Exp Mol Med.  1998 Jun;30(2):65-71.

Expression of a recombinant branched chain alpha-oxo acid dehydrogenase complex E2 (BCOADC-E2) in insect cells and its immunoreactivity to autoimmune sera

• Lee SM
• Ko SH
• Park JE
• Cha SH

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.

Keyword

autoantigen; branched chain a-oxo acid dehydrogenase complex (BCOADC); idiopathic cardiomyopathy (IDCM); insect cells; primary biliary cirrhosis (PBC)

MeSH Terms

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*