Expression of a recombinant, 4'-Phosphopantetheinylated, active M. tuberculosis fatty acid synthase I in E. coli

Szilvia Baron, Yoav Peleg, Jacob Grunwald, David Morgenstern, Nadav Elad, Moshe Peretz, Shira Albeck, Yishai Levin, John T Welch, Kim A DeWeerd, Alon Schwarz, Yigal Burstein, Ron Diskin, Zippora Shakked, Oren Zimhony

Research output: Contribution to journalArticlepeer-review

Abstract

Fatty acid synthase 1 (FAS I) from Mycobacterium tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4'-Phosphopantetheine (4'-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4'-PP transferase, termed acyl carrier protein synthase (AcpS). In order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid. We induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography. Activation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM. Purified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structure-function analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.
Original languageEnglish
Article numbere0204457
Number of pages13
JournalPLoS ONE
Issue number9
DOIs
StatePublished - 24 Sep 2018

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