In Vitro Study On The Detection Of Cross-linking Reagents By Bimolecular Fluorescent Proteins

Proteins are the functional material basis for life,the organic macromolecules that constitute cells and the main undertakers life activities.However,the execution of life activities can't be accomplished by a single protein.Almost all biological processes in living cells are mediated by the complex interactions of proteins.Therefore,the identification and analysis of protein-protein interactions is critical to determine and understand the function of proteins in biological systems.At present,there are many techniques to obtain information of protein-protein interactions,among which chemical cross-linking has been widely used in the study of protein-protein interactions because it is applicable to stable true interactions and frozen transient or unstable covalent bond interactions.Chemical cross-linking also has significant advantages for the structural characterization of individual purified proteins and protein complexes.The identified cross-linked residues/peptides in the cross-linking provide distance constraints that help to determine three-dimensional structural models.The specificity of chemical cross-linking agents is one of the most important characteristics to be considered in cross-linking experiments.On the basis of traditional cross-linking agents,screening and developing new chemical cross-linking agents is still a hot research field,especially for protein cross-linking agents.However,at present,protein electrophoresis of SDS-PAGE or mass spectrometry is still the main method to test whether chemical cross-linking agents can occur between proteins and the efficiency of detecting protein cross-linking after using chemical cross-linking agents.Protein electrophoresis of SDS-PAGE is a complicated and time-consuming method to detect protein cross-linking,while mass spectrometry requires expensive equipment.Therefore,we conducted this study to develop a rapid,simple and high-throughput method for preliminary screening of protein chemical cross-linking reagents in vitro.This study constructed with histidine labels and red fluorescent protein(m Cherry)gene plasmid vector p ET29(b)-m Cherry,with red fluorescence protein(m Cherry)and no labels of soluble green fluorescent protein(soluble GFP,s GFP)as experiment material,with the aid of histidine labels and solid medium(Ni NTA Beads)adsorption characteristics,two chemical cross-linking agents of formaldehyde and glutaraldehyde was carried out on the two kinds of fluorescent protein crosslinking.In the study of cross-linking of fluorescent protein mixture with equal molar concentration,the optimal cross-linking concentration of cross-linking agent was preliminarily determined by detecting the fluorescence intensity of s GFP in the remaining solution after adding the same amount of Ni NTA Beads.By simultaneously turning on the laser confocal microscope at 594 nm and 488 nm excitation wavelengths,the Ni NTA Bead was visually detected to check whether the cross-linking effect occurred,and the software Cellsens Standard was used.The software calculates the cross-linking efficiency of the results captured by the microscope.In order to further verify the sensitivity and stability of this method,we designed a trace detection of non-equal proportion of fluorescence protein molar concentration cross-linking.Under the premise of relatively small amount of one fluorescent protein,the addition amount of the other fluorescent protein was changed to observe whether the research technique could still detect the cross-linking results quickly and sensitively.Finally,we used the traditional SDS-PAGE technology to assist detection,and the cross-linking results of glutaraldehyde and formaldehyde at different concentrations on the two fluorescent proteins were further verified providing the accuracy of this research technology.The experimental results showed that the p ET29(b)-m Cherry plasmid vector was successfully constructed,and the red fluorescent m Cherry and the high soluble green fluorescent protein s GFP were induced and purified.The experimental results of optimal cross-linking concentration showed that the optimal cross-linking concentration of glutaraldehyde was 1.0%,and the optimal cross-linking concentration of formaldehyde was1.5%.At this time,the fluorescence intensity of s GFP in the corresponding protein solution had the lowest value.Laser confocal microscope observation and statistical results show that the efficiency at the same time at the excitation wavelength of 594 nm and 488 nm,the experimental samples present dual fluorescence(yellow),two kinds of cross-linking agents of all experimental samples have been cross-linked,and the control group containing only m Cherry displayed in red fluorescence,and the concentration of 1.0% glutaraldehyde cross-linking efficiency and 1.5% formaldehyde concentration of cross-linking are highest groups,in which the efficiency are 95.6%,90.9% respectively.The non-proportional study showed that the cross-linking effect could be detected sensitively even when the fluorescence protein solution was 3 ?L and the concentration difference between the two was 27 times.SDS-PAGE results showed that there were specific bands in the samples of the two kinds of cross-linking agents,and cross-linking occurred in all samples,which was consistent with the above experimental results.In conclusion,this study provides a simple and efficient visualization technique for the study of protein chemical cross-linking reagents in vitro,and also provides a technical possibility for the preliminary rapid screening of a large number of protein chemical cross-linking reagents in vitro.