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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.

SUBMITTER: Kitazono A

PROVIDER: S-EPMC207526 | biostudies-other | 1992 Dec

REPOSITORIES: biostudies-other

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Cloning, sequencing, and high expression of the proline iminopeptidase gene from Bacillus coagulans.

Kitazono A A Yoshimoto T T Tsuru D D

Journal of bacteriology 19921201 24

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 f ...[more]

PMID: 1459939

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Project description:The psd gene of Bacillus subtilis Marburg, encoding phosphatidylserine decarboxylase, has been cloned and sequenced. It encodes a polypeptide of 263 amino acid residues (deduced molecular weight of 29,689) and is located just downstream of pss, the structural gene for phosphatidylserine synthase that catalyzes the preceding reaction in phosphatidylethanolamine synthesis (M. Okada, H. Matsuzaki, I. Shibuya, and K. Matsumoto, J. Bacteriol. 176:7456-7461, 1994). Introduction of a plasmid containing the psd gene into temperature-sensitive Escherichia coli psd-2 mutant cells allowed growth at otherwise restrictive temperature. Phosphatidylserine was not detected in the psd-2 mutant cells harboring the plasmid; it accumulated in the mutant up to 29% of the total phospholipids without the plasmid. An enzyme activity that catalyzes decarboxylation of 14C-labeled phosphatidylserine to form phosphatidylethanolamine was detected in E. coli psd-2 cells harboring a Bacillus psd plasmid. E. coli cells harboring the psd plasmid, the expression of which was under the control of the T7phi10 promoter, produced proteins of 32 and 29 kDa upon induction. A pulse-labeling experiment suggested that the 32-kDa protein is the primary translation product and is processed into the 29-kDa protein. The psd gene, together with pss, was located by Southern hybridization to the 238- to 306-kb SfiI-NotI fragment of the chromosome. A B. subtilis strain harboring an interrupted psd allele, psd1::neo, was constructed. The null psd mutant contained no phosphatidylethanolamine and accumulated phosphatidylserine. It grew well without supplementation of divalent cations which are essential for the E. coli pssA null mutant lacking phosphatidylethanolamine. In both the B. subtilis null pss and psd mutants, glucosyldiacylglycerol content increased two- to fourfold. The results suggest that the lack of phosphatidylethanolamine in the B. subtilis membrane may be compensated for by the increases in the contents of glucosyldiacylglycerols by an unknown mechanism.

Dataset Information

Cloning, sequencing, and high expression of the proline iminopeptidase gene from Bacillus coagulans.

Publications

Cloning, sequencing, and high expression of the proline iminopeptidase gene from Bacillus coagulans.

Similar Datasets

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