High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2.
The diverse biological activities of the insulin-like growth factors (IGF-1 and IGF-2) are mediated by the IGF-1 receptor (IGF-1R). These actions are modulated by a family of six IGF-binding proteins (IGFBP-1-6; 22-31 kDa) that via high affinity binding to the IGFs (K(D) approximately 300-700 pM) both protect the IGFs in the circulation and attenuate IGF action by blocking their receptor access. In recent years, IGFBPs have been implicated in a variety of cancers. However, the structural basis of their interaction with IGFs and/or other proteins is not completely understood. A critical challenge in the structural characterization of full-length IGFBPs has been the difficulty in expressing these proteins at levels suitable for NMR/X-ray crystallography analysis. Here we describe the high-yield expression of full-length recombinant human IGFBP-2 (rhIGFBP-2) in Escherichia coli. Using a single step purification protocol, rhIGFBP-2 was obtained with >95% purity and structurally characterized using NMR spectroscopy. The protein was found to exist as a monomer at the high concentrations required for structural studies and to exist in a single conformation exhibiting a unique intra-molecular disulfide-bonding pattern. The protein retained full biologic activity. This study represents the first high-yield expression of wild-type recombinant human IGFBP-2 in E. coli and first structural characterization of a full-length IGFBP.
Swain
Monalisa
M
NMR Research Center, Indian Institute of Science, Bangalore 560012, India.
Slomiany
Mark G
MG
Rosenzweig
Steven A
SA
Atreya
Hanudatta S
HS
D001483:Base Sequence; D002460:Cell Line; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism
D017931:DNA Primers; D018971:Insulin-Like Growth Factor Binding Protein 2; D008024:Ligands; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D013936:Thymidine