Expression, purification, and characterization of secreted recombinant human insulin-like growth factor-binding protein-6 in methylotrophic yeast Pichia pastoris

The mitogenic and metabolic activities of insulin-like growth factors (IGF) are modulated by a family of six high-affinity IGF-binding proteins (IGFBPs). This study describes the secretion and purification of the recombinant human IGFBP-6 expressed in methylotrophic yeast Pichia pastoris. In this research, a multicopy expression plasmid pA-O815/3xIGFBP-6 containing 3 copies of human IGFBP-6 expression cassette was constructed and transformed into P. pastoris GS115. The encoding sequence of α-factor leading peptide fused in-frame at the 5′ end of human IGFBP-6 open reading frame and led expressed IGFBP-6 into the secretory pathway. After transformed cells were induced with methanol, medium supernatant was analyzed by SDS-PAGE and Western blotting. The two major protein bands of ∼30 and ∼18 kDa were detected. The protein of ∼30 kDa was confirmed to be the glycosylated recombinant human IGFBP-6 (rhIGFBP-6), which was partially proteolyzed by protease Kex2 to produce a ∼18 kDa fragment. Approximately 95% homogeneity of the soluble form of 30 kDa rhIGFBP-6 were achieved by two-step purification procedure using ion-exchange chromatography and then hydrophobic-interaction chromatography. The rhIGFBP-6 could be distributed to all of the cell body when cultured MDA-MB-231 cell with rhIGFBP-6 and the activities of rhIGFBP-6 were assayed by [3H]thymidine incorporation, which revealed that rhIGFBP-6 inhibited IGF-II-stimulated cell proliferation. Our results demonstrated that functional rhIGFBP-6 can be produced in sufficient quantities by using P. pastoris for further structural and functional studies.