In native chemical ligation, a peptide containing a C-terminal thioester (often derived from an unnatural amino acid like phenylalanine) is reacted with another peptide bearing an N-terminal cysteine residue. The reaction proceeds through a transthioesterification step, followed by a spontaneous S→N acyl shift, resulting in the formation of a native peptide bond. This method preserves the native conformation of the protein, making it particularly useful for generating proteins with post-translational modifications or unnatural amino acids.
Protein ligation has several advantages over traditional recombinant methods. It allows for the incorporation of non-natural amino acids, post-translational modifications such as glycosylation or phosphorylation, and the assembly of proteins from synthetic or semi-synthetic components. Additionally, it can be used to study protein folding, function, and interactions by enabling the creation of chimeric or hybrid proteins.
Beyond NCL, other ligation techniques include **expressed protein ligation (EPL)**, where a protein fragment is expressed in bacteria with an N-terminal cysteine and then ligated to a synthetic peptide. This method combines the efficiency of recombinant expression with the versatility of chemical ligation. Other variants, such as **oxidative ligation** and **trans-splicing ligation**, have also been developed to expand the scope of protein engineering.
Applications of protein ligation span drug development, vaccine design, and the study of protein structure-function relationships. It has also been instrumental in creating enzymes with improved stability or specificity for biotechnological and industrial applications. Despite its utility, challenges remain, including scalability, yield optimization, and the potential for side reactions that may affect protein integrity.