Expression of a recombinant, 4’-Phosphopantetheinylated, active M. tuberculosis Fatty acid Synthase I in E. coli

Baron, S.; Peleg, Y.; Grunwald, J.; Morgenstern, D.; Elad, N.; Peretz, M.; Albeck, S.; Levin, Y.; Welch, J. T.; Deweerd, K. A.; Schwartz, A.; Burstein, Y.; Diskin, R.; Shakked, Z.; Zimhony, O.

doi:10.1101/400622

bioRxiv · 10.1101/400622

Expression of a recombinant, 4’-Phosphopantetheinylated, active M. tuberculosis Fatty acid Synthase I in E. coli

Baron, S.·Peleg, Y.·Grunwald, J.·Morgenstern, D.·Elad, N.·Peretz, M.·Albeck, S.·Levin, Y.·Welch, J. T.·Deweerd, K. A.·Schwartz, A.·Burstein, Y.·Diskin, R.·Shakked, Z.·Zimhony, O.

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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.\n\nWe 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 structurefunction analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.

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Baron, S., Peleg, Y., Grunwald, J., Morgenstern, D., Elad, N., Peretz, M., Albeck, S., Levin, Y., Welch, J. T., Deweerd, K. A., Schwartz, A., Burstein, Y., Diskin, R., Shakked, Z., Zimhony, O.. 2018-08-28. Expression of a recombinant, 4’-Phosphopantetheinylated, active M. tuberculosis Fatty acid Synthase I in E. coli. https://doi.org/10.1101/400622

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