Rv0100, a proposed acyl carrier protein in Mycobacterium tuberculosis: expression, purification and crystallization

Abstract

Acyl carrier proteins (ACPs) are important components in fatty-acid biosynthesis in prokaryotes. Rv0100 is predicted to be an essential ACP in Mycobacterium tuberculosis, the pathogen that is the causative agent of tuberculosis, and therefore has the potential to be a novel antituberculosis drug target. Here, the successful cloning and purification of Rv0100 using Mycobacterium smegmatis as a host is reported. Crystals of the purified protein were obtained that diffracted to a resolution of 1.9 Å. Overall, this work lays the foundation for the future pursuit of drug discovery and development against this potentially novel drug target.

Keywords: Mycobacterium smegmatis; Mycobacterium tuberculosis; Rv0100; acyl carrier protein.

Figure 1

Sequence comparison of identified ACPs in the M. tuberculosis genome (Cole et al., 1998 ▸). The phosphopantetheine-binding site has been identified and is indicated (Haines et al., 2013 ▸). The sequences of Rv0100, Rv1344, Rv2244 and Rv0033 were taken from MycoBrowser (Kapopoulou et al., 2011 ▸). Rv0100 has 33.3%, 32.5% and 38.9% sequence similarity to Rv1344, Rv2244 and Rv0033, respectively (Rice et al., 2000 ▸). The alignment was created using Clustal Omega (Sievers et al., 2011 ▸).

Figure 2

Samples of purified Rv0100 after several washes with imidazole and elution with an Ni–NTA column were subjected to SDS–PAGE. The left column contains EZ-Run Prestained Rec Protein Ladder (Fisher BioReagents; molecular-weight markers are labeled in kDa). The expected molecular weight of Rv0100 is approximately 11.1 kDa.

Figure 3

The eluted protein was subjected to size-exclusion chromatography. A UV detector measured the absorbance at 280 nm and the protein eluted into a fraction collector. Fractions dispensed during peaks were considered to contain Rv0100. The first peak is likely to be a dimeric form and the second peak is likely to be the monomeric form.

Figure 4

Crystals of Rv0100 obtained using reagents from the Index screen in hanging-drop trays. Crystals were found in 0.2 M ammonium sulfate, 0.1 M bis-Tris pH 5.5, 25%(w/v) PEG 3350 (left), 0.2 M ammonium acetate, 0.1 M bis-Tris pH 5.5, 25% PEG 3350 (center) and 0.2 M lithium sulfate monohydrate, 0.1 M bis-Tris pH 5.5, 25%(w/v) PEG 3350 (right).