4. Discussion

The recombinant proteins produced in E.coli often aggregate or degrade rapidly because of their inability to form correct tertiary structures due to anomalies in protein folding (Kim, Kweon, Lee, Park, & Seo, 2005; Nishihara, Kanemori, Yanagi, & Yura, 2000). In this study, we used the chaperone protein pTf16 to increase the expression of VP3 in supernatant and the results showed that the solubility of the protein was significantly improved. In fact, it has been demonstrated that chaperone over-production can slow the rate of protein expression in E.coli by sequestering nascent polypeptides for prolonged periods (Hu et al., 2007). The effect of the combination of the chaperone protein pTf16 with the recombinant vector is similar to the effect of the low temperature and reduced inducer concentration which slows down the production but increases the proportion of functional heterologous protein in the process of recombinant protein production (Hu et al., 2007; Shibui & Nagahari, 1992).
The most important chaperones in E.coli include pTf16, GrpE, GroEL, DnaK, DnaJ, and GroES. The molecular chaperone TF expressed by pTf16 was initially identified as a protein that binds to certain precursor proteins and facilitates their transport to membrane vesicles (Crooke & Wickner, 1987). Some studies have shown that TF may play a role in protein folding because it is related to nascent peptides and 50S ribosomes (Hartl & Hayer-Hartl, 2002; Hesterkamp, Hauser, Lütcke, & Bukau, 1996). In addition, TF is related to GroEL, which can enhance the binding of GroEL substrate and promote protein folding or degradation (Kandror, Sherman, Moerschell, & Goldberg, 1997; Kandror, Sherman, Rhode, & Goldberg, 1995). The effectiveness of these chaperones on protein folding, stability, and aggregation has also been demonstrated (Maeng, Nam, & Kim, 2011; Nishihara, Kanemori, Kitagawa, Yanagi, & Yura, 1998; Veisi et al., 2015). In addition to chaperone proteins, fusion tagging technology also facilitates the expression of soluble proteins in E.coli . Davis et al (Davis, Elisee, Newham, & Harrison, 1999). used the NusA solubilizing label to increase the solubility of bovine growth hormone by up to 90%, De (De Marco, Stier, Blandin, & de Marco, 2004) and his colleagues showed that the fusion protein purified by NusA fusion tag was more stable and higher in content than GST fusion tag.
When selecting the optimal expression vector, we found that the NusA fusion protein also affect the solubilization . Previous studies have reported that the NusA solubilizing tag can significantly improve the solubility of recombinant proteins in E.coli (De Marco et al., 2004; Zacharchenko, Barsukov, Rigden, Bennett, & Mayans, 2016). Considering the size of the fusion protein and the difficulty of subsequent purification, the chaperone pTf16 was finally selected for later experiments.
In addition, during protein purification, we found that the mounting effect of VP3 is not ideal. This may be due to the fact that VP3 is too large, which prevents the enrichment of VP3 on Ni-NTA, resulting in VP3 not fully binding with Ni-NTA. We then incubated the supernatant with the Ni-NTA for 2 h and reduced the flow rate during elution, which improved the VP3 bounding to Ni-NTA to a certain extent. The purity of the VP3 could reach 70% after purification, and it can produce certain immunogenicity. This study lays the foundation for further research on the structure and function of VP3 and other proteins of bluetongue virus. Apart from this, it provided materials for the preparation of BTV-1 VP3 monoclonal antibody.
In conclusion, we expressed four recombinant proteins in E. coli,including His-VP3, His-VP3-Tf, His-VP3-NusA and TRX-His-VP3. Compared with His-VP3 without molecular chaperone, His-VP3 with molecular chaperone pTf16 increased its expression in the supernatant by about 30%. The VP3 purified by Ni-NTA has certain immunogenicity by Dot-ELISA and IFA test.
ACKNOWLEDGEMENTS
This work was supported by the National Key Research and Development Program (2017YFD0502304-4); ‘1125 talent gathering plan’ project of Zhengzhou. We thank Dr. Yankai Liu for revising the grammatical errors and reorganizing this manuscript. Dr. Liu held a doctorate from Queen Mary University of London and now he is an associate professor of bioinformatics at Zhengzhou University.
CONFLICT OF INTEREST
The authors declare that they have no competing interests.
ETHICAL APPROVAL
Ethical statement is not applicable because sample collection has been gathered.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.