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.
Kim
June-Hyung
JH
Department of Chemical Engineering, Dong-A University, Busan 604-714, Republic of Korea.
Jang
Kyoung-Soon
KS
Yang
Yung-Hun
YH
Kim
Yun-Gon
YG
Lee
Ji-Hye
JH
Oh
Min-Kyu
MK
Kim
Byung-Gee
BG
Lee
Chang-Soo
CS
D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001681:Biological Assay; D043182:Carboxylesterase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000378:metabolism; D004950:Esterases / Q000378:metabolism; D005240:Feasibility Studies; D023281:Genomics / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008066:Lipolysis; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D014176:Protein Biosynthesis; D013379:Substrate Specificity; D014280:Triglycerides / Q000737:chemistry
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C079999:LipA protein, Bacteria; D014280:Triglycerides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004950:Esterases; C526138:ybfF protein, E coli; D043182:Carboxylesterase; C416523:LipB protein, Bacillus subtilis