PMID:18258174
| Citation |
Kim, JH, Jang, KS, Yang, YH, Kim, YG, Lee, JH, Oh, MK, Kim, BG and Lee, CS (2008) Rapid functional identification of putative genes based on the combined in vitro protein synthesis with mass spectrometry: a tool for functional genomics. Anal. Biochem. 375:11-7 |
|---|---|
| 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 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. |
| Links |
PubMed Online version:10.1016/j.ab.2008.01.007 |
| Keywords |
Bacillus subtilis/enzymology; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Biological Assay; Carboxylesterase/metabolism; Escherichia coli/enzymology; Escherichia coli Proteins/metabolism; Esterases/metabolism; Feasibility Studies; Genomics/methods; Green Fluorescent Proteins/metabolism; Lipolysis; Mass Spectrometry/methods; Molecular Weight; Protein Biosynthesis; Substrate Specificity; Triglycerides/chemistry |
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