Customizing Native Chemical Ligation: A Case Study on Histone Variant H2A.B from Toolbox to Target

Chemical protein synthesis is a powerful tool that combines solid‐phase peptide synthesis with protein ligations to join synthetic protein segments together. This technology allows for the incorporation of site‐specific native and artificial modifications required for chemical biology research and therapeutic applications. Native chemical ligation (NCL), in which protein segments are joined together through a native amide bond, is the most widely used protein ligation strategy and has been employed in the synthesis and semi‐synthesis of many modified proteins. Whilst recent advances have extended the range of chemical strategies and ligation junctions available, the selection and optimization of an efficient route to a target protein can be challenging and time‐consuming. For most target proteins, several different approaches are possible, and their efficiency and sequence compatibility, as well as the protein properties and purpose of the target protein, must be evaluated. Here, several approaches to the synthesis of a target case study protein, the histone H2A.B variant are demonstrated, illustrating some of the potential challenges and troubleshooting options. This work provides a demonstration of the leading tools for NCL, to guide new users in the selection and optimization of appropriate strategies for targeting a diverse range of protein sequences and properties.