Novel Analytical Methods For Proteases And Protein Kinase Detection Based On Fluorescence Proteins

Protease and protein kinases are widespread in a living body and participate invarious activities of life. Developing protease and protein kinases detection methodswill not only enable us have a better understanding of life, but also have significantmeanings to human life and treatment of disease. Green Fluorescence Protein (GFP) isa kind of autofluorescent protein, wich consists of238amino acids and can exit greenfluorescence after excited by ultraviolet light. In cell and molecular biology study, GFPis always fused to other proteins or peptides, through molecular cloning, to formvarious biosensors. Compared with other organic fluorescent dyes, such as FITC, GFPis less toxical and better biocompatible. This leads a wider application of GFP inanalytical detection, such as fluorescent imaging, DNA/RNA labeling and proteininteraction reporter. However, recent reports of protease activity detection based onfluorescence proteins are scare, but is in geat need.In this thesis, we developed two label-free protease and protein kinases detectionmethods based on a recombinant enhanced green fluorescence protein (EGFP) andself-assembling Bimolecular fluorescence complementation (self-assembling BiFC)pairs, respectively. These two methods are summarized as below.(1) Here, a novel label-free and easy-conducting fluorescent assay has beendeveloped to detect the activity of thrombin by combining the autofluorescentproperties of EGFP and the selective collection ability of Ni2+-NTA modified magneticnanoparticles (Ni2+-NTA MNPs). The EGFP, containing a thrombin cleavage site and ahexahistidine sequence (His-tag) at its N-terminal, was adsorbed onto Ni2+-NTA MNPsthrough Ni2+-hexahistidine interaction, and dragged out of the solution by magneticseparation. Thrombin can selectively digest EGFP accompanied by His-tag leaving,and the resulting EGFP cannot be captured by Ni2+-NTA MNPs and keeps insupernatant. Hence the fluorescence change of supernatant can clearly represent theactivity of thrombin. Under optimized conditions, such assay showed a relatively lowdetection limit (3.0×10-4U mL-1), and was also used to detect the thrombin inhibitor,Hirudin, and further applied to detect thrombin activity in serum. Combined with thesatisfactory reusability of Ni2+-NTA MNPs, our method presents a promising candidatefor simple, sensitive, and cost-saving protease activity detecting and inhibitorscreening. (2) We got the truncated GFP with mature chromophore through digesting theentire green fluorescent protein and removing10thβ-sheet (S10). After being digestedwith trypsin, the entire green fluorescent protein was denatured with different agents,such as guanidine hydrochloride, SDS, Urea. In this work, we compared theirdenaturing and separating ability, as well as the outcome of the resultant truncatedGFPs recombined with synthetical S10.(3) Based on the self-assembling pairs got in the second work, we modified S10by adding cAMP-dependent protein kinase (PKA) recognized peptide at the C-terminal,and extended this self-assembling BiFC pairs to PKA activity detection. PKA inducedprotein phosphorylation prohibits the digestion of S10by carboxypeptidase Y (CPY).This enables S10attach to truncated GFP and reform entire fluorescence protein withrecovered fluorescence. Hence, the recovery of the fluorescence of GFP can indicatethe activity of PKA in reaction system.