In vivo system for the detection of low level activity barnase mutants.
We report the design of a new tightly controlled barnase system which allows the existence of the barnase gene in host cells without a signal sequence. When expression of barnase is turned on by gene inversion in vivo, the lethal effect of barnase (or its mutants) is not compromised either by coexpression of its polypeptide inhibitor (barstar), or by extracellular secretion. This serves as a rapid, sensitive in vivo test for the detection of any very low residual activity of the barnase mutants. Active-site mutants His102Lys, Glu73Asp and Arg87Lys, and a mutant which greatly reduces the stability and yield of protein, Arg83Lys, produce enough activity to be detectable by this test. In contrast, when expressed on a secretion vector, these mutants do not yield detectable activity in a solution assay. Truly inactive mutants, such as those of His102 to Gly, Ala or Leu, were completely harmless when expressed in this system.
Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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Jucovic
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Hartley
D001426:Bacterial Proteins; D001483:Base Sequence; D001665:Binding Sites; D007446:Chromosome Inversion; D017931:DNA Primers; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D012260:Ribonucleases / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D017931:DNA Primers; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease