High-yield bacterial expression and structural characterization of recombinant human insulin-like growth factor binding protein-2

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• 作者：Monalisa Swain ; Mark G. Slomiany ; Steven A. Rosenzweig ; Hanudatta S. Atreya
• 关键词：Recombinant human IGF- binding protein-2 (rhIGFBP-2) ; E. coli expression ; Protein purification ; Protein structure ; Secondary structure ; G-matrix Fourier transform (GFT) NMR
• 刊名：Archives of Biochemistry and Biophysics
• 出版年：2010
• 出版时间：15 September 2010
• 年：2010
• 卷：501
• 期：2
• 页码：195-200
• 全文大小：522 K

文摘

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 (Kub>Dub>  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.