Detection of Escherichia coli enoyl-ACP reductase using biarsenical-tetracysteine motif.
Although the tetracysteine (TC) motif has been used as a tag, the binding stability between TC motif and biarsenical reagent against extreme conditions as well as its capacity as a quantitative tag remains not well developed. To reveal these problems, we chose enoyl-acyl carrier protein reductase (FabI), which was involved in the final step of elongation in the bacterial fatty acid biosynthesis, to be tagged by the TC motif. Taking enhanced green fluorescent protein (EGFP) tagged FabI as a control, we investigated the activities of various TC tagged FabIs (N-terminus, C-terminus, or both N- and C-terminus TC motif). The results showed that all the TC tagged FabIs had high enzyme activities while the EGFP tagged FabI exhaustively lost the activity. Beside this, the characteristics of the tag, including labeling stability against extreme conditions, capacity for quantitative analysis, and ability for in-cell labeling, were also investigated. We demonstrated for the first time that the binding between FlAsH reagent and TC motif was stable against high pressure, high field strength, high temperature, and ultrasound. Furthermore, we verified the potential of TC motif for quantitative analysis of target protein by different approaches, including SDS-PAGE, spectrofluorometry (SPF), and capillary zone electrophoresis (CZE).
Yang
Hang
H
State Key Laboratory of Agricultural Microbiology and National Engineering Research Center of Microbial Pesticides, Huazhong Agricultural University, Wuhan, Hubei 430070, P. R. China.
He
Jin
J
Hu
Fen
F
Zheng
Cao
C
Yu
Ziniu
Z
D001152:Arsenicals / Q000737:chemistry*; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH) / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D054889:Fatty Acid Synthase, Type II; D007700:Kinetics; D013752:Tetracycline / Q000737:chemistry*
D001152:Arsenicals; D029968:Escherichia coli Proteins; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); C493924:fabI protein, E coli; D054889:Fatty Acid Synthase, Type II; D013752:Tetracycline