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Updated: May 14, 2025

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
Published on: June 20, 2019
Protocol for the purification, analysis, and handling of acyl carrier proteins from type I fatty acid and polyketide
Christian Gusenda1, Svenja Berlage2, Ines Burkhart3
1Institute of Organic Chemistry and Chemical Biology, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany; Buchmann Institute of Molecular Life Sciences (BMLS), Goethe University Frankfurt, 60438 Frankfurt am Main, Germany.
Abstract:
Acyl carrier proteins (ACPs) are key domains in fatty acid and polyketide synthases (PKSs), shuttling intermediates to catalytic partners. Here, we present a protocol for purifying and analyzing ACPs from fatty acid synthases (FASs) and PKSs, using four model ACPs from Mus musculus, Saccharopolyspora erythraea, Streptomyces venezuelae, and Streptomyces hygroscopicus. We describe steps for recombinant ACP production in E. coli; purification via chromatography or precipitation; ACP modification (holo/acyl forms); and analysis using urea PAGE, high-performance liquid chromatography (HPLC), and NMR spectroscopy. For complete details on the use and execution of this protocol, please refer to Gusenda et al.1.

