Biosensing And Protein Self-assembly Based On Recombinant Fluorescent Proteins

Proteins are essential components of organisms,and also the key elements of cell physiological activities,molecular evolution,and the pathogenetic processes.Fluorescent proteins possess intrinsic fluorescence and genetic encoding properties,playing an important role in biological tracing,sensing,and biomaterials researches.With the rapid development of genetic engineering and protein engineering technology,the rational design and modification of fluorescent protein s can endow fluorescent proteins core structure with unprecedentedly signal-switch properties and functions,and expand their application ranges in biosensing and protein assembly.Resurfacing is an important protein engineering technology,which enables proteins with different properties and functions.For instance,resurfacing of fluorescent protein with high surface net charges generates supercharged GFP(ScGFP).Positive ScGFPs can interaction with oppositely charged macromolecules via electrostatic interaction,including negatively charged nucleic acids,peptides,polymers and nanomaterials,and further trigger specific recognition for target analysis,or structural assembly between different biomolecules.Near-infrared fluorescent proteins with reduced autofluorescence,weak light scattering,strong tissue penetration,and low phototoxicity,are promising biosensing and bioimaging probes for the complex biological samples,and become the current research hotspot and development trend of fluorescent molecular imaging.However,the applications of near-infrared fluorescent proteins are mainly focused on biological processes,protein-protein interactions,and whole body imaging,and their research in biosensing remains to be developed.In this paper,we carried out the following studies of biosensing and protein assembly based on ScGFP and near-infrared fluorescent proteins:(1)A simple and universal sensing method based on ScGFP and functional peptide was developed for the detection of caspase-3 enzyme activity.Caspase-3 is a key enzyme in apoptosis,and its activity measurement can be an important indicator for related diseases and for drug screening.Taking ScGFP as the signal reporter,a simple"switch on"method for the detection of caspase-3 activity was developed based on the electrostatic interaction between negatively charged functional peptides and ScGFP.The functional peptide D2 contains seven consecutive aspartic acid s(D)at the N-terminus,a recognition and cleavage substrate sequence DEVD of caspase-3in the middle,and a sequence with the Dabcyl quenching group at the C-terminus.When the peptide and ScGFP are mixed in the buffer at p H 7.4,peptide D2 carries10 net negative charges,and the ScGFP/peptide complex is formed due to electrostatic interaction,which closes the distance between Dabcyl and ScGFP.Through fluorescence resonance energy transfer,Dabcyl can effectively quench the fluorescence of ScGFP.When caspase-3 is present in the solution,it recognizes and cleaves the peptide at the DEVD site,thereby releasing the short pepti de GGK-Dabcyl.The short peptide with the quenching group Dabcyl has no negative charge and will not get close to ScGFP,and the fluorescent signal of ScGFP can be recovered.Besides its high sensitivity and selectivity,this method can be applied for a general sensing platform for the detection and analysis of proteolytic enzymes.(2)Design,construction,expression and self-assembly of a new ScGFP.By resurfacing of GFP,a new recombinant fluorescent protein pGFP with a net positive charge(+15)at the top and a net negative charge(-10)at the bottom of the cylinder was developed.The protein maintains the folding and the fluorescence spectral properties of the original protein GFP roughly,and has good stability.It was found that the self-assembly of pGFP can be reversibly regulated by the ion strength.When Na Cl<500 m M,the assembly is formed between adjacent pGFP molecules due to electrostatic interactions.With the decrease of ions strength,pGFPs will produce several micrometer-scale assemblies.Once the assembly was formed,the absorbance of pGFP enhanced significantly,the fluorescence intensity increased by?26%,and the maximum fluorescence emission peak was red-shifted by 4 nm.This may be because the surrounding microenvironment of the pGFP mol ecule changed.As homo-FRET occurred between the adjacent pGFP molecules in the assembly,its fluorescence anisotropy value continued to decrease as the concentration of salt decreased.This work illustrates a new way for the redesign of protein surfaces and further for protein self-assembly into ordered microstructures controllably,which not only provides novel biomaterials,but also paves the way for kinetic/thermodynamic study of the self-assembly process.(3)Cloning,expression,purification and chara cterization of near-infrared fluorescent proteins.The plasmid p UCK-mifp and p UCK-irfp carrying the m IFP and iRFP gene respectively were obtained by whole gene synthesis,and the plasmids p ET28-mifp and p ET28-irfp for prokaryotic expression were further co nstructed.Then the target genes were induced and expressed in E.coli,and m IFP and iRFP with high purity were obtained after affinity purification.It was found that these two proteins can effectively bind with biliverdin,and show the typic absorption and emission peaks of m IFP and iRFP,respectively.Among them,Ex_{m IFP} is 683 nm and Em_{m IFP} is 704 nm;Ex_iRFP is 692 nm and Em_iRFP is 712 nm.In addition,their quantum yields were determined,Qy_{m IFP} was 0.93,and Qy_iRFP was 0.68,respectively.Moreover,the fluorescence of m IFP and iRFP in the buffer at p H 6.5-9 and p H 6-9can be well maintained above 80%,respectively.Cu²⁺,Zn²⁺,Hg²⁺,Ag⁺can quench the fluorescence of m IFP and iRFP.In addition,we also constructed plasmids pc DNA 3.1-mifp and pc DNA 3.1-irfp for protein expression in mammalian cells.m IFP and iRFP can not only be expressed in Hela cells,but also directly utilize the endogenous biliverdin,indicating that they possess potential and bright application in cell imaging and other fields.(4)Biomineralization synthesis of nanoparticles based on near-infrared fluorescent protein and H₂O₂ detection.Protein-templated synthesis of nanomaterials has received considerable attention in the fields of biomedicine and biosensing,due to the advantages of mild reaction conditions,size controllability,and mutiple functions.Among them,biomimetic biomineralization synthesis of manganese dioxide(Mn O₂)nanomaterials that uses protein as template can avoid the strong oxidizing reagents and surfactants,which are usually involved in hydrothermal methods,and have good biocompatibility.We selected the near-infrared fluorescent protein iRFP as the template to generate biomineralized nanoparticles iRFP-Mn O₂(iRMs),and studied the physicochemical properties of iRMs through various characterization methods(TEM,DLS,XPS,etc).The results suggested that iRMs was successfully synthesized with an average size of 36.0±15.8 nm.In the XPS spectra,four peaks that corresponded to the C 1s,N 1s,O 1s and Mn 2p were observed,respectively.The Mn 2p spectrum consisted of the two characteristic peaks of Mn(IV)2p_3/2 and Mn(IV)2p_1/2 respectively,which confirms the Mn⁴⁺manganese species and the formation of Mn(IV)O₂nanosheets.In addition,iRMs particles have strong broad absorption band at 300-800 nm,and it can effectively quench the near-infrared fluorescence of iRFP through energy transfer.Hydrogen peroxide(H₂O₂),one of the most siginificant chemicals,is widely used in clinical and pharmaceutical areas.At the same time,as an important reactive oxygen species,it is the intermediate product of various physiological processes.High concentration of H₂O₂ will cause cell damage and is highly relate to many diseases such as Alzheimer's disease,myocardial infarction,cancer,etc.Therefore,it is necessary to develop new methods and strategies for the detection of H ₂O₂.In the mild acidic condition,H₂O₂ reacted with Mn O₂ to produce manganese ions(Mn²⁺),resulting in the decomposition of iRMs,as well as the release of free iRFP.As a rusult,the near-infrared fluorescence of iRFPs was recovered,providindg a facile"turn on"assay for the detection of H₂O₂with the detection limit of about 0.06 m M.Meanwhile,iRMs exhibited good selectivity and stability.(5)Biomineralization synthesis of a near-infrared fluorescent nanoprobe for direct sensing of glucose in whole blood.The nanoprobe iRFP-GOx-Mn O₂(iRGMs)were prepared through biomineralization synthesis of the Mn O₂ nanoparticles that employed near-infrared fluorescent protein iRFP and glucose oxidase(GOx)as the templates.Similarly,the fluorescence of iRFP in iRGMs is effectively quenched by Mn O₂ through energy transfer.When iRGMs are added into whole blood samples,blood glucose is converted into gluconic acid by GOx in iRGMs,accompanied by the in situ generation of H₂O₂,which will reduce Mn O₂to produce Mn²⁺,decompose iRGMs and release the free iRFP.As a rusult,the near-infrared fluorescence of iRFPs was restored.The near-infrared nanoparticles exhibited the advantages of high efficiency of cascade reaction and the low background interference of the near-infrared fluorescence signal.With the detection limit of 0.03 m M and the excellent selectivity,the nanoprobe enabled rapid and accurate measurement of glucose levels in whole blood samples.Furthermore,a simple paper device based on iRGMs was designed for glucose detection.With this paper device,only 5microliters of sample was required for the direct assay of glucose in whole blood without any pretreatment.Our work afforded an alternative approach for the rapid and accurate monitoring of blood glucose levels.