Figure 4
Purification of recombinant VanY n . SDS-PAGE Analysis (staining with Coomassie brilliant blue) of protein samples collected during purification of VanYn from S. venezuelae carrying pIJ86ΩN-His6-vanY n or pIJ86ΩC-His6-vanY n . Crude fractions from S. venezuelae carrying pIJ86ΩN-His6-vanY n (lane 1) and S. venezuelae carrying pIJ86ΩC-His6-vanY n (lane 2). Fractions from HiTrap Chelating chromatography: N-His6-VanYn flow through (lane 3), C-His6-VanYn flow through at 6 M (lane 4), 4 M (lane 5) and 1 M (lane 6) urea. Purified N-His6-VanYn (lane 7, 2 μg) and C-His6-VanYn (lane 8, 4 μg) after desalting gel permeation. The purified VanYn migrated as a single band at a molecular mass of ~ 25 kDa and with > 90% purity. Standard reference protein: C-His6-VanYn from E. coli BL21(DE3) Star (5 μg, 25 kDa). LMW, molecular mass standard proteins (Amersham, GE-Healthcare).