PURIFICATION AND CHARACTERIZATION OF RECOMBINANT OSTEOCALCIN FUSION PROTEIN EXPRESSED IN ESCHERICHIA-COLI

Human osteocalcin (hOC) is a 49-amino-acid peptide produced mainly by bone osteoblasts. The amount of hOC in the circulation reflects the st atus of bone metabolism and it is used to monitor various bone-related diseases. The aim of this study was to produce recombinant human oste ocalcin (rhOC) in Escherichia coli and use it for designing new osteoc alcin fluorescence immunoassays. Recombinant DNA technology was used t o fuse synthetic hOC coding sequences to an affinity handle system bas ed on glutathione S-transferase (GST) gene. GST-rhOC fusion protein wa s produced in a bacterial intracellular expression system mainly in a soluble form. The affinity-purified fusion protein was cleaved with ac tivated protease factor X releasing the rhOC portion. The structure of rhOC was confirmed by mass spectrometry and amino acid sequencing. Th e fusion protein and its proteolytic cleavage product proved to be imm unoreactive as shown by Western blotting analysis and by a new osteoca lcin immunoassay based on time-resolved fluorescence. When osteocalcin was tested for its ability to bind to hydroxyapatite, there were no d ifferences between the recombinant forms and native human osteocalcin purified from bone, suggesting that the Gla residues might be importan t only in oriented high-affinity binding. (C) 1996 Academic Press, Inc .