The isolation and purification of biologically active recombinant and native autoantigens for the study of autoimmune disease.
The expression of recombinant, biologically active mouse myeloperoxidase (MPD) and the recombinant non-collagenous (NC1) domain of mouse collagen alpha 3 Type IV was achieved for the first time in Sf21 cells (Spodoptera frugiperda ovarian insect cells) using a baculovirus expression system. Following purification, the proteins were identified by reducing and non-reducing SDS-PAGE electrophoresis. Recombinant mouse MPO has a molecular weight of approximately 90 kDa and mouse alpha3(IV)NC1 approximately 32 kDa. In addition, milligram quantities of native mouse myeloperoxidase were purified from 32Dcl3 cells. Both native and recombinant myeloperoxidase were biologically active. This study also demonstrated that the immunization of myeloperoxidase deficient (Mpo-/-) mice with purified recombinant mouse myeloperoxidase induced a significant antibody response to native myeloperoxidase.
Apostolopoulos
Jim
J
Centre for Inflammatory Diseases, Monash University Department of Medicine, Level 5 Block E, Monash Medical Centre, 246 Clayton Rd., Clayton, 3168 Victoria, Australia. jim.apostolopoulos@med.monash.edu.au
Ooi
Joshua D K
JD
Odobasic
Dragana
D
Holdsworth
Stephen R
SR
Kitching
A Richard
AR
D000818:Animals; D001324:Autoantigens / Q000235:genetics / Q000302:isolation & purification*; D001327:Autoimmune Diseases / Q000209:etiology / Q000276:immunology*; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D024141:Collagen Type IV / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D018076:DNA, Complementary / Q000235:genetics; D005921:Glomerulonephritis / Q000209:etiology / Q000276:immunology; D007114:Immunization; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D009195:Peroxidase / Q000172:deficiency / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D018411:Spodoptera
D001324:Autoantigens; D024141:Collagen Type IV; D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D009195:Peroxidase