Demonstration of the interaction of transforming growth factor beta 2 and type X collagen using a modified tandem affinity purification tag

Like other members of the transforming growth factor beta (TGF-β) family of growth factors, the biological activity of TGF-β2 is believed to be regulated by the formation and dissociation of multiprotein complexes. To isolate the molecular complex formed by TGF-β2 secreted by hypertrophic chondrocytes we have used expression of TGF-β2 fused with the humanized, tandem affinity purification (hTAP) tag and mass spectrometry for the identification of interacting proteins. The hTAP synthetic gene was assembled by systematically replacing the rare codons of the original TAP tag with codons most preferred in highly expressed human genes to circumvent the poor translation efficiency of the original TAP tag in animal cells. TGF-β2 was shown to interact with Type X collagen and this interaction confirmed using V5 tagged TGF-β2. Functional interaction was suggested by the inhibition of TGF-β2 activity by type X collagen in culture and the influence of a mutation in type X collagen on the distribution of TGF-β2 in growth cartilage.