Tandem affinity purification tagging of fatty acid biosynthetic enzymes in Synechocystis sp. PCC6803 and Arabidopsis thaliana.
De novo fatty acid synthesis in plants occurs primarily in the plastids and is catalysed by a type-II fatty acid synthase (FAS) in which separate enzymes catalyse sequential reactions. Genes encoding all of the plant FAS components have been identified, following enzyme purification or by homology to Escherichia coli genes, and the structure of a number of the individual proteins determined. There are several lines of biochemical evidence indicating that FAS enzymes form a multi-protein complex and both in vitro and in vivo strategies can be used to investigate the association and interactions between them. To investigate protein interactions in vivo, tandem affinity purification-tagged FAS components are being used to purify complexes from both Arabidopsis thaliana and Synechocystis PCC6803. Here, the development of the tandem affinity purification method, its modification, and its use in plants is described and the experimental results achieved so far are reported.
Brown
Adrian P
AP
School of Biological and Biomedical Sciences, University of Durham, Science Laboratories, South Road, Durham DH1 3LE, UK. a.p.brown@durham.ac.uk
Affleck
Valerie
V
Fawcett
Tony
T
Slabas
Antoni R
AR
D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000302:isolation & purification* / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D064429:Fatty Acid Synthases / Q000302:isolation & purification* / Q000378:metabolism; D005227:Fatty Acids / Q000096:biosynthesis*; D018087:Plastids / Q000201:enzymology; D025941:Protein Interaction Mapping / Q000379:methods; D021122:Protein Subunits / Q000378:metabolism; D040901:Proteomics / Q000379:methods / Q000639:trends; D046939:Synechocystis / Q000201:enzymology*
D029681:Arabidopsis Proteins; D001426:Bacterial Proteins; D005227:Fatty Acids; D021122:Protein Subunits; D064429:Fatty Acid Synthases