ABSTRACT
The conditions for heterologous expression of recombinant bovine adrenodoxin in E. coli have been optimized, thus reaching expression levels up to 12-14 micromoles per liter of culture medium. A highly efficient method for purification of this recombinant ferredoxin from the E. coli cells has been developed. The structural-functional properties of the highly purified recombinant protein have been characterized and compared to those of natural adrenodoxin purified from bovine adrenocortical mitochondria. In contrast to natural adrenodoxin, which is characterized by microheterogeneity, the recombinant adrenodoxin is homogeneous as judged by N- and C-terminal amino acid sequencing, and its sequence corresponds to the full-length mature form of adrenodoxin containing 128 amino acid residues. The interactions of the natural and recombinant adrenodoxins with cytochrome P450scc have been studied and compared with respect to: the efficiency of their enzymatic reduction of cytochrome P450scc in a reconstituted system; the ability of the immobilized adrenodoxins to bind cytochrome P450scc; the ability of the adrenodoxins to induce a spectral shift of cytochrome P450scc and to effect the average polarity of exposed tyrosines in the low-spin cytochrome P450scc. The recombinant adrenodoxin is functionally active and in the reduced state as well as at low ionic strength it displays higher affinity to cytochrome P450scc as compared to the natural bovine adrenocortical adrenodoxin. The possible role of the C-terminal sequence of the adrenodoxin molecule in its interaction with cytochrome P450scc as well as the advantages of using the recombinant protein instead of the natural one are discussed.
Subject(s)
Adrenodoxin/chemistry , Adrenodoxin/metabolism , Cholesterol Side-Chain Cleavage Enzyme/metabolism , Adrenodoxin/isolation & purification , Amino Acid Sequence , Animals , Cattle , Cholesterol Side-Chain Cleavage Enzyme/isolation & purification , Chromatography, Ion Exchange , Cloning, Molecular , Escherichia coli , Ferredoxin-NADP Reductase/isolation & purification , Ferredoxin-NADP Reductase/metabolism , Kinetics , Molecular Sequence Data , Peptide Fragments/chemistry , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Structure-Activity RelationshipABSTRACT
Three monoclonal antibodies to human spleen ferritin were produced and their interaction with soluble and immobilized ferritins studied. An immunoassay was developed to monitor the interaction of soluble biotinylated ferritin and the antibody with subsequent separation of the soluble complexes on streptavidin-cellulose. Analysis of immunoreactivities of a series of isoferritins (human liver, spleen, heart and equine spleen ferritins) revealed that all the three monoclonal antibodies bound to only human L-type ferritins (spleen and liver ferritins), suggesting a high species- and tissue specificity of these antibodies. The monoclonal antibodies were specifically directed against conformation-dependent antigenic determinants as could be evidenced from the lack of their binding to the subunits of the dissociated ferritin. The affinity of the monoclonal antibody for the ferritin deprived of iron (apoferritin) was higher than that for native ferritin due to the greater conformational flexibility of the apoferritin molecule. The latter property may underly a complete loss of immunoreactivity by the apoprotein adsorbed on the polystyrene surface as a result of conformational changes induced in the apoferritin molecule by adsorption. These findings provide additional support for recognition by all of the three antibodies of conformational antigenic epitopes in the ferritin molecule.
Subject(s)
Antibodies, Monoclonal/immunology , Apoferritins/immunology , Ferritins/immunology , Spleen/metabolism , Antibodies, Monoclonal/isolation & purification , Antibody Specificity , Antigen-Antibody Reactions , Cross Reactions , Ferritins/metabolism , HumansABSTRACT
A radioimmunoassay to determine a titer of autoantibodies to thyroglobulin in human blood serum was developed. The scheme of analysis included a reaction between 125I-thyroglobulin and autoantibodies to thyroglobulin with subsequent separation of the produced immune complex and free polyethylene glycol-labeled thyroglobulin. A high specificity of the radioimmunoassay is ensured by the use of highly purified thyroglobulin for obtaining the labeled agent. A possibility of the use of the method for the diagnosis of autoimmune thyroid diseases was shown.