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.
Bibliographical noteFunding 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).
- Enzyme activity
- In vitro protein synthesis
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology