A new member of the bacterial ribonuclease inhibitor family from Saccharopolyspora erythraea

Abstract 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 13 059 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 5×10 −12 or 7×10 −7 , respectively.