Sufficient quantities of homogeneous samples of post-translationally modified proteins are often not readily available from biological sources to facilitate structure–function investigations. Native chemical ligation (NCL) is a convenient method for the production of biologically active proteins from smaller fragments. Such an approach allows protein modifications to be introduced in a controlled fashion into smaller peptide fragments which are amenable to total chemical synthesis. These fragments of defined sequence and structure can be elaborated to full-length proteins through NCL reactions with suitable components derived from bacterial origin. This report describes methods for the bacterial production of components for NCL and their use in typical reactions.