부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.bbrc.2005.05.164 Byline: Pierre Douette (a), Rachel Navet (a), Pascal Gerkens (b), Moreno Galleni (c), Daniel Levy (d), Francis E. Sluse (a) Keywords: Fusion protein; Maltose-binding protein; N-utilizing substance A; Uncoupling protein 1; Solubility; Aggregation; GroEL/ES Abstract: Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein. Author Affiliation: (a) Laboratory of Bioenergetics, Centre of Oxygen Research and Development, Institut de Chimie B6, Universite de Liege, Sart Tilman, B-4000 Liege, Belgium (b) Laboratory of Mass Spectrometry, Institut de Chimie B6, Universite de Liege, Sart Tilman, B-4000 Liege, Belgium (c) Centre for Protein Engineering, Institut de Chimie B6, Universite de Liege, Sart Tilman, B-4000 Liege, Belgium (d) Institut Curie, UMR-CNRS 168 and LRC-CEA 34V, 11 Rue Pierre et Marie Curie, 75231 Paris Cedex 05, France Article History: Received 21 May 2005 Article Note: (footnote) [star] Abbreviations: Hsp, heat shock protein; IPTG, isopropyl-[beta]-d-thiogalactopyranoside; MBP, maltose-binding protein; MS, mass spectrometry; NusA, N-utilizing substance A; UCP1, uncoupling protein 1.