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Figure 2.
Expressed protein ligation (EPL)
The amino-terminal peptide of the target protein is expressed in vivo as a fusion protein with an intein that has been modified to eliminate the branched ligation. Thiols (R = benzyl, phenyl, alkyl, CH2CH2SO3Na) are used to cleave the intein and generate a reactive carboxy-terminal thioester. This construct is then reacted with a peptide generated by solid phase peptide synthesis that contains an amino-terminal cysteine and an unnatural amino acid(s) in the position of interest. Reaction between the two constructs results in thiotransesterification, followed by S to N acyl shift to regenerate a peptide. There are many variations on this general theme. |
