Protein conjugates are very important molecules for the treatment and detection of diseases. Chemical methods to attach new functionality to proteins can be very effective but often suffer from lack of specificity, instability, or harsh conjugation conditions. Chemoenzymatic modification of proteins is an attractive option to create highly specific conjugates in gentle, biological conditions; however, these methods often require expensive specialized substrates, bulky fusion tags, and extra purification steps to achieve the desired conjugate, sometimes in intractably low yield. Staphylococcus aureus sortase A and its engineered variants are used to attach oligoglycine derivatives to the C-terminus of proteins expressed with a minimal LPXTG tag. This strategy has been used extensively to make valuable conjugates in vitro, and to make protein-protein conjugates in living cells. Despite the simplicity of sortase-based labeling, several hurdles exist to limit the practicality of widespread use and scaling of sortase-based conjugation. Here we demonstrate that by altering reaction conditions, engineered variants of this enzyme can have activity that allow proteins to be tagged using a diverse set of non-biological functional groups, including several adaptors for bioorthogonal “Click” labeling. We show this labeling works on several model proteins, including medically relevant antibody derivatives for imaging or therapeutic use. Importantly, this technique also works in living Escherichia coli, enabling simple, inexpensive production of chemically functionalized proteins with no additional purification steps. We are currently further engineering this promiscuous enzyme for higher activity on specific substrates and effective bioconjugation in eukaryotic cells.

In vivo site-specific protein tagging with diverse amines using an engineered sortase variant

253rd National Meeting (2017)

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