Preparation:
4 different bacterial species were tested by cloning and expressing the S components of their ECF transporter for riboflavin. the RibU S-component was then eluted via affinity chromatography. After the protein was obtained it was used to create crystals for protein crystallisation.
RibU-T-A-A’ complex:
The RibU components and A component were purified separately. A His-tag (6 histidines residues) was added to the C-terminus of the A-component and to the N-terminus of the RibU component. This allowed them to be purified. However it was apparent that the T-component was not purified in the absence of all 3 other components (all 3 components were needed to purify the T-component).
In vivo experiments:
2 E.coli riboflavin-auxotrophic strains were used. These 2 mutant strains were unable to grouw in normal medium, but were able to grow after the addition of 20mgl-1 of Riboflavin. the mutant E.coli strains were transformed individually with plasmids expressing RibU, T, RibU+T, A+A’ and RibU + T + A + A’ and cultured with 20mgl-1 of Riboflavin and the effects observed.
Mass spectrometry:
The purified RibU protein is yellow, suggesting a bound substrate molecule. Therefore the RibU was centrifuged and the yellow supernatant was diluted and subjected to liquid chromatography mass spectrometry.
