Heterologous Expression And Purification Of Enterovirus 71 2C Soluble Protein

Purpose: Bio-informatics methods and different expression vectors were used to express the EV71 2C protein. Soluble proteins with high purity, uniform quality and corrected folding were obtained for further crystal screening.Methods:1. Analysis of bioinformatics characteristics: EV71(200804 strain) 2C protein amino acid and gene sequence were downloaded from the NCBI database. Softwares such as Protparam, Protean, Top Pred 2 service, Pfam and SMART were used to analyze the basic characteristics and secondary class structure, hydrophilicity and core domains as well. Six different lengths of fragments were determined and designed according to the analysis of hydrophobic property and distribution of core domain.2. Gene clone of the recombinant 2C truncated fragments of EV71: EV71 full-length genome, A12 plasmid, was used as a template. The corresponding primers were designed as follows: 2C-91aa-Bam HI-5, 2C-118aa-Bam HI-5, 2C-256aa-Xho I-3T, 2C-296aa-Xho I-3T,2C-316aa-Xho I-3T. The six intercepted 2C fragments were amplified and then ligated onto p Gl01 and psj8 vectors, respectively. Twelve recombinant plasmids comprised of p Gl01vector-2C truncated fragments and psj8 vector-2C ones were constructed successfully which named as P1 to P6 and M1 to M6 respectively. Following the identification of enzyme digestion and exact sequencing, the engineering bacteria obtained after transforming into E.Coli BL21(DE3) and inducing by IPTG.3. Expression, purification and crystal screening of EV71 truncated 2C protein: The engineering bacteria were cultivated to the optical density value between 0.6 to 0.8, the0.6m M IPTG was used to induce the plasmid expression at 20℃ for 16 hours. After the bacteria were collected, ultra-sonication and high-speed centrifugation were performed at low temperature to obtain supernatant. The crude extracts of p Gl01-2C truncated proteins were loaded onto the nickel ion affinity chromatography column for a basic purification. The samples were eluted by elution buffer containing imidazole. The crude extracts of psj8-2C ones were loaded onto the MBP Trap?HP column and then eluted by elution buffer containing imidazole and 10 m M maltose. The samples were collected as follows: I. The ultrasonic broken bacteria(CE). II. High speed centrifugal supernatant(S). III. Protein samples flowed through the nickle column(FL). IV. Protein samples washed by suspension buffer(W). V. Protein samples eluted by elution buffer(ELU). SDS-PAGE electrophoresis was used for identifying and analyzing the expression as well as solubility of proteins. The targeted proteins on SDS-PAGE with the single and distinct bands of CE and ELU were figured out as M1 and M4, and then these protein isolates were further purified by gel filtration affinity chromatography. The final purified proteins were screened for crystals growing using sitting drop method.Results:1. The designation of EV71 2C fragments: Six fragments were intercepted according to the bioinformatics analysis. Core domains such as ATPase and RNA helicase were included all truncated ones. AWS domain was also designed in the truncated ones. Unless the above described, some special domains such as N terminals and C terminals including middle amino acid region were distributed among different fragments. For the detailed information please see table 2.2. Heterologous expression of the recombinant 2C truncated fragments of EV71: Six fragments of EV71 2C were amplified by PCR successfully and then ligated onto p Gl01 vector and psj8 vector respectively. Twelve recombinant plasmids comprised of six p Gl01-2C and six psj8-2C plamids were successfully constructed. The engineering bacteria were obtained following transforming into E.coli BL21(DE3) and inducing by IPTG.3. Purification and crystal screening of EV71 truncated 2C protein:According to the SDS-PAGE results, the 2C proteins containing p Gl01 and psj8 vector were all expressed in E.coli BL21(DE3). The molecular weight is conformed to expected values. The p Gl01-2C proteins were hard dissolved, while the psj8-2C proteins fused with maltose binding proteins were of solubility. The result of gel filtration affinity chromatography showed that the truncated proteins with fragment M1 and M4 are of higher purity and homogeneity. Unfortunately, we could not see a crystal growing by fine screening.Conclusions:In the present study, gene clone, heterologous expression and purification were successfully performed. Twelve recombinants of 2C intercepted fragments were constructed. Proteins with high purity, homogeneity and solubility were obtained. We conclude that the truncated proteins of psj8 vector-2C: M1 and M4(91aa-256 aa and 118aa-256aa) are more stable than the other ones used in this study. Additionally, maltose binding protein plays a critical role in improving the solubility of 2C protein.