The Antigenic Activity And Soluble Expression Of Recombinant Fusion Protein In E.coli

E.coli expression system is the most frequently-used expression system which can express the foreign protein with clear genetic background, reproductive speed, low cost and stable heredity and other advantages, but the foreign proteins are often expressed in inclusion bodies with low activity for the reason of the lack of some post-translational modification enzymes.Ferritin, a soluble protein, is widely presented in vivo. Ferritin can self-assemble into a nanoparticle with 24 subunits. The particle is 10 – 12 nm in diameter. Nanoscale macromolecules have a very strong antigenicity, related studies show that a virus epitope fused with ferritin could self-assemble to a nanoscale epitope protein, the antigen epitope are on the surface of the polymer, meanwhile, the immune activity is enhanced.Heat shock protein 65 is a kind of molecular chaperone proteins. It can maintain and change the conformation of the precursor protein to form a loose structure to help the foreign proteins to assemble correctly and expressed in solubility. Meanwhile, HSP65 is always used for the carrier protein of vaccine with its strong immunological specificity. HSP65 can also transfer the antigen polypeptide to CTL to induce specific immune response. To construct the fusion protein with insoluble target protein and the chaperone protein of HSP65 is a method to increase the solubility of proteins in E.coli.In this study, a recombinant epitope protein fused with ferritin FF was expressed as inclusion bodies in E.coli. We renatured the inclusion bodies of FF by the method of dialysis with arginine or titration with alkaline solution to get the target proteins named FA or FZ. The soluble FF was purified by nickel affinity chromatography to get FN. Three different particles were observed by TEM. FA was appeared as compact hollowed nanoparticles in diameter of 12 nm. FZ was the small loose shape but the nanoscale particles. And FN was formed a particle structure with a diameter slightly larger than the 12 nm, being similar to the natural ferritin nanoparticles. We used three different recombinant proteins to immunize mice / guinea pig, FA can induce high levels antibodies against FMDV in mice and it also has a high immunogenicity. But we did not detect high levels of anti-FMDV antibodies in guinea pigs immunized with FA, it may be due to the differences between the animals or the manipulation of immunization.Because we did not make the epitope soluble with ferritin, we took advantage of HSP65 and its variants to fuse the epitope peptide PO. According to the predicting of 3D structures, we designed and constructed several recombinant proteins. Only the peptide fused with full-length HSP65 or N- and C- terminal are soluble expression, which named HPO or NPOC. We purified the use soluble fraction HPO and NPOC by nickel affinity chromatography and gel filtration chromatography, and also treated the inclusion bodied of NPOC by the method of urea-denaturation to refolding with dialysis or gel filtration chromatography, the pure soluble NPOC proteins purified with diffirent pathway were both obtained and recognized by FMDV specific antiserum in western and ELISA, notably, the signal of NPOC was slightly higher than the groups of PO and HPO. To test whether the antibodies stimulated by these recombinant proteins can recognize nature FMDV, three groups with six female ICR mice each were immunized with 10 ug/mouse, and the serum were collected at the 14 days post second immunization. An ELISA was used to detect immunaogenicity. The result showed that the virus was recognized by all three kinds of sera. Compared with PO group, the results of average value of HPO were substantially the same, but there is larger individual difference in it. The highest average and the minimum individual difference were all appeared in the group of NPOC, showing that the antibodies stimulated by NPOC recognized FMDV most and NPOC may simulate na?ve FMDV most. In order to analyze the better immune effect of NPOC, the 3D structures of natural HSP65 and NPOC were both predicted by SWISS MODEL software, There were mostly α- helix structure in the N terminal and C-terminal part of HSP65, two terminations attracted each other by intermolecular forces, while the middle part was supported in the other side of the protein by two β- sheets. Remarkably, the terminations of NPOC were interacted similarly with that in HSP65. And the mid part replaced with PO epitope is also analogous to that in HSP65, maintaining a separate structure in a side of NPOC. Meanwhile an RGD loop is exposed on the surface of PO epitope. The structure was also modified by www.predictprotein.org website, showing that majority of amino acids exposed on the surface of NPOC was the mid part, corresponding to the 3D structure above.In summary, as a molecular chaperone, ferritin can not make an epitope peptide soluble in E.coli, but through the renaturation process, the ferritin fusion protein can be formed as nanoparticles, what has a good antigenic activity. The fusion protein NPOC flanked with the N- and C- terminal of HSP65 has solved the problem PO inclusion body expression, but also can show the epitope surface helps exert their biological functions. The fusion mode can also be used as an E. coli expression system in recombinant protein solubilization platform, while not affecting the target protein structure and function.