Expression and purification of integral membrane fatty acid desaturases

• Published in: PloS one Vol. 8; no. 3; p. e58139
• Main Authors: Chen, Haiqin, Gu, Zhennan, Zhang, Hao, Wang, Mingxuan, Chen, Wei, Lowther, W Todd, Chen, Yong Q
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 08-03-2013; Public Library of Science (PLoS)
• Subjects: Amino acids; Aspergillus oryzae; Biology; Cancer; Chemical properties; Cloning; Crystal structure; Crystallography; Culture; Dehydrogenation; Desaturase; Enzymes; Escherichia coli; Fatty acid desaturases; Fatty Acid Desaturases - biosynthesis; Fatty Acid Desaturases - chemistry; Fatty Acid Desaturases - genetics; Fatty Acid Desaturases - isolation & purification; Fatty acids; Food science; Fungal Proteins - biosynthesis; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - isolation & purification; Fungi; Genomes; Integrals; Laboratories; Lipids; Medicine; Membrane Proteins - biosynthesis; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - isolation & purification; Metastasis; Mortierella - enzymology; Mortierella - genetics; Mortierella alpina; Mucoromycotina; Pichia pastoris; Plasmids; Polyunsaturated fatty acids; Protein expression; Proteins; Purification; Recombinant; Recombinant proteins; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Saccharomyces cerevisiae; Unsaturated fatty acids; Yeast; North Carolina; United States > US; China
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0058139

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.