Purification Of An E.coli Expressed Recombinant Protein Consisting Of Multiple Epitopes Derived From Bovine Asial FMDV

Foot-and-mouth disease (FMD) is a acute, fevered and highly contagious disease affecting cloven-hoofed animals and can long-distance transmission. The incidence rate of FMD is 100 percent, can result in death of young stock, but recovery of adult animals. Because foot and mouth disease spreads rapidly, difficult to control, and has less remedial measures, so be called "number one killer of animal husbandry", a very serious harm. Therefore, FMD is listed on the A list of animals infectious diseases by the Office International des E'pizooties (OIE).FMD is caused by infection of Foot-and-mouth disease virus (FMDV) which gives FMD transmission capability because of its great viability and infectious ability. FMDV is the type species of the Aphthovirus genus of the Picornaviridae family. FMDV consists of a single-stranded, plus-sense RNA genome surrounded by four structural proteins to form an icosahedral capsid. Seven serotypes (A, O, C, AsiaⅠ, and SATⅠ,Ⅱ,Ⅲ) and multiple subtypes have been identified. And there is no cross immune reaction among these serotypes and subtypes. It is found that the VP1, a structural protein on the surface of FMDV, determines the antigenicity. The conserved triplet Arg-Gly-Asp (RGD) motif in a G-H loop on the VP1 is the core structure for the virus to host cells. In addition, VP1 contains B and T cells epitope that can induce protective neutralizing antibodies in susceptible animals against FMDV.At present, the disease prevention method is immunizing susceptible animals with vaccines. Our group has been developing a novel bovine recombinant protein containing B and T epitopes as AsiaⅠFMDV vaccine, called rA7. Protein rA7 expresses in E.coli as inclusion body. By the prediction, the molecular weight of rA7 is 48KD and isoelectric point is 11.04. 421 amino acids contained in rA7, but no cysteine. In addition, 6×His-tag exists on both of N-terminal and C-terminal to facilitate the use of nickel affinity chromatography for purification. Protein rA7 has been successfully expressed and purified, but couldn't protect bovine suffering from challenge with FMDV after immunization. The reason maybe that protein rA7 is at a state of denaturation or not complete refolding, so couldn't fold into its spatial structure and exert its biological activity.Inclusion body protein refolding in vitro is the most critical and most difficult step in genetically engineered protein production. Refolding methods of inclusion body commonly include dilution, dialysis, ultrafiltration and chromatography method. Protein refolding by chromatography has merits of velocity, easy amplification and little sample dilution factor. So it was frequently studied and successfully applied in production recombinant protein. The chromatography methods to refold protein divided into affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, expanded bed adsorption and gel filtration chromatography. Refolding by gel filtration chromatography (SEC) is selecting different molecular weight materials through the voids between the gel particles. Macromolecular protein gradually separate with denaturant because of faster transfer. And the protein diffuse in gel at a slower rate as proteins start folding. weakened interactions between protein molecules inhibit protein aggregation and to be helpful for protein renaturation. SEC method is applied to refold variety denatured proteins because it's easy to operate and amplify, and the recovery of activated protein is higher. So, the SEC method developed relatively fast in recent years.There are many factors influences the refolding of proteins. Apart from the nature of protein itself, factors such as protein concentration, denaturant concentration and removal rate, pH, temperature, additives and other factors all affect the protein folding for the right active conformation . Therefore, it is important to explore the best refolding conditions for protein renaturation. In this study, in order to obtain activated recombinant protein rA7, we observe the extent of protein polymerization to investigate the best conditions for protein refolding via diluting denatured proteins. Then use Ni2 + affinity chromatography to purify histidine-containing protein rA7 and applicate gel filtration chromatography to refold purified protein rA7. Finally the activity of the recombinant protein rA7 is tested by detecting FMDV neutralizing antibody in bovine serum after immunization.The dilution experiment indicated that: 1) Protein rA7 tend to polymerize when the urea decreases at a lower concentration; 2) Protein rA7 containing KCl at a lower concentration can reduce protein aggregation; 3) low pH values can reduce protein rA7 aggregation; 4) Protein rA7 is stable when the dilute solutions contain less anionic charge. Therefore, we determined the refolding solution as 20mmol/L Tris, 50mmol/L NaCl, 0.5mol/L urea, pH 7.0 to refold protein rA7. The lower flow rate is also taken in the course of protein renaturation by SEC to assist protein rA7 refolding. Finally, we obtain the protein solution contains 0.5mol/L urea, and purity of protein is above 95%.To estimate the activity of protein rA7, we inoculated cattle at doses of 2400μg. the experiment simultaneously set up Bovine AsiaⅠ/O FMDV bivalent inactivated vaccine and protein diluent as positive and negative control, respecticvely. Then sera were collected on day 21 after the first and second inoculation, and neutralizing antibodies were detected by cell protective assays and liquid phase blocking ELISA. The results demonstrated that proten rA7 containing 0.5mol/L urea could elicit strong anti-FMDV neutralizing antibodies, and the titers of the vaccine induced antibody were positive to correlated to the time of immunization. On day 28 after the first and second inoculation, cattle were challenged by AsiaⅠFMDV; And on day 10 after post-challenge, cattle with first inoculation were all protected, and cattle with second inoculation developed vesicles on the feet, the characteristic FMD syndrome. the results indicated that the protein rA7 containing 0.5mol/L urea had strong biological activity and could protect cattle against FMDV challenge with immunized once.On the basis of experimental results about rA7 function, we rethought about refolding and purification of protein rA7 and found that to obtain activated protein, we should pay close attention to two pionts bellow. First, The concentration of denaturant should be low enough. In this study, the protein rA7 exposed poor activity at 1mol/L urea concentration, while the urea concentration fell to 0.5mol/L, protein rA7 showed a strong protection to the cattle immunized. The results indicated that the denaturant should be at a suitable concentration in protein solution to make better solubility and refolding completely. Second, protein should be in a suitable environment helpful for refolding. Before using the best refolding conditions, we could only produced clarified protein rA7 solution containing 1mol/L urea, but through the dilution method to determine the best refolding conditions, we have been purified clarified protein solution containing 0.5 mol/L urea and confirmed its good activity by functional experiments.The study presents a simple dilution method to determine the best refolding conditions, and simultaneously study the main factors influencing protein refolding. We successfully purified and refolded protein rA7 by Ni2 + affinity chromatography and gel filtration chromatography at the conditions ditermined, respectively. Hope that the method proposed in this study can be useful for protein expressed in the form of inclusion bodies or even provide some ideas for that.