Rapid functional identification of putative genes based on the combined in vitro protein synthesis with mass...

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.