An orthogonal purification strategy for isolating crosslinked domains of modular synthases.
Chemo-enzymatic methods for covalently crosslinking carrier proteins with partner enzymes within modular synthases hold promise for elucidating and engineering metabolic pathways. Our efforts to crystallize the ACP-KS complexes of fatty acid synthases have been complicated by difficulties in the purification of the crosslinked complex from the other proteins in the reaction. Here we present a solution that employs an orthogonal purification strategy to achieve the quantity and level of purity necessary for further studies of this complex.
Haushalter
Robert W
RW
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA.
Worthington
Andrew S
AS
Hur
Gene H
GH
Burkart
Michael D
MD
D000179:Acrylates / Q000737:chemistry*; D000213:Acyl Carrier Protein / Q000737:chemistry* / Q000302:isolation & purification; D003065:Coenzyme A / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D003432:Cross-Linking Reagents / Q000737:chemistry; D004925:Escherichia / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D064429:Fatty Acid Synthases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D008958:Models, Molecular; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000737:chemistry*; D048630:Polyketide Synthases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D000179:Acrylates; D000213:Acyl Carrier Protein; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D010204:Pantetheine; D048630:Polyketide Synthases; D064429:Fatty Acid Synthases; D003065:Coenzyme A