Expression And Refolding Of The Extracellular Domain Of The Human Acetylcholine Receptor α1 Subunit

The nicotinic acetylcholine receptor (nAChR) molecule is an oligomeric transmembrane glycoprotein (Mr~290,000) consisting of five homologous subunits in the stoichiometryα2βγδ(embryonic) orα2βεδ(adult), which form the cation channel. nAChR found at the neuromuscular junction (NMJ) is the autoantigen involved in the autoimmune disease myasthenia gravis (MG).The N-terminal extracellular domain (amino acids 1-210) of theαchain contains both the binding sites for cholinergic ligands and the main immunogenic region (MIR). Anti-MIR antibodies have a high pathogenetic potential, because they are very efficient in causing AChR loss in muscle cell cultures and their injection into rats causes severe myasthenic symptoms. The MIR is clearly not a single conformationally dependent antigenic region in antigen modulation, but rather a group of mutually overlapping epitopes. Because of too few amino acid residues, only approximately half of all anti-MIR mAb bind and the weak immunogenicity, injection of the MIR epitope is not a good choice for the development of experimental MG. Usually, EAMG can be induced by passive transferring of serum from MG patients or by active immunization with Torpedo Californica AChR. Because of both the limit amount of the serum collected from the MG patients, and the huge difficulty of catching Torpedo Californica, as well as the high complexity and low efficiency of the AChR purification from Torpedo Californica, it is so difficult to induce quantities of animal models of EAMG. Therefore, it is urgent and significant to develop a simpler method to induce EAMG.Early studies demonstrated that anti-AChR Ab are high affinity IgG: therefore both B and CD4+ T cells are involved in the pathogenesis of the disease. The ECD of AChR, which includes both the binding sites for MIR and T and B cells epitopes, can be utilized as immunogen to stimulate the growth of Ag-specific T cells and fix the complement. So it is realizable to induce EAMG by the extracellular regionα1-207 of the human muscle AChR.The human neuromedulloblastoma cell line TE671 is shown to express functional nicotinic acetylcholine receptors (AChRs) that they do react with autoantibodies to muscle AChRs from myasthenia gravis patients and with mAbs to muscle AChRs. Since that theαsubunit of AChRs purified from TE671 cells corresponds to that of muscle AChR, the ECD protein of TE671 AChRαsubunit predicted from a genomic clone can be used to induce EAMG, instead of human muscle AChRs. At the beginning of 1990s, Talib obtained the coding sequence representing the ECD of AChRαsubunits (aa 1-210) by genic technology and, further, express the denes in Escherichia coli. With the expression product, Lennon induced successfully the Lewis rats of EAMG. Currently, the increasing development of molecular biology has driven the highly-developed molecular cloning and recombinant expressing methods. In our study, on the basis of Talib's work, the experiment methods were improved to clone aimed fragment by directly purifying the cDNA sequences of theα subunit of AChRs from TE671 cells.The full length cDNA of human skeletal muscle AChRα1 subunit was amplified by RT-PCR from TE671 cells. The DNA fragment encoding ECD was amplified by PCR and inserted into the prokaryotic expression vector pET-16b. The reconstructed plasmid was transformed into the host strain BL21(DE3)pLysS. Protein expression was induced by IPTG. The expressed protein was purified by immobilized Ni2+ affinity chromatography and refolded by dialysis. The refolded protein was identified by ELISA.The results demonstrated that a 650bps band was amplified which was as expected. Sequencing results of the reconstructed plasmid showed no mutation or frame shift. Comparing the amount of the protein induced by IPTG with the initial one without induction using SDS-PAGE method, we found that the IPTG method increased obviously the amount of the protein. Furthermore, the inclusion bodies were resolved and refolded. The identity of the refolded protein was verified by ELISA probed with a specific antibody mab35.In conclusion, it is feasible to obtain large quantities of correctly folded recombinant ECD of human muscle AChR, which contributes to a much simpler, faster and cheaper EAMG inducing process, and further, greatly facilitates the studies on the therapy of MG.