title: Improved methodology to obtain large quantities of correctly folded recombinant N-terminal extracellular domain of the human muscle acetylcholine receptor for inducing experimental autoimmune myasthenia gravis in rats.
abstract: BACKGROUND
  Human myasthenia gravis (MG) is an autoimmune disorder of the neuromuscular system. Experimental autoimmune myasthenia gravis (EAMG) is a well-established animal model for MG that can be induced by active immunization with the Torpedo californica-derived acetylcholine receptor (AChR). Due to the expensive cost of purifying AChR from Torpedo californica, the development of an easier and more economical way of inducing EAMG remains critically needed.
  
  
  METHODS
  Full-length cDNA of the human skeletal muscle AChR α1 subunit was obtained from TE671 cells. The DNA fragment encoding the extracellular domain (ECD) was then amplified by polymerase chain reaction (PCR) and inserted into pET-16b. The reconstructed plasmid was transformed into the host strain BL21(DE3)pLysS, which was derived from Escherichia coli. Isopropyl-β-D-thiogalactopyranoside (IPTG) was used to induce the expression of the N-terminal ECD. The produced protein was purified with immobilized Ni(2+) affinity chromatography and refolded by dialysis.
  
  
  RESULTS
  The recombinant protein was efficiently refolded to soluble active protein, which was verified by ELISA. After immunization with the recombinant ECD, all rats acquired clinical signs of EAMG. The titer of AChR antibodies in the serum was significantly higher in the EAMG group than in the control group, indicating successful induction of EAMG.
  
  
  CONCLUSIONS
  We describe an improved procedure for refolding recombinant ECD of human muscle AChR. This improvement allows for the generation of large quantities of correctly folded recombinant ECD of human muscle AChR, which provides for an easier and more economical way of inducing the animal model of MG.
authors.affiliation: Department of Neurology, Tangdu Hospital, the Fourth Military Medical University, Xi'An, China ; Department of Neurology, PLA Navy General Hospital, Beijing, China.
authors.forename: Chenjing
authors.initials: C
authors.lastname: Sun
authors.affiliation: Department of Neurology, First Hospital of Jilin University, Changchun, China.
authors.forename: Hongliang
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Neurology, Tangdu Hospital, the Fourth Military Medical University, Xi'An, China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Xu
authors.affiliation: Department of Neurology, Tangdu Hospital, the Fourth Military Medical University, Xi'An, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Gao
authors.affiliation: Department of Neurology, PLA Navy General Hospital, Beijing, China.
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Qi
authors.affiliation: Department of Neurology, Tangdu Hospital, the Fourth Military Medical University, Xi'An, China.
authors.forename: Zhuyi
authors.initials: Z
authors.lastname: Li
keywords: acetylcholine receptor; experimental autoimmune myasthenia gravis; extracellular domain; prokaryotic expression; refolding