(666f) In Vitro Expression and Post-Translational Tagging of Proteins with Azide Functionalized Myristic Acid Via Endogenous N-Myristoyl Transferase

We have recently described a post-translational protein tagging mechanism carried out in vivo in Escherichia coli that enables selective and site-specific labeling of a protein of interest[1].The eukaryotic post-translational modification enzyme, N-myristoyl transferase (NMT), selectively attaches myristic acid and myristic acid analogues to the N-terminus of proteins that carry a short amino acid NMT recognition peptide. We have demonstrated selective NMT-mediated tagging of recombinant proteins, both natural NMT substrates and proteins engineered to carry the NMT recognition peptide, with an azide functionalized myristic acid analog, 12-azidododecanioic acid (12-ADA). Greater than ninety percent of the recombinantly expressed protein substrate gets tagged with 12-ADA, and the tagging method can be engineered such that the tagged proteins remain active at wild-type levels. Additionally, the bioorthogonality of the 12-ADA enables bioconjugation of the tagged protein directly to alkyne or cyclooctyne functionalized surfaces directly from clarified cell lysates without prior protein purification.

In the current work we demonstrate protein expression and enzymatic tagging in a cell-free translation/transcription (TL/TX) system. Briefly, insect cell extracts were used to express two genes, luciferase and cAMP-dependent protein kinase A (PKA), in the presence and absence of an azide myristic acid analog (12-ADA). PKA is a naturally myristoylated protein; luciferase does not carry the recognition sequence and therefore should not be labeled with 12-ADA by the NMT that is endogenous to the TL/TX system. We find that luciferase and PKA are synthesized at similar levels in the TL/TX system. TL/TX cell extracts containing expressed luciferase and PKA with and without 12-ADA, were treated with an alkyne-TAMRA fluorophore that reacts with azide-labeled protein to produce a covalent fluorescent-protein conjugatevia copper-catalyzed azide-alkyne cycloaddition (copper-catalyzed click chemistry). Only PKA expressed in the presence of 12-ADA is selectively conjugated to alkyne-TAMRA. This result indicates that natural NMT substrate proteins do not get expressed during the TL/TX reaction, and thus do not get tagged with 12-ADA. Significantly, TAMRA conjugation to 12-ADA-PKA occurs within the TL/TX expression volume without the removal of excess free 12-ADA.

In future work, we seek to use our novel protein engineering methods to enable specific capture and interrogation of proteins of interest directly from complex biological mixtures. The successful demonstration of in vitroexpression and co/post-translational tagging of proteins of interest highlighted here will facilitate the development of high-throughput assay platforms to enable detailed studies of protein biochemistry and protein function.

1. Kulkarni, C, Kinzer-Ursem, TL, and Tirrell, DA, Selective Functionalization of the Protein N Terminus with N-Myristoyl Transferase for Bioconjugation in Cell Lysate. ChemBioChem, (2013). 10.1002/cbic.201300453: p. 1958.