FAQ: How can the C-terminal fusion vectors be used to label the C-terminus of the target protein?

The C-terminus of the target protein can be covalently labeled by using L-[35S]-cysteine or by using a biotinylated synthetic peptide with an N-terminal cysteine (see below), immediately following thiol-induced on-column cleavage and elution. The thiol-tagged protein is mixed with the label and incubated at 4°C overnight. The sulfhydryl group of the cysteine can initiate the attack at the thioester bond present at the C-terminus of the target protein. The cysteine residue then forms a stable peptide bond with the C-terminus of the target protein by a spontaneous S-N shift. The L-[35S]-cysteine sample can be analyzed by SDS-PAGE and autoradiography. The biotinylated protein sample can be analyzed by SDS-PAGE followed by a western blot with anti-biotin antibody. The choice of thiol for cleavage and labeling depends on the desired outcome. DTT causes efficient on-column cleavage of the precursor protein and leads to maximum protein yields. However, the percentage of total protein labeled is lower than another thiol, 2-mercaptoethanesulfonic acid (MESNA). The use of MESNA as the thiol to induce on column cleavage has been found to increase the percentage of the total protein that is labeled. However, MESNA is not as efficient at causing on-column cleavage as DTT and so the total yield (labeled and unlabeled) of protein may be lower. In the cases in which a high specific activity or high biotin incorporation is required the use of MESNA is recommended. The incorporation of a biotinylated peptide onto the C-terminus of a protein can be accomplished using freshly isolated target protein and a biotinylated peptide with an N-terminal cysteine. NEB supplies peptides to specifically label the C-terminus of a thioester tagged protein with either a fluorescein (NEB #P6606) or a biotin (NEB #P6607) conjugated molecule.