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 status of bone metabolism and it is used to monitor various bone-related diseases. The aim of this study was to produce recombinant human osteocalcin (rhOC) in Escherichia coli and use it for designing new osteocalcin fluorescence immunoassays. Recombinant DNA technology was used to fuse synthetic hOC coding sequences to an affinity handle system based on glutathione S-transferase (GST) gene. GST-rhOC fusion protein was produced in a bacterial intracellular expression system mainly in a soluble form. The affinity-purified fusion protein was cleaved with activated protease factor X releasing the rhOC portion. The structure of rhOC was confirmed by mass spectrometry and amino acid sequencing. The fusion protein and its proteolytic cleavage product proved to be immunoreactive as shown by Western blotting analysis and by a new osteocalcin immunoassay based on time-resolved fluorescence. When osteocalcin was tested for its ability to bind to hydroxyapatite, there were no differences between the recombinant forms and native human osteocalcin purified from bone, suggesting that the Gla residues might be important only in oriented high-affinity binding.
Käkönen
S M
SM
Department of Biotechnology, University of Turku, Turku, FIN-20520, Finland.
Hellman
J
J
Pettersson
K
K
Lövgren
T
T
Karp
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D001842:Bone and Bones / Q000378:metabolism; D017886:Durapatite / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005170:Factor X / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015675:Osteocalcin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017421:Sequence Analysis
D011993:Recombinant Fusion Proteins; D015675:Osteocalcin; D005170:Factor X; D017886:Durapatite