Related Experiment Video
Updated: Apr 23, 2026
Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD
Published on: December 30, 2016
Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease
Emmanuel Nji1, Dianfan Li1, Declan A Doyle1
1Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland.
Abstract:
The prokaryotic lysine-specific permease (LysP) belongs to the amino acid-polyamine-organocation (APC) transporter superfamily. In the cell, members of this family are responsible for the uptake and recycling of nutrients, for the maintenance of a constant internal ion concentration and for cell volume regulation. The detailed mechanism of substrate selectivity and transport of L-lysine by LysP is not understood. A high-resolution crystal structure would enormously facilitate such an understanding. To this end, LysP from Pseudomonas aeruginosa was recombinantly expressed in Escherichia coli and purified to near homogeneity by immobilized metal ion-affinity chromatography (IMAC) and size-exclusion chromatography (SEC). Hexagonal- and rod-shaped crystals were obtained in the presence of L-lysine and the L-lysine analogue L-4-thialysine by vapour diffusion and diffracted to 7.5 Å resolution. The diffraction data were indexed in space group P21, with unit-cell parameters a = 169.53, b = 169.53, c = 290.13 Å, γ = 120°.
