Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein
Figure 2
Protein C-terminal labeling with a fluorophore.
A. Schematic illustration of the labeling reaction. IN and IC: components of the Ssp GyrB S11 split-intein. M: maltose binding protein as the target protein to be labeled. C: chitin-binding domain as the affinity binder for purification of the precursor protein. L: 5-carboxyfluorescein as the labeling group. In the synthetic peptide IC-L, IC is connected to L by the sequence SAGSGK, with L attached to the side chain of the K (lysine) residue. B. Analysis of the labeling results. The purified precursor protein was incubated at room temperature for 16 hours, with or without the peptide and the reducing agent TCEP, as indicated. The reaction products were resolved through SDS-PAGE and visualized either by Coomassie staining, by Western blotting using anti-C antibodies, or by fluorescence scan (excitation at 488 nm, filter for 520 nm). Positions of the precursor protein (MINC), the labeled protein (ML), and the excised N-intein (INC) are indicated. In lane 1, a minor protein band at the same position as ML is the endogenous E. coli maltose binding protein that is known to be co-purified in the amylose affinity chromatography. C. Time-course of the reaction between MINC precursor protein and IC-L peptide in the presence or absence of TCEP. Labeling efficiency was calculated from densitometry analysis on anti-C Western blots. Error bars represent standard deviations from triplicate experiments.