High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2 |
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Authors: | Monalisa Swain Mark G Slomiany Steven A Rosenzweig |
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Institution: | a NMR Research Center, Indian Institute of Science, Bangalore 560012, India b Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore 560012, India c Department of Cell and Molecular Pharmacology & Experimental Therapeutics, Medical University of South Carolina, Charleston, SC 29425, USA |
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Abstract: | 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 (KD ∼ 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. |
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Keywords: | Recombinant human IGF- binding protein-2 (rhIGFBP-2) E coli expression Protein purification Protein structure Secondary structure G-matrix Fourier transform (GFT) NMR |
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