Rapid functional identification of putative genes based on the combined in vitro protein synthesis with mass spectrometry: A tool for functional genomics

June Hyung Kim, Kyoung Soon Jang, Yung Hun Yang, Yun Gon Kim, Ji Hye Lee, Min Kyu Oh, Byung Gee Kim, Chang Soo Lee

    Research output: Contribution to journalArticlepeer-review

    7 Citations (Scopus)

    Abstract

    For the rapid identification of functional activity of unknown genes from a sequence database, a new method based on in vitro protein synthesis combined with mass spectrometry was developed. To discriminate their subtle enzymatic activity, in vitro synthesized and one-step purified lipolytic enzymes, such as lipA and lipB from Bacillus subtilis and an unknown protein ybfF from Escherichia coli, were reacted with a mixture of triglycerides with different carbon chain lengths. Using direct matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of reaction product, all three enzymes were revealed to have strong esterase activity rather than true lipase activity, which has no reactivity on long-chain fatty acids such as triolein. These results were also confirmed by classical color assay using p-nitrophenyl butyrate (pNPB) and p-nitrophenyl palmitate (pNPP) as representative lipolytic substrates.

    Original languageEnglish
    Pages (from-to)11-17
    Number of pages7
    JournalAnalytical Biochemistry
    Volume375
    Issue number1
    DOIs
    Publication statusPublished - 2008 Apr 1

    Bibliographical note

    Funding Information:
    This work was supported partly by the Basic Research Program of the Korea Science and Engineering Foundation (grant R01-2005-000-10558-0) and partly by the ERC program of MOST/KOSEF (grant R11-2000-075-03001-0).

    Keywords

    • Enzyme activity
    • Esterase
    • In vitro protein synthesis
    • Lipase
    • MALDI-MS

    ASJC Scopus subject areas

    • Biophysics
    • Biochemistry
    • Molecular Biology
    • Cell Biology

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