Molecular cloning and sequencing of the sppA gene and characterization of the encoded protease IV, a signal peptide peptidase, of Escherichia coli.
Clones carrying a gene causing overproduction of protease IV, a signal peptide peptidase of Escherichia coli, were isolated from the Clarke and Carbon's collection. Restriction mapping analysis revealed that pLC7-10 and pLC40-13, thus isolated, shared the same chromosomal DNA region. The 2.3-kilobase RsaI-SalI fragment in this region, which was found to carry the gene, was subjected to nucleotide sequence determination. Only one long open reading frame was found. The hypothetical polypeptide sequence deduced from the DNA sequence has a molecular mass of 67,241 daltons. The putative gene was named sppA. Protease IV was purified to homogeneity from the cytoplasmic membrane of an overproducing strain harboring a sppA gene-carrying plasmid. The purified enzyme gave a single polypeptide band of 67,000-dalton molecular mass on sodium dodecyl sulfate-polyacrylamide gel. This molecular mass and the amino acid composition of the purified enzyme were consistent with the deduced primary structure of the sppA gene product. The molecular mass thus determined was almost twice as large as that previously reported by Pacaud (Pacaud, M. (1982) J. Biol. Chem. 257, 4333-4339). A cross-linking study revealed that protease IV is a tetramer of the polypeptide. From these results, we conclude that protease IV is a tetramer of the sppA gene product.
D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D046911:Macromolecular Substances; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids; D021382:Protein Sorting Signals / Q000378:metabolism
D000596:Amino Acids; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D021382:Protein Sorting Signals; D010447:Peptide Hydrolases; C030732:protease IV