Molecular cloning of genes for proteases A and B from streptomyces griseus ATCC10137

Abstract

Proteases A and B are extracellular proteins, which are secreted by Streptomyces griseus. Since the oligonucleotide was designed from an identical amino acid sequence of both proteases, the probe hybridized equally to two fragments generated by either BamHI(8.4 and 6.8 kbp) or BamHI-BglII(3.2 and 2.8 kbp). By using the oligonucleotide probe, plasmids containing sprA and sprB were isolated from a genomic DNA library prepared from S. griseus DNA. Of 3,500 E. coli transformants, 12 were detected by the oligonucleotide probe and isolated for further characterization. These colonies contained two distinct classes of plasmids, based on restriction analysis. As expected based on the hybridization of genomic DNA, the plasmids contained either a 2.8-or 3.2-kbp BamHI-BglII fragment. The DNA fragments isolated by hybridization screening were tested for expression of proteolytic activity. The 2.8-and 3.2-kbp KpnI-XbaI fragments were ligated into the KpnI-XbaI site of the vector pUJ718-2, to allow transformation of S. lividans with selection for thiostrepton or ampicillin resistance. Transformants containing these constructions were then tested on a skim milk plate for secretion of proteases. A clear zone, representing the degradation of milk protein, surrounded each transformant that contained the 2.8-or 3.2-kbp KpnI-XbaI fragment. The clear zones were not found around S. lividans colonies which contained vector pUJ718-2. The functional limits of the genes were determined by subcloning restriction fragment into pUJ718-2, transforming S. lividans and testing for proteolytic activity. The intact protease genes could be delimited to a 1.4 kbp SalI fragmant for sprA and a 1.4 kbp BssHII fragment for sprB.