Purification, crystallization and preliminary X-ray crystallographic studies of KstR2 (ketosteroid regulatory protein) from Mycobacterium tuberculosis

Abstract

KstR2 (Rv3557c) is one of two TetR-family transcriptional repressors of cholesterol metabolism in Mycobacterium tuberculosis. The ability to degrade cholesterol fully is important for pathogenesis, and therefore this repressor was expressed, purified and crystallized. Crystals of KstR2 diffracted to better than 1.9 Å resolution and belonged to space group C2, with unit-cell parameters a = 72.3, b = 90.3, c = 49.7 Å, α = γ = 90, β = 128.2°.

Keywords: Mycobacterium tuberculosis; TetR family; cholesterol; steroid.

Figure 1

Size-exclusion chromatography (SEC) of KstR2. (a) SDS–PAGE of SEC fractions. Lane 1, molecular-weight marker (labelled in kDa); lane 2, KstR2 cleaved with rTEV; lanes 3–15, fractions from SEC. Cleaved and uncleaved protein are indicated with arrows; KstR2* = His₆ tag + S tag + KstR2. Fractions 11–13 were pooled and concentrated. (b) SEC-MALS analysis of purified KstR2 showing the expected refractive index of the dimer (dotted line) and the measured refractive index of the SEC peak (circles) at two concentrations of KstR2, as well as the elution volume of the peak.