Chemoenzymatic Semisynthesis of Proteins

Abstract

Protein semisynthesisdefined herein as the assembly of a protein from a combination of synthetic and recombinant fragmentsis a burgeoning field of chemical biology that has impacted many areas in the life sciences. In this review, we provide a comprehensive survey of this area. We begin by discussing the various chemical and enzymatic methods now available for the manufacture of custom proteins containing noncoded elements. This section begins with a discussion of methods that are more chemical in origin and ends with those that employ biocatalysts. We also illustrate the commonalities that exist between these seemingly disparate methods and show how this is allowing for the development of integrated chemoenzymatic methods. This methodology discussion provides the technical foundation for the second part of the review where we cover the great many biological problems that have now been addressed using these tools. Finally, we end the piece with a short discussion on the frontiers of the field and the opportunities available for the future.

Development and application of ubiquitin-based chemical probes

“…Protein semisynthesis methods may be a useful alternate. [70][71][72][73][74] 3.2.1 Recombinant production of Ub thioesters. Ub thioesters can be produced through intein splicing, wherein an expressed protein undergoes an intramolecular N-to-S acyl transfer to produce a protein thioester with the expulsion of the intein 75,76 (Fig.…”

“…Ub conjugates incorporating isopeptide bond mimetics can also be synthesized from the expressed Ub [70][71][72][73]84,85 (Fig. 7).…”

Chemical Biology of Autophagy-Related Proteins With Posttranslational Modifications: From Chemical Synthesis to Biological Applications

“…Alternatively, enzyme-mediated ligations such as Sortase Amediated ligation (SML) and thiol-independent peptide ligations techniques such as serine/threonine ligation (STL) and αketoacid-hydroxylamine (KAHA) ligation were developed and applied for protein chemical synthesis (Bode, 2017;Schmidt et al, 2017;Liu and Li, 2018;Thompson and Muir, 2020). Sortase A is a calcium-dependent transpeptidase isolated from Staphylococcus aureus in the 1990s (Mazmanian et al, 1999).…”

Site-Specific Modification and Segmental Isotope Labelling of HMGN1 Reveals Long-Range Conformational Perturbations Caused by Posttranslational Modifications

“…38,39 Recent developments and combinations with enzymatic ligations have greatly expanded the scope of protein synthesis and semi-synthesis and allow for incorporation of site-specific PTMs and combinations of PTMs, in principle, in any part of the protein. 38,40 Furthermore, protein semi-synthesis also allows for introduction of site-specific and segmental isotope labels that are often essential for studying structural and binding properties of proteins by NMR spectroscopy. 41 NMR is a powerful technique for studying dynamic and intrinsically disordered proteins like HMGN1, which are not amenable to X-ray crystallography or cryo-electron microscopy.…”