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 alpha-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 approximately 30 and approximately 18kDa were detected. The protein of approximately 30kDa was confirmed to be the glycosylated recombinant human IGFBP-6 (rhIGFBP-6), which was partially proteolyzed by protease Kex2 to produce a approximately 18kDa fragment. Approximately 95% homogeneity of the soluble form of 30kDa 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 [(3)H]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.
Chen
Zhaoli
Z
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, 5 Dong Dan San Tiao, Beijing 100005, PR China.
Chen
Hong
H
Wang
Xin
X
Ma
Xiaoli
X
Huang
Bingren
B
D015870:Gene Expression / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D018976:Insulin-Like Growth Factor Binding Protein 6 / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D018976:Insulin-Like Growth Factor Binding Protein 6; D011994:Recombinant Proteins