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
Related Questions
- In the case of the C-terminal fusion vectors (pTYB1,2,3 and 4), which residues at the C-terminal of the target protein may inhibit cleavage or cause in vivo cleavage?
- In the case of C-terminal fusion vectors, if the EcoRI site is used as the 3´ cloning site, which vector should be chosen?
- How can the C-terminal fusion vectors be used to label the C-terminus of the target protein?
