Adenoviral-Mediated Expression of Human Insulin-like Growth Factor-Binding Protein-3 Sue M. Firth,* ,1 Usha Ganeshprasad,* Philip Poronnik,† David I. Cook,† and Robert C. Baxter* *Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, NSW 2065; and †Department of Physiology, University of Sydney, Sydney, NSW 2006, Australia Received January 22, 1999, and in revised form March 10, 1999 Insulin-like growth factors (IGFs) in the circulation are predominantly sequestered into ternary com- plexes comprising IGF, IGF-binding protein-3 (IGFBP- 3), and the acid-labile subunit (ALS). Besides its role in regulating IGF bioavailability in the circulation, IGF- BP-3 has both IGF-dependent and IGF-independent actions on cell proliferation. As part of our studies into the structure–function relationships of the multifunc- tional IGFBP-3, we have evaluated the efficiency of an adenovirus-mediated expression system for rapid, me- dium-scale production of functional, glycosylated IGF- BP-3. Replication-deficient adenovirus containing hu- man IGFBP-3 cDNA was generated using standard techniques. Secreted, recombinant IGFBP-3 (IGFBP- 3 Ad ) was purified from the culture medium of virus- infected cells by IGF-I affinity chromatography fol- lowed by reverse-phase HPLC. When analyzed by SDS–PAGE, IGFBP-3 Ad was similar in size (43- to 45- kDa glycoform doublet) to IGFBP-3 Pl derived from plasma. In addition, IGFBP-3 Ad was detected by immu- noblot using an antibody specific for human IGFBP-3 and by ligand blot using radiolabeled IGF-I. IGFBP-3 Ad had similar affinities for IGF-I and ALS and an approx- imately 25% decreased affinity for IGF-II compared to IGFBP-3 Pl . IGFBP-3 Ad showed no significant difference in its susceptibility to an IGFBP-3 protease present in medium conditioned by MCF-7 breast cancer cells compared to IGFBP-3 Pl , but appeared more resistant to the IGFBP-3 protease present in pregnancy serum. IGFBP-3 Ad also exhibited increased binding to T47D cells which may be related to the glycosylation state of the protein. © 1999 Academic Press Insulin-like growth factors, IGF-I and IGF-II, are potent mitogenic peptides found in high concentrations in the circulation. The IGFs are sequestered into high- affinity ternary complexes by insulin-like growth fac- tor-binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS); these complexes regulate both the sta- bility of the IGFs in the circulation and their egress from the vasculature to target tissues (1). At the cellu- lar level, IGFBP-3 has been shown to modulate IGF activity in a paradoxical fashion. It has been suggested that IGFBP-3 in the extracellular milieu inhibits IGF activity by forming high-affinity binary complexes with IGFs, while IGFBP-3 associated with the cell surface potentiates IGF action, possibly by concentrating IGFs to their cognate receptors (2). In the past few years, attention has also turned to the cellular actions of IGFBP-3 which may be independent of IGFs. It has been shown that IGFBP-3 has growth inhibitory func- tions that are independent of the type I IGF receptor (3), perhaps related to the recent discovery that IGF- BP-3 can be translocated into the nuclei of cells where it could exert a direct influence on gene expression (4). IGFBP-3 function is thought to be modulated by post- translational events such as phosphorylation and lim- ited proteolysis (5). The diversity of functions attrib- uted to IGFBP-3 has made it a challenging prospect in protein structure–function studies. There are few re- ports on the elucidation of structural determinants involved in protein–protein or protein– cell interactions required for IGFBP-3 function (4, 6 – 8); these studies are usually limited by the nonavailability of large quantities of purified protein. Traditionally, heterologous protein expression is usually achieved in bacterial cells by taking advantage of plasmid vectors, containing powerful promoters, which are easy to manipulate in Escherichia coli (9, 10). However, the limitations in using these systems for eukaryotic protein expression include inappropri- ate or lack of complex posttranslational modifications, the accumulation of insoluble aggregated products in inclusion bodies as a result of inefficient secretion, and 1 To whom correspondence should be addressed. Fax: 612-9926- 8484. E-mail: sfirth@med.usyd.edu.au. Protein Expression and Purification 16, 202–211 (1999) Article ID prep.1999.1075, available online at http://www.idealibrary.com on 202 1046-5928/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.