Cloning, overexpression, purification, crystallization, and preliminary X-ray studies of SP-0149, the substrate binding protein of an ABC transporter from Streptococcus pneumoniae

The substrate binding protein (SP-0149) of an ABC transporter from Streptococcus pneumoniae was molecularly cloned, overexpressed and purified. Diffraction quality crystals were grown using the hanging-drop vapour-diffusion technique. A truncated (29 residues from the N-terminus) and N-terminal His-tagged form of SP-0149 from pneumococcal strain ATCC BAA-334 was overexpressed and purified to homogeneity using affinity and gel-filtration chromatography. Diffraction quality crystals were grown at 293 K using the hanging-drop vapour-diffusion technique. X-ray diffraction data were collected to 2.3 Å resolution from a single-crystal that belonged to the orthorhombic space group P2₁2₁2₁ with the unit-cell parameters a = 54.56, b = 75.61, c = 75.52 Å. The calculated values of the Matthews coefficient assuming one molecule (with calculated molecular weight of 30 400 Da) in the crystal asymmetric unit and the corresponding solvent content were 2.56 ų Da⁻¹ and 52.0%, respectively