Expression, purification and preliminary X-ray analysis of the C-terminal domain of an arginine repressor protein from Mycobacterium tuberculosis

Abstract

The gene product of an open reading frame Rv1657 from Mycobacterium tuberculosis is a putative arginine repressor protein (ArgR), a transcriptional factor that regulates the expression of arginine-biosynthetic enzymes. Rv1657 was expressed and purified and a C-terminal domain was crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 2.15 A. The crystals belong to space group P1 and the Matthews coefficient suggests that the crystals contain six C-terminal domain molecules per unit cell. Previous structural and biochemical studies on the arginine repressor proteins from other organisms have likewise shown the presence of six molecules per unit cell.

Figure 1

16% SDS–PAGE analysis of the purification stages of full-length Rv1657 (17 kDa). Lane 1, protein molecular-weight standards (kDa); lane 2, GST-Rv1657 fusion protein; lane 3, fusion protein cleaved with TEV protease; lane 4, purified Rv1657 fraction from the flowthrough fraction from the GSTrap column.

Figure 2

20% SDS–PAGE analysis of the cleavage during crystallization. Lane 1, protein molecular-weight standards (kDa); lane 2, dissolved crystals that had been grown at 295 K for two months; lane 3, protein solution corresponding to that in crystallization drops stored at 277 K for two months; lane 4, a second Rv1657 preparation containing full-length protein also stored at 277 K for two months. The sample in lane 4 is overloaded and appears to be greater than 19.4 kDa; however, the molecular weight of this sample was shown to be 17 456.7 Da by mass spectrometry.

Figure 3

Sequence alignment of arginine repressor proteins from four different organisms. The numbering above the alignment corresponds to the sequence of Rv1657 from M. tuberculosis. Sequence alignment was performed using the program ClustalW (Thompson et al., 1994 ▶) and the figure was generated using the program ESPript (Gouet et al., 1999 ▶).

Figure 4

Rv1657 crystals. The approximate dimensions of the diffraction-quality crystals were 100 × 100 × 50 µm and the crystals were grown in 20% PEG 10 000, 0.1 M HEPES buffer pH 7.5 over two months.