T7 RNA polymerase can direct expression of influenza virus cap-binding protein (PB2) in Escherichia coli.
Influenza virus cap-binding protein (PB2; Mr 85,000) is made in Escherichia coli when the cloned cDNA is transcribed by T7 RNA polymerase. Translation begins at the probable natural start codon and also from at least five internal sites in the same reading frame. The eukaryotic initiation site is not typical of protein initiation sites of E. coli, in that the closest potential Shine-Dalgarno sequence is far (15 nucleotides) from the start codon. Nevertheless, protein synthesis initiates efficiently at this site even in competition with a strong upstream prokaryotic initiation site. PB2 is somewhat unstable in the cell, but accumulates to a level where it is easily detectable in electrophoresis patterns of total cell protein. The full-length protein and various subfragments of it are insoluble in crude extracts, but have been useful for producing antibodies.
Rosenberg
A H
AH
Biology Department, Brookhaven National Laboratory, Upton, NY 11973.
Studier
F W
FW
D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005796:Genes; D005814:Genes, Viral; D009980:Influenza A virus / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps / Q000235:genetics*; D013604:T-Phages / Q000201:enzymology*; D014158:Transcription, Genetic
D002352:Carrier Proteins; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps; D012321:DNA-Directed RNA Polymerases