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

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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