title: Cloning, sequencing, and high expression of the proline iminopeptidase gene from Bacillus coagulans.
abstract: The gene coding for proline iminopeptidase in Bacillus coagulans was cloned and expressed in Escherichia coli. Nucleotide sequencing revealed an 861-bp open reading frame with an unusual TTG initiation codon, encoding a 287-amino-acid protein. The calculated molecular weight of the product was 32,415. The amino acid sequences of the amino-terminal region and those of some peptide fragments obtained by endoproteinase Asp-N digestion of the purified enzyme completely coincided with those deduced from the nucleotide sequence. The rare TTG initiation codon that normally codes for leucine was translated as a formal initiation codon; a methionine residue was found at the amino terminus of the enzyme. By using a vector bearing the strong tac promoter, an expression level as high as 200-fold that of the first clone was achieved. The replacement of the TTG initiation codon with ATG and a simultaneous reduction of the distance to the tac promoter resulted in a further increase of 2.5-fold. The expressed enzyme was easily purified to homogeneity by hydrophobic chromatography on a Toyopearl HW-65C column and crystallization, with a recovery of activity of 36%. The molecular weight was found to be 33,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on a Hi-Load 16/60 Superdex 200 fast protein liquid chromatography column. The expressed enzyme showed the same catalytic and physicochemical properties as those of the wild type, specifically cleaving the N-terminal proline from small substrates.
authors.affiliation: School of Pharmaceutical Sciences, Nagasaki University, Japan.
authors.forename: A
authors.initials: A
authors.lastname: Kitazono
authors.forename: T
authors.initials: T
authors.lastname: Yoshimoto
authors.forename: D
authors.initials: D
authors.lastname: Tsuru
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D015183:Restriction Mapping; D013379:Substrate Specificity
chemical_list: D004269:DNA, Bacterial; D000626:Aminopeptidases; C022450:prolyl aminopeptidase