Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein
Figure 6
C-terminal biotinylation using the Ssp GyrB S11 split-intein.
A. The three precursor proteins (MINC, EINH, and GST-ACP-INC) were incubated with (+) or without (−) the peptide IC-B in the presence of 0.1 mM TCEP for 18 h at room temperature, and the reaction products were analyzed by Western blotting using antibodies against biotin. From the three precursor proteins, the three target proteins for biotinylation were a maltose binding protein (M, 43 kDa), an enhanced green fluorecent protein (E, 27 kDa), and a glutathione-S-transferase/acyl carrier fusion protein (GST-ACP, 39 kDa), respectively. B. Efficiency of C-terminal biotinylation of the three target proteins as determined by densitometry analysis on Western blots using anti-C antibodies (for MINC and GST-ACP-INC) and anti-H antibodies (for EINH). Error bars represent standard deviations from triplicate experiments.