Highly purified oligo-His tagged human recombinant alpha(1)-AChR is immunogenic in vivo and suitable for T cell stimulation in vitro in experimental and human myasthenia gravis.
Using recombinantly expressed proteins for selection of antigen-specific T cell lines carries a high risk of selecting T cells specific for contaminating proteins. This risk is especially high for very hydrophobic proteins which are notoriously difficult to purify, such as the integral membrane protein acetylcholine receptor (AChR). We prepared a highly purified recombinant AChR by adding an oligo-histidine affinity-tag to the human alpha(1)-AChR and expressing it in E. coli. This allowed purification by Ni-NTA chromatography and subsequent electroelution from preparative SDS gel as purification steps, resulting in complete purity as assessed by silver stain on SDS-PAGE. This protein preparation induced fatal experimental allergic myasthenia gravis in Lewis rats. Furthermore, the protein could be used to select T cell lines from immunized Lewis rats and patients with myasthenia gravis. However, even with this highly purified protein, one of 8 Lewis rat T cell lines and 3 of 7 human T cell lines cross-reacted to E. coli control proteins. The results show that oligo-histidine tagged, highly purified human alpha(1)-AChR is highly immunogenic in vivo and in vitro.
Voltz
R
R
Department of Neuroimmunology, Max-Planck-Institute, Planegg-Martinsried, Germany. rvoltz@ski.mskcc.org
Kamm
C
C
Padberg
F
F
Malotka
J
J
Kerschensteiner
M
M
Spuler
S
S
Tzartos
S
S
Dornmair
K
K
D000345:Affinity Labels; D000818:Animals; D001323:Autoantibodies / Q000097:blood; D002460:Cell Line; D005260:Female; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D008213:Lymphocyte Activation / Q000187:drug effects / Q000235:genetics; D009157:Myasthenia Gravis / Q000209:etiology / Q000235:genetics / Q000276:immunology*; D015345:Oligonucleotide Probes / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew; D011950:Receptors, Cholinergic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000276:immunology* / Q000302:isolation & purification; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism
D000345:Affinity Labels; D001323:Autoantibodies; D015345:Oligonucleotide Probes; D011950:Receptors, Cholinergic; D011994:Recombinant Proteins; D006639:Histidine