Expressed Protein Ligation at Methionine: N-Terminal Attachment of Homocysteine, Ligation, and Masking

The synthesis of chemically-modified proteins is a valuable tool for the study of their function.[1] One of the most enabling protein synthesis technologies is native chemical ligation (NCL), in which a peptide thioester is reacted with a peptide bearing an N-terminal Cys to form a native amide bond.[2] In many cases, one of these two fragments can be expressed in E. coli and then ligated to a smaller synthetic peptide so that a large semi-synthetic protein can be made with a minimum of peptide synthesis. This sort of expressed protein ligation (EPL) can be implemented in two ways.[3] For a synthetic C-terminus, the N-terminal region can be expressed as a fusion to an intein and then converted to a thioester for ligation. Likewise, a protein can be expressed with an N-terminal Cys and ligated to a synthetic thioester. EPL is an extremely efficient way of making large quantities of synthetic proteins. However, there are limitations to the method, most notably, the need for Cys at the site of ligation.