Expression of a recombinant branched chain alpha-oxo acid dehydrogenase complex E2 (BCOADC-E2) in insect cells and its immunoreactivity to autoimmune sera.
- Author:
Sang Mi LEE
1
;
Soon Hee KO
;
Jeong Eu PARK
;
Sang Hoon CHA
Author Information
1. Kangwon National University, College of Agriculture & Life Sciences, Division of Food and Biotechnology, Chuncheon, Korea.
- Publication Type:Original Article ; Research Support, Non-U.S. Gov't
- Keywords:
autoantigen;
branched chain a-oxo acid dehydrogenase complex (BCOADC);
idiopathic cardiomyopathy (IDCM);
insect cells;
primary biliary cirrhosis (PBC)
- MeSH:
Acetyltransferases/metabolism;
Acetyltransferases/immunology;
Acetyltransferases/genetics*;
Animal;
Baculoviridae/genetics;
Cardiomyopathy, Congestive/immunology;
Cattle;
Human;
Immune Sera;
Insects/cytology*;
Ketone Oxidoreductases/metabolism;
Ketone Oxidoreductases/immunology*;
Ketone Oxidoreductases/genetics*;
Liver Cirrhosis, Biliary/immunology;
Multienzyme Complexes/metabolism;
Multienzyme Complexes/immunology*;
Multienzyme Complexes/genetics*;
Protein Engineering/methods;
Recombinant Proteins/isolation & purification;
Recombinant Proteins/immunology;
Recombinant Proteins/genetics*
- From:Experimental & Molecular Medicine
1998;30(2):65-71
- CountryRepublic of Korea
- Language:English
-
Abstract:
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.
