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.
Highlights
Branched α-oxo acid dehydrogenase complex, located in the matrix of mitochondria, is involved in the catabolism of the decarboxylation of alpha keto acids originated from transamination of the branched chain amino acids such as valine, isoleucine and leucine (Griffin et al, 1989), and composed of three catalytic subunits; branched αoxo acid decarboxylase (E1), dihydrolipoyl transaminase (E2) and dihydrolipoyl dehydrogenase (E3)
The recombinant baculovirus transfer vector was introduced with linearized baculovirus DNA into Sf9 insect cells using lipofectin as described in Materials and Methods. pAcGHLT baculovirus transfer vector was used since the recombinant protein can be produced as a fusion protein containing GST and poly (His) tag, thereby, facilitating the purification of a foreign protein based upon specific affinity binding
In order to determine the optimal multiplicity of infection (MOI) for the expression of the fusion protein, Sf9 insect cells were infected with the recombinant baculovirus at media containing 5ለ106 (MOI 5), 10, 15 or 20, and the cellular proteins were analyzed by immunoblot using mouse anti-branched chain α-oxo acid dehydrogenase complex (BCOADC)-E2 monoclonal antibody (5AC12C1) (Figure 3)
Summary
Branched α-oxo acid dehydrogenase complex, located in the matrix of mitochondria, is involved in the catabolism of the decarboxylation of alpha keto acids originated from transamination of the branched chain amino acids such as valine, isoleucine and leucine (Griffin et al, 1989), and composed of three catalytic subunits; branched αoxo acid decarboxylase (E1), dihydrolipoyl transaminase (E2) and dihydrolipoyl dehydrogenase (E3). Deficiency of this enzyme complex results in inherited maple syrup urine disease (MSUD) manifested by ketoacidosis, neurological disorders and mental retardation (Danner and Elsas, 1989).
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