A new member of the bacterial ribonuclease inhibitor family from Saccharopolyspora erythraea.
We have identified Sti, the gene of a ribonuclease inhibitor from Saccharopolyspora erythraea, by using a T7 phage display system. A specific phage has been isolated from a genome library by a biopanning procedure, using RNase Sa3, a ribonuclease from Streptomyces aureofaciens, as bait. Sti, a protein of 121 amino acid residues, with molecular mass 13059 Da, is a homolog of barstar and other microbial ribonuclease inhibitors. To overexpress its gene in Escherichia coli, we optimized the secondary structure of its mRNA by introducing a series of silent mutations. Soluble protein was isolated and purified to homogeneity. Inhibition constants of complex of Sti and RNase Sa3 or barnase were determined at pH 7 as 5 x 10(-12) or 7 x 10(-7), respectively.
Krajcikova
Daniela
D
Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Hartley
Robert W
RW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015698:Genomic Library; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D012260:Ribonucleases / Q000037:antagonists & inhibitors*; D016930:Saccharopolyspora / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D017931:DNA Primers; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D012260:Ribonucleases