Bifunctional proteins composed of fused domains are one of the most widely used tools in biological science. However, production of fusion proteins does not always proceed smoothly, as there may be difficulty in achieving both reasonable yield and proper folding and function of both domains. Some fusion proteins, such as bispecific antibodies comprised of a typical IgG with an scFv fused to the light chain, are very poorly expressed compared to their individual component domains. While other fusion proteins, such as immunotoxins—fusions of antibodies or antibody fragments to bacterial toxins, consist of domains that require production in different host cells. Here we describe a simple, site-specific enzymatic reaction which ligates independent protein domains to assemble functional fusion proteins in vitro in high yield. This method utilizes the sortase A enzyme to catalyze peptide bond formation between small peptide motifs (LPETG and GGG) that are easily expressed as tags on individual components of the prospective fusion protein. Reaction conditions are mild, and excess of either domain effectively pushes the reaction toward completion. We report successful ligation of 9 pairs of fusion partners, indicating the robust nature of this facile reaction—demonstrating that sortassembly is a broadly tractable approach for producing bifunctional proteins when conventional fusion methods fail.

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