Backbone Expansion via Enzyme-Mediated Side-Chain Transfer

There exists no straightforward strategy to generate protein materials containing internally spliced non-protein matter, especially at lengths and scales that support functional characterization. Here we describe Backbone Expansion via Enzyme-mediated Side-chain Transfer (BEST), a facile and general strategy for generating proteins containing internally spliced, non-protein matter at scale. Protein substrates for BEST contain a reactive glutamine (Gln), a backbone ester linkage, and a non-canonical side chain carrying a bioorthogonal reaction handle; the latter two elements are easily installed using a non-canonical ⍺-hydroxy acid. The bioorthogonal side chain is first extended via a click reaction with an element of non-protein matter that terminates in a primary amine. Treatment of this side-chain extended protein with transglutaminase (TGase) results in an intramolecular reaction between the glutamine side chain and the primary amine, generating an ester-containing macrocycle; selective cleavage of the macrocycle under basic conditions inserts the non-protein matter into the backbone with a minimal scar. Thus rather than excising an internal sequence element to restore polypeptide backbone continuity as in an intein-catalyzed reaction, BEST inserts diverse non-protein matter between two neighboring residues and subsequently cleaves the backbone between them. By transforming the protein backbone itself into an addressable locus for synthetic macromolecular integration, BEST establishes a general framework for constructing protein-polymer hybrids in which abiotic matter is not appended to a protein but structurally encoded within.