Expression and Purification of Integral Membrane Fatty Acid Desaturases |
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| Authors: | Haiqin Chen Zhennan Gu Hao Zhang Mingxuan Wang Wei Chen W Todd Lowther Yong Q Chen |
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| Institution: | 1. State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province, People’s Republic of China.; 2. Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America.; 3. Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America.; Max Delbrueck Center for Molecular Medicine, Germany, |
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| Abstract: | Fatty acid desaturase enzymes perform dehydrogenation reactions leading to the insertion of double bonds in fatty acids, and are divided into soluble and integral membrane classes. Crystal structures of soluble desaturases are available; however, membrane desaturases have defied decades of efforts due largely to the difficulty of generating recombinant desaturase proteins for crystallographic analysis. Mortierella alpina is an oleaginous fungus which possesses eight membrane desaturases involved in the synthesis of saturated, monounsaturated and polyunsaturated fatty acids. Here, we describe the successful expression, purification and enzymatic assay of three M. alpina desaturases (FADS15, FADS12, and FADS9-I). Estimated yields of desaturases with purity >95% are approximately 3.5% (Ca. 4.6 mg/L of culture) for FADS15, 2.3% (Ca. 2.5 mg/L of culture) for FADS12 and 10.7% (Ca. 37.5 mg/L of culture) for FADS9-I. Successful expression of high amounts of recombinant proteins represents a critical step towards the structural elucidation of membrane fatty acid desaturases. |
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