The Application Of Biarsenical-Tetracysteine System In Protein Labeling, Detection And Quantitative Analysis

The understanding of protein localization, distribution and content within living cell can give insight into its function and mechanism. Small fluorescent tags have great power in those fields, especially the green fluorescent protein (GFP), which gives rise to the flourishing of in-cell studying. However, due to its large size (up to 25 kDa), the GFP and its derivatives all suffer from shortcomings. Therefore, many small chemical tags have been developed and got widely used, among which was the success of biarsenical-tetracysteine (TC) system. The TC motif not only can be used in cell imaging within living cells, but also displays lots of advantages. Firstly, the smaller size of the TC motif faces a smaller chance to interference with the structure of target protein. Secondly, the biarsenical reagent can bind to its motif with high specificity and selectivity. Last, it's a powerful tool for quantitative analysis for target protein. Although the TC motif has been used as a tracing tag, the binding stability between TC motif and biarsenical reagent against extreme conditions as well as its capacity as a quantitative tag remain deserves attention.To reveal these problems, we chose enoyl-acyl carrier protein (ACP) reductase (FabI), which is involved in the final step of elongation in the bacterial fatty acid biosynthesis and is an important target for bacteriostat screening, 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 Fabls (N-terminus, C-terminus, or both N-and C-terminus TC motif, which are referred to as Nt-FabI, Ct-FabI, Bt-FabI, Gt-FabI and Wt-FabI, respectively). The results showed that all the TC tagged FabIs had high enzyme activities, while the EGFP tagged FabI exhaustively lost the activity. These findings indicate that the TC tag dose not affect the functions of target proteins. The enzyme activities after labeling with FlAsH for TC-tagged FabIs decline slightly, and it is most clear for the Wt-FabI. Additional, the binding stability between FlAsH reagent and its TC motif against extreme conditions, including high temperature, high pressure (supported by HPLC), high field strength (supported by CE), microwave, and ultrasonic were also investigated comprehensively. The results suggested that the binding between the two was stable against conditions of heating 70℃for 10 min, high pressure of up to 5.24 MPa, high field strength of 397 V/cm and ultrasonic at 80 W for 30 min. However, the microwave (750 W) exhibited a time-dependent destroy to the labeled complex. An obvious destroy can be seen after treating for 15 min, and when the time was extended to 25 min, the binding between FlAsH reagent and TC motif was destroyed completely. We also investigated the capacity of TC motif for quantitative analysis of target protein. The results showed that the TC motif exhibits a detection limit of 10-16 mol on CZE, and the detection was linear range from 10-12 to 10-10 mol. In analytical SDS-PAGE gel, an injection of only 1 ng (about 10-11 molar) labeled protein can be detected. And on spectrofluorometric (SPF), the detection limit was 10-12 mol. Those results show that the TC motif is a powerful tool for detection and quantitative analysis of target protein.Meanwhile, the over-expressed acyl carrier protein (holo-ACP) by Escherichia coli was quantitated by means of TC motif. In bacteria, the gene production directly from acpP was apo-ACP, a protein without function, and must be modified by acyl carrier protein synthase (AcpS) to convert to its active form (called holo-ACP). The holo-ACP can transfer all kinds of substrates in the cycle of fatty acid biosynthesis. Herein, four expression strains, Hh02:BL21(DE3)/pB-lu-ACP & pET-AcpS, HhO4: BL21(DE3)/pB-lu-ACP-AcpS, Hh06:BL21(DE3)/pB-lu-ACP-AcpS & pET-AcpS and Hh08:BL21(DE3)/pB-lu-ACP were constructed. The plasmids within each strain make a difference on the ratio between apo-ACP and holo-ACP from one another, supporting the analysis of the two styles of ACP on micellar electrokinetic capillary chromatography (MEKC) later. In the analytical condition, the two styles of ACP, which were difficult to be distinguished on SDS-PAGE gel, could get well separation directly from cell lysate after inducing. The over-expressed holo-ACP was quantitated using FITC as an internal reference. In vitro reaction analysis, the TC-tagged ACP was confirmed to have function and can join the elongation of fatty acid biosynthesis, which was also proved on MEKC.