Novel Analytical Methods Based On Supercharged Green Fluorescent Protein

Supercharged green fluorescent protein (scGFP) is a sort of green fluorenscent protein with high net charge. They show several advantages such as fluorescence-stability and aggregation-resistance. In addition, they are quite stable in different pH and exhibit well salt-tolerance. Superpositively charged green fluorescent protein is cell penetrating and can deliver DNA, siRNA and other biological macromolecules into mammalian cells. Due to the above advantages, scGFP has attracted more and more attention in the field of biochemistry, and exhibits great promise for applications in biosensing. Here we developed a novel label-free method for PARP-1 detection based on +36GFP/GO interaction. In addition, we constructed a MMP-2-sensitive fluorescent probe using -30GFP as the fluorescent reporter. The specific works are summarized as follows:(1) Our previous work demonstrated that +36GFP could bind to GO through electrostatic interaction and hydrophobic interaction and GO quench the fluorescence of +36GFP efficiently. DNA with appropriate length can restore the fluorescence of +36GFP via forming +36GFP/DNA nano-complex. Taking the advantage of large negatively charged poly(ADP-ribose) polymer (PAR), a novel, convenient and label-free fluorescence assay strategy for PARP-1 activity detection was developed based on the PAR-mediated +36GFP/GO interaction and the fluorescence intensity responded to the activity of PARP-1.This proposed method exhibits a wide linear range (0.002-0.07 U/?L) and a low detection limit of 0.002 U/?L, and has been successfully applied to assay the PARP-1 activity inhibition. Compared with other PARP-1 assays, this method is convenient, low-cost and sensitive. Furthermore, the present approach would be potentially extended to the detection of caspase 3.(2) A novel automatic and integrated micro-enzyme assay (Al?EA) platform was proposed to detect thrombin online in our previous work. The recombinant enchanced green fluorescent protein (EGFP) possesses a His-tag and the thrombin-specific recognition sequence (LVPRGS). Taking the advantage of the unique structure and fluorescence property of recombinant EGFP, we have constructed a poly (GMA-EDMA)-ANTA-Ni2+ monolithic capillary column to form a micro enzyme reaction system through the interaction between His-tag and Ni2+. Thrombin recognized and digested the thrombin-specific recognition sequence to release EGFP, and then online thrombin detection was achieved with capillary electrophoresis with laser-induced fluorescence detection technique (CE-LIFD). Matrix metalloproteinase-2 (MMP-2), one of the most important proteolytic enzymes that can degrade type IV collagen and promote tumor invasion and migration, was reported to be closely related to tumorifenesis. Here, we constructed a MMP-2-sensitive fluorescent probe named MMP-2S-30GFP. The fluorescent probe consists of three parts:N terminal His-tag, MMP-2 specific recognition sequence (MMP-2S), C terminal-30GFP. The full length gene inserted in plasmid pUCK was obtained by whole gene synthesis. We obtained the prokaryotic expression vector containing the gene with "two-step" and "one-pot" methods, and then transformed the plasmid pET28a-mmp-2s-30gfp into E.coli and added IPTG to induce the expression of the probe. Finally, the purification of MMP-2S-30GFP was performed with Ni-NTA agarose and desalinating column. MMP-2S-30GFP with high purity was characterized by SDS-PAGE, ultraviolet absorption spectrum and fluorescence spectrum. It paves the way for developing a highly sensitive method for detecting MMP-2 online with the combination of capillary electrophoresis technology. Besides, it provides a guideline for detection different enzymes at the same time online based on the difference in charge to mass ratio of varying scGFP.