Expression of a recombinant branched chain alpha-oxo acid dehydrogenase complex E2 (BCOADC-E2) in insect cells and its immunoreactivity to autoimmune sera.
Preparation of a pure autoantigen by way of recombinant DNA technology has an important value in an accurate diagnosis or prognosis of an autoimmune disease. BCOADC-E2 subunit, a mitochondrial protein, has been known to be the autoantigen of primary biliary cirrhosis (PBC), a chronic autoimmune liver disease, as well as idiopathic dilated cardiomypathy (IDCM), a chronic autoimmune heart disease. Recombinant form of this molecule had been expressed in E. coli but with low yield and severe degradation. Furthermore, sera from IDCM patients failed to recognized BCOADC-E2 molecule produced in prokaryotic expression system. In this study, a recombinant bovine BCOADC-E2 fusion protein has been expressed in insect cells using baculovirus expression system and analyzed anti-BCOADC-E2 reactivity in sera from patients with PBC or with IDCM. Optimal production of the recombinant fusion protein has been achieved at 20 multiplicity of infection (MOI), and the protein was affinity-purified using metal-binding resins. The affinity-purified BCOADC-E2 protein was successfully recognized by sera from PBC patients, but not by sera from IDCM patients suggesting that the different auto-immune response against BCOADC-E2 is needed to be elucidated in terms of epitope recognition.
Lee
S M
SM
Kangwon National University, College of Agriculture & Life Sciences, Division of Food and Biotechnology, Chuncheon, Korea.
Ko
S H
SH
Park
J E
JE
Cha
S H
SH
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000123:Acetyltransferases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002311:Cardiomyopathy, Dilated / Q000276:immunology; D002417:Cattle; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D006801:Humans; D007106:Immune Sera; D007313:Insecta / Q000166:cytology*; D007658:Ketone Oxidoreductases / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D008105:Liver Cirrhosis, Biliary / Q000276:immunology; D009097:Multienzyme Complexes / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
D007106:Immune Sera; D009097:Multienzyme Complexes; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase