Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein
Figure 7
Immobilization of biotinylated enhanced green fluorescent protein (EB) to a solid surface.
A, The biotinylation-purification method. The precursor protein (EINC) in a cell lysate is first bound to chitin beads through its chitin binding domain (C). After washing away the unbound proteins, the beads are incubated with the peptide IC-B that consists of IC followed by the sequence SAGSK with biotin linked to the lysine (K) side chain. A trans-splicing reaction produces the biotinylated target protein (EB) that can bind to streptavidin-coated beads (S). In the experimental proof, streptavidin-coated beads were incubated either with the EINC precursor in a cell lysate (panels 1 and 2) or with the biotinylated and purified EB protein (panels 3 and 4). Panels 1 and 3 are differential interference contrast images, while panels 2 and 4 are fluorescence micrographs (45 ms exposure time). B, The biotinylation-fixation method. The IC-B peptide is first bound to streptavidin-coated beads and then incubated with the precursor protein EINC in a cell lysate to achieve trans-splicing, and the resulting biotinylated target protein (EB) is automatically fixed to the beads. In the experimental demonstration, streptavidin-coated beads with (panels 3 and 4) or without (panels 1 and 2) the pre-bound IC-B peptide were incubated with the EINC protein in an E. coli cell lysate for trans-splicing. After washing away unbound proteins, the beads were photographed as differential interference contrast images (panels 1 and 3) or as fluorescence images (panels 2 and 4, 600 ms exposure time).