Adenoviral-mediated expression of human insulin-like growth factor-binding protein-3.
Insulin-like growth factors (IGFs) in the circulation are predominantly sequestered into ternary complexes comprising IGF, IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS). Besides its role in regulating IGF bioavailability in the circulation, IGFBP-3 has both IGF-dependent and IGF-independent actions on cell proliferation. As part of our studies into the structure-function relationships of the multifunctional IGFBP-3, we have evaluated the efficiency of an adenovirus-mediated expression system for rapid, medium-scale production of functional, glycosylated IGFBP-3. Replication-deficient adenovirus containing human 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 followed 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 immunoblot 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 approximately 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.
Firth
S M
SM
Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, NSW, Australia. sfirth@med.usyd.edu.au
Ganeshprasad
U
U
Poronnik
P
P
Cook
D I
DI
Baxter
R C
RC
D000260:Adenoviruses, Human / Q000235:genetics*; D017077:Culture Media, Conditioned; D010450:Endopeptidases / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D008024:Ligands; D011247:Pregnancy; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014407:Tumor Cells, Cultured
D017077:Culture Media, Conditioned; D018972:Insulin-Like Growth Factor Binding Protein 3; D008024:Ligands; D011994:Recombinant Proteins; D010450:Endopeptidases; C080473:insulin-like growth factor binding protein-3 protease