Expression of bovine pancreatic ribonuclease A coded by a synthetic gene in Bacillus subtilis.
Two different hybrid genes were constructed which fuse the Bacillus amyloliquefaciens alkaline protease gene (apr[BamP]) promoter and signal peptide coding region to a synthetic bpr gene coding for the mature bovine pancreatic RNase A. The first gene fusion (apr-bpr1) contained the apr[BamP] signal peptide coding region fused to mature bpr through a linker coded 3-amino acid region and retained the signal processing site ala-ala of the alkaline protease. The second fusion (apr-bpr2) joined the end of the apr[BamP] signal peptide coding sequence to the mature bpr resulting in a hybrid signal processing site ala-lys. B. subtilis strains harboring these gene fusions secreted bovine pancreatic RNase A into the growth medium. Cleavage at the hybrid signal processing site ala-lys resulted in the secretion of bovine pancreatic RNase A from B. subtilis which had an N-terminal amino acid sequence that was identical to the native RNase A. Bovine pancreatic RNase A contains four disulfide bonds and the proper formation of these bonds is required for activity. RNase activity could be detected in the culture supernatants of strains carrying the apr-bpr gene fusions, which suggests that the proper disulfide bonds have formed spontaneously.
Vasantha
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Genex Corporation, Gaithersburg, MD 20877.
Filpula
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D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics; D005813:Genes, Synthetic; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012259:Ribonuclease, Pancreatic / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D004247:DNA; D012259:Ribonuclease, Pancreatic; D010450:Endopeptidases; D012697:Serine Endopeptidases; C032923:microbial serine proteinases