title: Expression and purification of integral membrane fatty acid desaturases.
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.
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu Province, People's Republic of China.
authors.forename: Haiqin
authors.initials: H
authors.lastname: Chen
authors.forename: Zhennan
authors.initials: Z
authors.lastname: Gu
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.forename: Mingxuan
authors.initials: M
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
authors.forename: W Todd
authors.initials: WT
authors.lastname: Lowther
authors.forename: Yong Q
authors.initials: YQ
authors.lastname: Chen
mesh_terms: D044943:Fatty Acid Desaturases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D020094:Mortierella / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D044943:Fatty Acid Desaturases