A new method for the purification of bioactive insulin-like growth factor-binding protein-3.
We present a novel efficient procedure for high level purification of human IGFBP-3. Insulin-like growth factor-binding proteins (IGFBPs) are key regulators of insulin-like growth factor mediated signal transduction and thereby can profoundly influence cellular phenotypes. Certain IGFBPs, including IGFBP-3, have also been described to possess additional IGF-independent activities, which rely, at least in part, on their nuclear localization. However, the mechanisms of IGF-independent biological activities of IGFBP-3 are not well understood. For the study of these functions, recombinant IGFBP-3 is used. However, it has traditionally been difficult to obtain recombinant protein in sufficient quality and quantity. Here we describe a new procedure for the purification of recombinant IGFBP-3 from cell culture supernatants involving a two-step affinity purification procedure. Using this new protocol, we obtained pure IGFBP-3 free of any visible contaminants. We also provide evidence that the protein purified in this way retains biological activity, to bind IGF and modulate IGF-dependent signal transduction. We also show that the purified protein produced by the new procedure is readily internalized by human fibroblasts, suggesting that this protein is also suitable to study intracellular trafficking of IGFBP-3.
Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, Innsbruck, Austria.
Haymo
H
Pircher
Andrea
A
Matscheski
Andreas
A
Laich
Martin
M
Hermann
Barbara
B
Moser
Hans-Peter
HP
Viertler
Lucia
L
Micutkova
Herbert
H
Lindner
Bettina
B
Sarg
Werner
W
Zwerschke
Pidder
P
Jansen-Dürr
D002846:Chromatography, Affinity; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism*; D018969:Insulin-Like Growth Factor Binding Proteins / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013002:Somatomedins / Q000378:metabolism*
D018972:Insulin-Like Growth Factor Binding Protein 3; D018969:Insulin-Like Growth Factor Binding Proteins; D011994:Recombinant Proteins; D013002:Somatomedins