli cells. We report the cloning of their genes using PCR techniques and their efficient expression from pET vectors upon induction with IPTG (isopropyl-beta-D-thiogalactopyranoside). Moreover, some of these proteins, e.g., 3AB, 3A and 2B, are quite toxic for E. coli cells and lysed them upon production. Our results demonstrate the usefulness of this inducible system using the pET vectors to express these toxic poliovirus proteins.
Lama
J
J
Centro de Biología Molecular, Universidad Autónoma de Madrid, Spain.
Guinea
R
R
Martinez-Abarca
F
F
Carrasco
L
L
D000086042:3C Viral Proteases; D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D017955:Poliovirus / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins
C520978:3AB protein, poliovirus; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D007544:Isopropyl Thiogalactoside; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases; C057400:picornain 2A, Picornavirus