Molecular cloning and sequence analysis of a gene encoding an extracellular proteinase from Lactobacillus helveticus CP790.
A 2.6-kilobase HaeIII DNA fragment corresponding to an extracellular proteinase gene (prtY) was cloned from chromosomal DNA of Lactobacillus helveticus CP790 in Escherichia coli using a pKK223-3 vector. The transformant expressed a 48-kDa protein that reacts with monoclonal antibodies specific to the proteinase and seemed to be a pre-proproteinase, but had no proteolytic activity. About 1.6 kilobases of the 2.6-kilobase DNA fragment, which contained the complete gene for the proteinase was sequenced. Sequence analysis found an open reading frame with a capacity to encode a protein of 449 amino acids. The coding region contained a Gram-positive-type signal peptide of 30 amino acids. The N-terminal sequences of the proproteinase and the mature proteinase have been observed in the polypeptide at position + 31 and + 38. The putative amino acid sequence showed a significant similarity to a surface layer protein of L. helveticus and Lactobacillus acidophilus in the amino terminal signal sequence and carboxyl terminus.
R&D Center, Calpis Co, Ltd, Fuchinobe, Sagamihara, Kanagawa, Japan.
N
N
Yamamoto
T
T
Shinoda
T
T
Takano
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics; D005110:Extracellular Space / Q000201:enzymology; D005798:Genes, Bacterial; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D010450:Endopeptidases