Improvement Of Substrate Selectivity Of DNAzyme Based On The Recombinant AfFtn-DCV

In recent years,with the development of genetic engineering and protein engineering,lots of recombinant proteins have been developed and widely used,where different functional protein modules were combined together to obtain new proteins with specific new functions.Artificial enzymes have the advantages of easy preparation,low price,and low environmental interference,but they have low substrate selectivity.Here,we fused ferritin?AfFtn?with duck circovirus?DCV?proteins.AfFtn protein presented pH-dependent self-assembly,and DCV protein can be specifically covalently linked with DNA.DCV works as the medium to link different functional DNAs?such as DNAzyme?with AfFtn,and the DNA can be packaged in the assembled structure of AfFtn.Electrostatic repulsive force as well as the structure barrier may improve the substrate selectivity of DNAzyme.Split fluorescent proteins,as sensoring elements,have the advantage of being label-free and artificial synthesized,but it most of them have high background.Herein,based on the yellow-green fluorescent protein mNeonGreen2 and the red fluorescent protein sfCherry2 and protein recombination technology,we constructed mNG2 _1-10/11 and sfCherry2_1-10/11 split fluorescent proteins with low background,high-signal ratio.The specific research contents are as follows.?1?Cloning,expression and characterization of the recombinant protein of AfFtn-DCV.Firstly,the recombinant plasmid pET28a-afftn-dcv was successfully constructed,and the recombinant protein of AfFtn-DCV was obtained by prokaryotic expression and purification in vitro.Subsequently,the purified AfFtn-DCV was characterized,including the protein molecular weight and purity by gel electrophoresis,and the size of the assembled protein by size exclusion chromatography,transmission electron microscopy and dynamic light scattering.These results demonstrated that the recombinant protein can assemble into a nanocage structure at pH=8.0 and disassemble at pH=5.8,and it can covalently link to DNA.In summary,the recombinant protein containing the functions of both AfFtn and DCV,laying the foundation for the subsequent applications.?2?Improvement of the substrate selectivity of DNAzyme.First,we optimized the conditions for the covalent connection of AfFtn-DCV to DNA.Then the relative location of DNA in the assembled structure was explored through the protective effect of AfFtn-DCV on the covalently linked DNA,and it was confirmed that DNA was encapsulated inside the cavity of AfFtn-DCV cage.Finally,the catalytic activity of the DNAzyme covalently linked to AfFtn-DCV was investigated,and it was found that the DNAzyme preferred the positively charged substrate TMB than the negatively charged one ABTS.The protein cage barrier and the electrostatic repulsive force may improve the substrate selectivity of DNAzyme..?3?Construction and preliminary exploration of 2 split fluorescent proteins.First,we constructed pET28a-mng_1-10 and pET28a-sfcherry2_1-10 recombinant plasmids and successfully expressed active mNG2_1-10 and sfCherry2_1-10 in vitro.Then we optimized the recovery conditions and found that sfCherry2_1-10/11 split fluorescent protein showed a high recombinant efficiency of 40.6%.Further,sfCherry2_1-10/11 and sfGFP_1-10/11 split fluorescent proteins were verified as a pair of orthogonal proteins.Therefore,our preliminary exploration of the mNG2_1-10/11 and sfCherry2_1-10/11 split fluorescent proteins lay the foundation for future multicolor imaging applicat ions.