Cloning, sequencing, and high expression of the proline iminopeptidase gene from Bacillus coagulans
Journal of Bacteriology, ISSN: 0021-9193, Vol: 174, Issue: 24, Page: 7919-7925
1992
- 50Citations
- 17Captures
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Metrics Details
- Citations50
- Citation Indexes50
- 50
- CrossRef39
- Captures17
- Readers17
- 17
Article Description
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.
Bibliographic Details
http://www.scopus.com/inward/record.url?partnerID=HzOxMe3b&scp=0026690122&origin=inward; http://dx.doi.org/10.1128/jb.174.24.7919-7925.1992; http://www.ncbi.nlm.nih.gov/pubmed/1459939; https://journals.asm.org/doi/10.1128/jb.174.24.7919-7925.1992; https://dx.doi.org/10.1128/jb.174.24.7919-7925.1992; https://jb.asm.org/content/174/24/7919
American Society for Microbiology
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