Identification and characterization of inosine monophosphate dehydrogenase from Halobacterium salinarum

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

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    10 Citations (Scopus)

    Abstract

    Extremely halophilic Archaea, Halobacterium salinarum live in hypersaline habitats and maintain an osmotic balance of their cytoplasm by accumulating high concentrations of salt (mainly KCI). Therefore, their enzymes adapted to high NaCl concentrations offer a multitude of acutal or potential applications such as biocatalysts in the presence of high salt concentrations. In this study, the protein expression profile of H. salinarum cultured under different NaCl concentrations (3.5 M, 4.3 M, and 6.0 M) was investigated using two-dimensional gel electrophoresis (2-DE). As a result of 2-DE, the protein spots concentrated in acidic range at pH 3-10 were separated effectively using pH 3.5-4.5 ultrazoom IPG DryStrips. The proteins which proved to be upregulated or downregulated in 2-DE gel were digested with trypsin and identified with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and electrospray ionization quadrupole (ESI-Q) TOF-mass spectrometry. Most proteins were identified as known annotated proteins based on sequence homology and few as unknown hypothetical proteins. Among proteins identified, an enzyme named inosine monophosphate dehydrogenase (IMPDH) was selected based on the possibility of its industrial application. IMPDH gene (1.6 kb fragment) expected to exist in H. salinarum was amplified by polymerase chain reaction (PCR) and expressed in Escherichia coli strain, BL21 (DE3) using a pGEX-KG vector. Recombinant IMPDH purified from H. salinarum has a higher activity in the presence of salt than in the absence of salt.

    Original languageEnglish
    Pages (from-to)3632-3641
    Number of pages10
    JournalProteomics
    Volume4
    Issue number11
    DOIs
    Publication statusPublished - 2004 Nov

    Keywords

    • Electrospray ionization-quadrupole time of flight-mass spectrometry
    • Halobacterium salinarum
    • Inosine monophosphate dehydrogenase
    • Matrix-assisted laser desorption/ionization-time of flight
    • Two-dimensional gel electrophoresis

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology

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