An efficient proteomics based strategy for the functional characterization of a novel halophilic enzyme from Halobacterium salinarum

Jiyeon Choi, Won A. Joo, Soo Jin Park, So Hee Lee, Chan Wha Kim

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

The extremely halophilic archaeon, Halobactenum salinarum grows in environments containing over 25% NaCl. The enzymes of this organism have thus been adapted to be active and stable in hypersaline conditions, which makes them strong candidates as robust industrial enzymes. In this study, the proteomics approach was applied to screen novel halophilic enzymes. We focused initially on proteins that are differentially expressed under different salt concentrations in culture media. After two-dimensional gel electrophoresis over a pH 3.5-4.5 range, 29 differentially expressed protein spots were identified by tandem mass spectrometry and six of these had no similarity to preexisting genes of known function. To predict the function of them, we used various bioinformatic methods. Among other proteins, we selected Vng0487h, which showed a high similarity to acetyltransferases. As a step toward assaying the enzymatic activity of this protein, we cloned the Vng0487h gene of H. salinarum and expressed and purified the recombinant protein with a glutathione-S-transferase (GST) tag in Escherichia coli. Using a GST-pulldown assay, a protein fragment derived from E. coli could interact with recombinant Vng0487h, and was identified to be the ribosomal protein L3. This protein showed high sequence homology with ribosomal protein L7/12 from E. coli and ribosomal protein L13p from H. salinarum. This suggests that Vng0487h acetylates a subunit of ribosomal protein, possibly L13p, in H. salinarum. During the present study, an efficient procedure was established to screen novel halophilic enzymes, and to predict and assess their functions.

Original languageEnglish
Pages (from-to)907-917
Number of pages11
JournalProteomics
Volume5
Issue number4
DOIs
Publication statusPublished - 2005 Mar

Keywords

  • Acetyltransferase
  • Characterization
  • Halophilic enzyme

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Fingerprint Dive into the research topics of 'An efficient proteomics based strategy for the functional characterization of a novel halophilic enzyme from Halobacterium salinarum'. Together they form a unique fingerprint.

Cite this