A new method for the purification of bioactive insulin-like growth factor-binding protein-3 Haymo Pircher a,1 , Andrea Matscheski a,1 , Andreas Laich a , Martin Hermann b , Barbara Moser a , Hans-Peter Viertler a , Lucia Micutkova a , Herbert Lindner c , Bettina Sarg c , Werner Zwerschke a , Pidder Jansen-Dürr a, * a Institute for Biomedical Aging Research, Austrian Academy of Sciences, Rennweg 10, A-6020 Innsbruck, Austria b KMT Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Center of Operative Medicine, Innsbruck Medical University, Innrain 66, A-6020 Innsbruck, Austria c Medical University of Innsbruck, Innsbruck Biocenter, Division of Clinical Biochemistry, Fritz-Preglstrasse 3, A-6020 Innsbruck, Austria article info Article history: Received 28 October 2009 and in revised form 1 February 2010 Available online 11 February 2010 Keywords: IGF-binding protein Purification Affinity chromatography abstract 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 trans- duction 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, suggest- ing that this protein is also suitable to study intracellular trafficking of IGFBP-3. Ó 2010 Elsevier Inc. All rights reserved. Introduction Insulin-like growth factors (IGFs) play an important role in reg- ulating cell growth, apoptosis 2 and differentiation in a variety of cell types [1–3], and have been implicated in tumorigenesis (reviewed by [4]). The activity of IGF-I is mainly mediated by the IGF type I receptor. The IGF system also comprises six secreted IGF-binding proteins (IGFBP-1 to 6), which serve as transport vehicles for IGFs and control their biological availability [5]. The expression of the various IGFBPs is differentially regulated in a tissue-dependent fash- ion, suggesting that each IGFBP exerts a specific role. Insulin-like growth factor-binding protein-3 (IGFBP-3), a key component of the IGF/IGFBP-axis, which plays an important role in tumorigenesis [5], can bind to IGF-I and thereby regulate its mitogenic activity in the extracellular environment. Increased expression of IGFBP-3 contrib- utes to both p53-dependent and -independent apoptosis [6]. IGFBP-3 can induce apoptotic cell death by several pathways and modulate the anti-apoptotic effects of IGF-I by regulating the IGF-I/IGF-R–I interaction (reviewed by [5,7]). In addition to IGF-related functions of IGFBP-3, IGF-indepen- dent actions of IGFBP-3 contribute to its anti-proliferative and proapoptotic activities [7]. For example, IGFBP-3 can induce pro- grammed cell death in immortalized IGF-R-negative mouse fibro- blasts [6], and IGFBP-3 mutants that do not bind IGFs can stimulate apoptosis in both prostate cancer [8] and breast cancer [9] cells. Moreover, the expression of an IGF-non-binding IGFBP-3 mutant induces apoptosis and inhibits prostate tumor progression in a transgenic mouse model of prostate cancer [10]. IGF-indepen- dent actions of IGFBP-3 may involve the activation of cell-surface receptor kinases or may be mediated by intracellular IGFBP-3 [5]. It was demonstrated that IGFBP-3 is internalized by a variety of cell types in a process involving endocytic pathways [11], and a recent study showed the endocytic uptake of proteins fused to a 18 amino acids peptide sequence corresponding to the heparin-binding do- main in the C-terminus of IGFBP-3 [12]. Purified human IGFBP-3 (hIGFBP-3) can be used for biological studies of its cellular uptake which will shed new light on its intra- 1046-5928/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2010.02.005 * Corresponding author. Fax: +43 512 583919 8. E-mail address: p.jansen-duerr@oeaw.ac.at (P. Jansen-Dürr). 1 These two authors contributed equally to the manuscript. 2 Abbreviations used: IGFBPs, insulin-like growth factor-binding proteins; IGF, insulin-like growth factor; IGFBP-3, insulin-like growth factor-binding protein-3; hIGFBP-3, human insulin-like growth factor-binding protein; HDF, human diploid fibroblast; DMEM, Dulbecco’s modified Eagle’s medium. Protein Expression and Purification 71 (2010) 160–167 Contents lists available at ScienceDirect Protein Expression and Purification journal homepage: www.elsevier.com/locate/yprep