Development of an enzyme-linked immunoreceptor assay (ELIRA) for quantification of the biological activity of recombinant human bone morphogenetic protein-2.
Human bone morphogenetic protein-2 is a representative of the transforming growth factor-beta (TGF-beta) superfamily of cytokines. It was produced in high-cell-density cultivations of recombinant Escherichia coli leading to the formation of inclusion bodies with aggregated inactive protein so that the protein had to be solubilized and renatured. Thus, the biological activity of the recombinant protein had to be determined. To avoid time-consuming cell-based assays or radioactive labelling of proteins enzyme-linked immunoreceptor assays were developed. They were based on the specific interaction between the biologically active protein and its receptors, of which the extracellular ligand binding domains were tagged with the Fc part of human IgG and expressed in insect cells. The amount of bound ligand, corresponding to the biologically active recombinant protein, was determined via enzyme-labelled antibodies. Application to various batches of protein showed that not only the amount of active protein could be quantified but also the quality of the protein preparations could be evaluated in significantly shorter analysis times than with conventional cell-based assays.
Wendler
Janine
J
Department of Natural Product Biology, National Research Centre for Biotechnology (GBF), Mascheroder Weg 1, 38124 Braunschweig, Germany.
Hoffmann
Andrea
A
Gross
Gerhard
G
Weich
Herbert A
HA
Bilitewski
Ursula
U
D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*
C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; C499469:recombinant human bone morphogenetic protein-2