Expression and purification of biologically active IGF-binding proteins using the LCR/Mel expression system.
The anabolic effects and bioavailability of insulin-like growth factors I and II (IGF-I, IGF-II) 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 IGFBP-3 and IGFBP-4 using the LCR/Mel expression system. Using this system, we found that recombinant IGFBP-3 was secreted by Mel cells and had a glycosylation pattern similar to that of native IGFBP-3. Recombinant IGFBP-4 secreted from Mel cells had a molecular size identical to that of non-glycosylated native IGFBP-4. 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. IGF-I bound with high affinity to recombinant IGFBP-3 and IGFBP-4 with K(D)s of <0.25 nmol. As reported for native IGFBPs, IGF-II bound with affinity higher than IGF-I to recombinant IGFBP-3 and IGFBP-4 (K(D) of <0.05 nmol). Recombinant IGFBP-3 and IGFBP-4 were found to inhibit the IGF-induced proliferation of an NIH3T3 cell line engineered to overexpress the IGF-I receptor. We have compared the binding kinetics of Mel cell-expressed IGFBPs with that of recombinant protein expressed in Escherichia coli and found them to be equivalent. Here, we show that the LCR/Mel expression system represents an effective route for expression of biologically active IGFBPs.
Bagnall
Wendy
W
Respiratory and Inflammation Research Department, AstraZeneca Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK.
Sharpe
Paul M
PM
Newham
Peter
P
Tart
Johnathan
J
Mott
Richard A
RA
Torr
Vanessa R
VR
Forder
Robert A
RA
Needham
Maurice R C
MR
D015152:Blotting, Northern; D002460:Cell Line; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology; D018969:Insulin-Like Growth Factor Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D007700:Kinetics; D011485:Protein Binding; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology
D018970:Insulin-Like Growth Factor Binding Protein 1; D018971:Insulin-Like Growth Factor Binding Protein 2; D018972:Insulin-Like Growth Factor Binding Protein 3; D018974:Insulin-Like Growth Factor Binding Protein 4; D018969:Insulin-Like Growth Factor Binding Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins