title: Trigger factor assisted soluble expression of recombinant spike protein of porcine epidemic diarrhea virus in Escherichia coli.
abstract: 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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea.
authors.forename: Da-Chuan
authors.initials: DC
authors.lastname: Piao
authors.affiliation: Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea.
authors.forename: Do-Woon
authors.initials: DW
authors.lastname: Shin
authors.affiliation: Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea.
authors.forename: In-Seon
authors.initials: IS
authors.lastname: Kim
authors.affiliation: Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea.
authors.forename: Hui-Shan
authors.initials: HS
authors.lastname: Li
authors.affiliation: Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea.
authors.forename: Seo-Ho
authors.initials: SH
authors.lastname: Oh
authors.affiliation: Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea.
authors.forename: Bijay
authors.initials: B
authors.lastname: Singh
authors.affiliation: Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea.
authors.forename: S
authors.initials: S
authors.lastname: Maharjan
authors.affiliation: Institute of Green-Bio Science & Technology, Seoul National University, Pyeongchang-gun, 25354, Republic of Korea.
authors.forename: Yoon-Seok
authors.initials: YS
authors.lastname: Lee
authors.affiliation: Institute of Green-Bio Science & Technology, Seoul National University, Pyeongchang-gun, 25354, Republic of Korea.
authors.forename: Jin-Duck
authors.initials: JD
authors.lastname: Bok
authors.affiliation: Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea.
authors.forename: Chong-Su
authors.initials: CS
authors.lastname: Cho
authors.affiliation: Department of Animal Science, Tianjin Agricultural University, Tianjin, 300-384, People's Republic of China.
authors.forename: Zhong-Shan
authors.initials: ZS
authors.lastname: Hong
authors.affiliation: Institute of Green-Bio Science & Technology, Seoul National University, Pyeongchang-gun, 25354, Republic of Korea. kangsk01@snu.ac.kr.
authors.forename: Sang-Kee
authors.initials: SK
authors.lastname: Kang
authors.affiliation: Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 08826, Republic of Korea. cyjcow@snu.ac.kr.|Department of Animal Science, Tianjin Agricultural University, Tianjin, 300-384, People's Republic of China. cyjcow@snu.ac.kr.
authors.forename: Yun-Jaie
authors.initials: YJ
authors.lastname: Choi
mesh_terms: D000818:Animals; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000235:genetics*; D005260:Female; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D019696:Peptidylprolyl Isomerase / Q000235:genetics*; D053485:Porcine epidemic diarrhea virus / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D012995:Solubility; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*
chemical_list: D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; C403324:trigger factor, E coli; D019696:Peptidylprolyl Isomerase
keywords: Chaperone co-expression system; Inclusion bodies; PEDV; Spike glycoprotein; Subunit vaccine; Trigger factor