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

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 lip A and lip B 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.

Citation

June-Hyung Kim, Kyoung-Soon Jang, Yung-Hun Yang, Yun-Gon Kim, Ji-Hye Lee, Min-Kyu Oh, Byung-Gee Kim, Chang-Soo Lee. Rapid functional identification of putative genes based on the combined in vitro protein synthesis with mass spectrometry: a tool for functional genomics. Analytical biochemistry. 2008 Apr 1;375(1):11-7

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PMID: 18258174

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