Application of protein engineering strategies to the redesign of the active site of subtilisin has successfully generated an efficient peptide ligase, subtiligase. The novel enzyme subtiligase has been shown to have many uses, from the total synthesis of RNase A to the semisynthesis of a variety of other proteins. Although the enzyme is in an early stage of development, it shows great promise. Subtiligase will certainly be a useful and important addition to the available strategies for the synthesis of proteins via segment condensation.