Trigger factor assisted soluble expression of recombinant spike protein of porcine epidemic diarrhea virus in Escherichia coli
Posted on 2016-05-04 - 05:00
Abstract Background Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. The spike glycoprotein (S) of PEDV is the major immunogenic determinant that plays a pivotal role in the induction of neutralizing antibodies against PEDV, which therefore is an ideal target for the development of subunit vaccine. In an attempt to develop a subunit vaccine for PEDV, we cloned two different fragments of S protein and expressed as glutathione S-transferase (GST)-tagged fusion proteins, namely rGST-COE and rGST-S1D, in E.coli. However, the expression of these recombinant protein antigens using a variety of expression vectors, strains, and induction conditions invariably resulted in inclusion bodies. To achieve the soluble expression of recombinant proteins, several chaperone co-expression systems were tested in this study. Results We firstly tested various chaperone co-expression systems and found that co-expression of trigger factor (TF) with recombinant proteins at 15 °C was most useful in soluble production of rGST-COE and rGST-S1D compared to GroEL-ES and DnaK-DnaJ-GrpE/GroEL-ES systems. The soluble rGST-COE and rGST-S1D were purified using glutathione Sepharose 4B with a yield of 7.5 mg/l and 5 mg/l, respectively. Purified proteins were detected by western blot using mouse anti-GST mAb and pig anti-PEDV immune sera. In an indirect ELISA, purified proteins showed immune reactivity with pig anti-PEDV immune sera. Finally, immunization of mice with 10 μg of purified proteins elicited highly potent serum IgG and serum neutralizing antibody titers. Conclusions In this study, soluble production of recombinant spike protein of PEDV, rGST-COE and rGST-S1D, were achieved by using TF chaperone co-expression system. Our results suggest that soluble rGST-COE and rGST-S1D produced by co-expressing chaperones may have the potential to be used as subunit vaccine antigens.
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Piao, Da-Chuan; Shin, Do-Woon; Kim, In-Seon; Li, Hui-Shan; Oh, Seo-Ho; Singh, Bijay; et al. (2016). Trigger factor assisted soluble expression of recombinant spike protein of porcine epidemic diarrhea virus in Escherichia coli. figshare. Collection. https://doi.org/10.6084/m9.figshare.c.3620201.v1
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AUTHORS (13)
DP
Da-Chuan Piao
DS
Do-Woon Shin
IK
In-Seon Kim
HL
Hui-Shan Li
SO
Seo-Ho Oh
BS
Bijay Singh
SM
S. Maharjan
YL
Yoon-Seok Lee
JB
Jin-Duck Bok
CC
Chong-Su Cho
ZH
Zhong-Shan Hong
SK
Sang-Kee Kang
YC
Yun-Jaie Choi