Expression and purification of biologically active IGF-binding proteins using the LCR/ XXXX expression system.

@entity10246 @entity526 @entity7423 @entity10247 @entity7424 @entity28690 @entity10250
The anabolic effects and bioavailability of insulin-like growth factors I and II ( @entity7424 , @entity10247 ) are regulated in part by a family of IGF-binding proteins (IGFBPs). There are six known members of the IGFBP family, which share distinct structural characteristics and functional activities. To study the binding properties of these proteins, we have expressed recombinant @entity10246 and @entity10250 using the LCR/ @entity28690 expression system. Using this system, we found that recombinant @entity10246 was secreted by @entity28690 cells and had a glycosylation pattern similar to that of native @entity10246 . Recombinant @entity10250 secreted from @entity28690 cells had a molecular size identical to that of non-glycosylated native @entity10250 . The binding kinetics of recombinant IGFBPs was measured using a solid-phase ligand-binding assay, an in vitro solution-binding assay, and a cellular proliferation assay. @entity7424 bound with high affinity to recombinant @entity10246 and @entity10250 with K(D)s of <0.25 nmol. As reported for native IGFBPs, @entity10247 bound with affinity higher than @entity7424 to recombinant @entity10246 and @entity10250 (K(D) of <0.05 nmol). Recombinant @entity10246 and @entity10250 were found to inhibit the IGF-induced proliferation of an NIH3T3 cell line engineered to overexpress the @entity7423 . We have compared the binding kinetics of @entity28690 cell-expressed IGFBPs with that of recombinant protein expressed in @entity526 and found them to be equivalent. Here, we show that the LCR/ @entity28690 expression system represents an effective route for expression of biologically active IGFBPs.
