The Recombinant Expression Of SNAP-tag And Its Research In Fluorescent Labeling Of Peroxisome

To explore the distribution, transportation and interaction of the intracellular active substance is of great importance for understanding complex physiological and biochemical processes. The continuous development of fluorescent technology provides technical means for exploring the complex intracellular processes and achieves intracellular visualization. As an important organelle in eukaryotic cells, Peroxisome lack fluorescent probe which can locate the target accurately to study its function up to now. In recent years, proteins specific labeling technologies have been developed and applied, which combines the advantages of a fluorescent protein and small molecule fluorescent probes. SNAP-tag is well approved in these technologies because of the particular advantages of high specificity and stability of the covalent labeling protein. In this paper, we based on SNAP-tag protein labeling technology using new fluorescent substrate to realize the labeling of peroxisomes and conducting a real-time monitoring of NO. To provide technical means for monitoring the distribution and changes of small molecule active substance in cells, and exploring their functions. Based on this, This paper mainly does the following works:(1) Using genetic engineering techniques, we built up prokaryotic expression vector pET28a-SNAP and obtained purified SNAP-tag protein (approximately 27 kD) at a concentration of 2.14 mg·mL-1 by prokaryotic expression. Using specific novel fluorescent substrates TMR-BG, BDP-BG to label SNAP-tag protein in vitro. The spectroscopy results showed that, compared with the control group (using the BSA protein), the fluorescence of the experimental group which joined SNAP-tag protein was significantly enhanced, and fluorescence intensity was growing with time up to balance. These results proved that novel fluorescent substrate can achieve high efficiency, specific binding with SNAP-tag protein, and lay the feasibility foundation for subsequent labeling experiments in cells.(2) Constructing eukaryotic expression vector pSNAP-PTS1/pCLIP-PTS1. Stably transfected into COS-7 cells, and realized the expression of SNAP-tag/CLIP-tag protein in peroxisomes. Using specific fluorescent substrates TMR-BG, BDP-BG to label SNAP-tag and BDP-V-BC to label CLIP-tag, imaging results showed all of the three fluorescent substrate can label the specific intracellular site, and the labeling positions showed spherical or oval distribution in the cytoplasm, but others without PTS1 signal peptide render the entire cell scatter mark. We can conclude that intracellular peroxisome marking has been realized preliminarily. pEGFP-PTS1 eukaryotic expression vector was built as well, it was transiently transfected to the cells stably expressing SNAP-PTS1. Through EGFP confocal imaging with TMR-BG, results showed that the colocalization effect is good, which can confirm the pSNAP-PTS1 specifically expressed in peroxisomes and realize the label of peroxisome In addition, we realize simultaneous labeling of peroxisomes by TMR-BG and BDP-BG.(3) NO fluorescent substrate TMR-NO-BG are used, which can also recognize the SNAP-tag to detect NO, which achieves a real-time monitoring of NO while labeling peroxisomes.In summary, we realize the fluorescent labeled of peroxisomes by using recombinant expression of SNAP-tag protein labeled with new fluorescent substrate, and conduct real-time monitoring of NO.