Construction Of Split GFP-based Sensing Systems And Detection Of Transpeptidase Activity

Green fluorescent protein（GFP）has been developed as a powerful molecular tool in the field of life science for its unique fluorescence and gene coding properties,and many biosensing methods based on green fluorescence protein have been developed.Based on its auto-fluorescence and the property that it can be divided into non-fluoresecing fragments,split GFP fluorescence complementary system was developed,which is highly suitable for the development of biosensing platform due to its label-free and high signal-to-background ratio.Our group has reported a sensing system for kinase activity detection based on the split GFP bimolecular system（GFP1-10/GFP10,10+1 system）.In this paper,we designed and constructed a simple and universal biosensing platform for sortase transpeptidase activity detection based on tripartite split GFP system by using GFP1-9 as a reporter.Moreover,we constructed a bimolecular split GFP system on mammanlian cell membrane for imaging.The research work is as follow:1.Acquisition of GFP1-9 fragment and optimization of assembly experimental conditions between GFP1-9 and GFP10-11.We obtained GFP1-9 with high purity after construction of the recombinant expression plasmid pCold-gfp1-9 and expression of it in E.coli BL21.Meanwhile,after verifying the assembly between GFP1-9 and its complementary peptide fragment GFP10-11,the reaction conditions were investigated and optimized.These experiments could lay a foundation for the design and experimentation of subsequent biosensing system.2.Tripartite split GFP serve as a sensor platform for the analysis of transpeptidase activity and its inhibitors screening.Herein,we developed a novel fluorescent biosensor to detect sortase transpeptidase activity based on a transpeptidation-triggered assembly of tripartite split GFP.Peptide P₁,composed GFP10 with the sortase A（SrtA）recognition sequence（LPETX）,and peptide P₂,GFP11 with oligoglycine at N-terminal,were designed and synthesized,respectively.Existence of SrtA enables P₁ and P₂ to ligate into one peptide,which could spontaneously bind to GFP1-9 and assemble into functional GFP.Thus,the sortase-catalyzed transpeptidation can switch on the fluorescence signal of GFP.The method was successfully applied to detect SrtA activity with a low detection limit of 0.16 nM,and for its inhibition measurement.Moreover,the feasibility of the proposed assay was further expanded to detect SrtA in human blood,and further gram-positive pathogens analysis in frozen food.3.Construction of a bimolecular split GFP system on mammanlian cell membrane.We obtained pDisplay-gfp1-10 recombinant plasmid with a mammalian expression vector pDisplayTM,that allows GFP1-10 to appear on the extracellular side of cell membrane,Then,GFP1-10,displayed on the membrane,was able to bind to GFP10-11 peptide to form intact GFP and recovery the fluorescence.It provides the potential for the detection of membrane surface proteases or secreted enzymes.