Cloning of the chandipura virus phosphoprotein encoding gene and its expression in Escherichia coli.
Chandipura (CHP) virus, a member of the vesiculovirus genus within the Rhabdoviridae family, was first isolated from human patients in India. The full length phosphoprotein P gene of CHP have been cloned and expressed in Escherichia coli using the T7 polymerase-based pET-3 series of expression vectors. Under optimal conditions of induction with IPTG, the recombinant P protein constituted 35% of the total bacterial protein. The bacterially expressed protein was found to be phosphate-free. Deletion analysis suggested that the anomalous mobility of the P protein was due to its high acidity. The expressed protein can be phosphorylated in vitro by the extracts prepared from baby hamster kidney cells or rabbit reticulocytes. The cellular kinase involved in phosphorylation appears to be casein kinase II.
Chattopadhyay
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Department of Biochemistry, University College of Science, Calcutta, India.
Chattopadhyay
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D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers; D004926:Escherichia coli; D015870:Gene Expression / Q000187:drug effects; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007668:Kidney; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D018116:Vesiculovirus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification
D017931:DNA Primers; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D007544:Isopropyl Thiogalactoside