Cloning and expression of the vesicular stomatitis virus phosphoprotein gene in Escherichia coli: analysis of phosphorylation status versus transcriptional activity.
The phosphoprotein (P, previously known as NS) genes of vesicular stomatitis virus serotypes New Jersey and Indiana have been cloned in the Escherichia coli expression vector pET-3a. Transcription of P genes in these clones initiated from a phage T7 RNA polymerase promoter, whereas translation was driven by the Shine-Dalgarno sequence and the initiator AUG codon of the T7 gene 10 message. The clones were introduced into an appropriate E. coli strain in which T7 RNA polymerase was expressed under the control of the lac promoter. Under optimal conditions of induction with isopropylthiogalactopyranoside, P protein made in these bacterial strains constituted 5 to 20% of total cellular protein. P protein expressed in bacteria was unphosphorylated and transcriptionally active in an in vitro reconstitution assay with viral L protein and an N-RNA template. However, the P protein was phosphorylated in vitro by the kinase activities associated with L and the N-RNA template.
Barik
S
S
Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44195.
Banerjee
A K
AK
D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000187:drug effects / Q000235:genetics*; D018116:Vesiculovirus; D014758:Viral Core Proteins / Q000096:biosynthesis / Q000235:genetics*; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins
D010750:Phosphoproteins; D012367:RNA, Viral; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D007544:Isopropyl Thiogalactoside; D011494:Protein Kinases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases