Arg-rich Peptide Facilitated Soluble Expression Of Recombinant Proteins In E.Coli

E. coli was widely used to express exogenous proteins as its definite genetic map and low cultivate costs, but the proteins expressed in E.coli often in the form of inclusion bodies with high density and insoluble. And they only could be dissolved in denaturing agents like urea or guanidine hydrochloride. After removing the proteins from the denaturing agents, the protein can recover to the normal structure from the degenerative fully extended state, but this process can not ensure the restoration of the biological activity of the protein. In our laboratory, several kinds of engineered porcine circovirus(PCV) capsid protein mutants were high level of soluble expressed in E.coli. By structure analysis, we found that there are large amounts of arginine in the N-terminal of these proteins. So we presumed that it may be the reason why these proteins soluble expression.In order to obtain soluble protein from E.coli, we tried to use the arginine-rich peptide in the N-terminal of the PCV2 b capsid(N41) as a protein antigen fusion peptide, fusing with epitope proteins and expressed in E. coli. Firstly, we obtained the encoding gene of N41 by PCR. Fused the short peptide N41 and PO which consisted of the epitopes of the foot-and-mouth disease virus(FMDV) VP1 capsid protein. And then subcloned into p ET28 a expression vector. PET28a-N41-PO recombinant plasmid was constructed. The fusion protein N41-PO was expressed in E. coli.We found that N41-PO can be partly soluble expressed in E.coli. The target purified protein by nickel affinity chormatography. Then we immunized the ICR mice with purified N41-PO mixed with oil-in-water emulsion at a ratio of 1:1. We detected the antibody in the serum from the mice immunized with N41-PO by indirect enzyme linked immunosorbent assay(ELISA) and found the serum can recognize the inactivity FMDV. The preliminary results showed the N41 peptide can enhance the solubleexpression of exogenous protein with high activity in E.coli.We engineered the N41 fusion protein soluble expression system, to prove the effect of N41 to facilitate the soluble expression of exogenous protein in E.coli. And conducted the fusion proteins consisting of N41 and New VP1 or VP1 OK93 which were expressed inclusion bodies in E.coli. The results showed that both N41-VP1 and N41-VP1 OK93 realized partly soluble expression in E.coli. Without procedure of denaturalization and renaturation in the purification, the exogenous protein could have higher immunological activity. All results indicated that N41 definitely facilitated the soluble expression of exogenous protein in E.coli and the fusion protein had better immunological activity. But there were a problem that expression level of N41 fusion protein in E.coli was low. All of proteins expressed in E.coli were lower than 20 percent. And compared with PO, the expression level of N41-PO was about 50 percent decreased.The Myanmar 98 strain of FMDV started a pandemic in many Asian countries since 2010, and it was the most serious in recent 50 years. It had taken seriously loss to the development of swine industry in China. In order to obtain a high activity and safety vaccine against Myanmar 98 strain of FMDV, we attained its encoding gene of the epitope peptide by PCR and constructed the epitope peptide vaccine N41-H1 with N41 fusion protein soluble expression system. N41-H1 showed completely soluble expression in E.coli and its immunological activity was detected. The results showed that the serum of N41-H1 immunized mice had lower recognition to the inactivity FMDV. To enhance the immunogenicity of the epitope peptide vaccine, we constructed a new epitope peptide vaccine N41-H3 by subclonig the three tandem repeats of H1 genes into the N41 fusion protein soluble expression plasmid. After detecting the antibody level of serum from immunized mice, we found that the serum from mice immunized by N41-H3 could recognize FMDV better.In a word, the arginine rich peptide N41 can promote the soluble expression of exogenous protein in E.coli, and the expressed foreign protein has better activity. But the expression level of N41 fusion proteins in E. coli is low, and there are still needed to make it better.