title: Efficient expression of influenza virus NS1 nonstructural proteins in Escherichia coli.
abstract: RNA segment 8 of the influenza A virus genome codes for two nonstructural proteins, NS1 and NS2, for which the functions are unknown. Cloned cDNA copies of this gene from three different influenza A virus strains were inserted into an Escherichia coli plasmid expression vector, pAS1, carrying the strong regulatable lambda phage promoter, PL. After induction, the NS1 proteins were overproduced to levels of 20-25% of total cellular protein. This was surprising in that the codon composition for these eukaryotic genes is similar to that for weakly expressed proteins in E. coli. Thus, under the appropriate conditions, it appears that high level expression of genes containing a relatively large proportion of minor codons can be obtained. The NS1 protein produced in bacteria from a cloned cDNA copy of the A/PR/8/34 virus NS gene was purified to apparent homogeneity and used to generate a high-titer monospecific rabbit antiserum. Immunoprecipitation studies showed this antibody to be crossreactive against the NS1 proteins produced by several different influenza A virus strains. Immunofluorescence experiments in Madin-Darby canine kidney cells showed the NS1 proteins to be located in the nucleoplasm early in infection for all strains examined. With some of the strains, NS1-specific immunofluorescence was observed predominantly in the nucleoli later in infection. This technology can be used to obtain other viral proteins in pure form for structural, functional, and immunological studies.
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004262:DNA Restriction Enzymes; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors; D009980:Influenza A virus / Q000235:genetics*; D007668:Kidney; D010957:Plasmids; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D004247:DNA; D004262:DNA Restriction Enzymes