Protein Modification With Nucleic Acid Based On A* Protein And Its Applications

Background Ordered immobilization of enzyme on the electrode surface can improve the reproducibility and stability of enzyme biosensor,and then promote the development of the novel and superior biosensor.Spatially-ordered protein assemblies can be constructed by using self-assembled DNA which lays the foundation for the controllable immobilization of enzyme on the electrode surface.There are many attentions paid to the method for modification of protein with nucleic acid.Although there are a variety of modification methods,there is a risk of loss of the inherent properties of protein or DNA nanostructure through the complicated chemical or biological modification process,and thus causing great limitation of their practical application.Modification method is a bottleneck for the development of DNA-protein conjugates.Thus,developing an easy-to-use and economical modification method for modification of protein with nucleic acid is necessary of constructing the DNA-protein conjugates,and the ordered immobilization of enzyme on the electrode surface.Objective Exploring an easy-to-use and economical method for modification of protein with nucleic acid based on enzymatic catalysis.Constructing spatially-ordered protein nanostructures,thus further to promote the ordered immobilization of enzyme on the electrode surface,and then finally fabricating generating superior enzyme biosensor.Methods Firstly,a fusion protein of target protein(enhanced green fluorescent protein as an example in this study)with A*protein from bacteriophage phi 174 was constructed,then the fusion protein was reacted with the single-stranded nucleic acid under the condition of 37°C,thus causing the nucleic acid covalently modified on the target protein via a stable phosphotyrosine linkage by using specific nucleic acid recognition ability of A*protein.After that,we analyzed the DNA-EGFP conjugate using spectrofluorometric and gel electrophoretic method to evaluate the impacts of this strategy on the properties of target protein and A*protein respectively,and estimate the reaction yields.In addition,we also tried to separate the DNA-EGFP conjugates from the mixed reaction by means of gel filtration chromatography,high performance liquid chromatography and magnetic beads.Secondly,we screened six different HUH endonucleases,then examined their nuclease activity in vitro after the heterologous expression in E.coli BL21,in an attempt to exploit other protein which has common specific nucleic acid recognition ability with A*protein.Thirdly,we used graphene oxide/Prussian blue/chitosan as functional materials to modify the electrode,and glucose oxidase as the bioactive element to optimize the electrode parameters and electrode properties,in an attempt to use the enzyme nanostructures prepared by the above method on this prepared electrodes,to realize ordered immobilization of enzyme on the electrode surface,and then finally fabricating novel efficient and highly stable enzyme sensors.Results Firstly,The A*protein-based method for modification of protein with nucleic acid does not affect the biological activity of both the green fluorescent protein and the A*protein,and the process does not require any chemical modification of the single-stranded nucleic acid.The reaction achieved a steady state after 30 min,and the yield is up to approximately 80%.But,it is difficult to obtain purified DNA-protein conjugate after various separation attempts.Secondly,among the six different HUH endonucleases,only part of the proteins can covalently bind to nucleic acid,and the binding yield is low.Thirdly,in the preparation of the electrode,we optimized the detection potential and working pH of the electrode,laying the foundation for the further application of this electrode.But due to the first half of this study of the enzyme immobilization method still needs to be improved,ordered immobilization of enzyme on the electrode is not complete.Conclusion An easy-to-use and economical method for modification of protein with nucleic acid based on enzymatic catalysis was developed based on A*protein,which laid a foundation for the ordered immobilization of enzyme on the electrode surface.On the other hand,we get some knowledge on the properties of A*protein and other HUH proteins,it is in the initial exploration stage and needs more effects to further exploration.