Oral Presentation The 44th Lorne Conference on Protein Structure and Function 2019

Protein engineering through tandem transamidation (#49)

Robert E. Thompson 1 , Adam J. Stevens 1 2 , Tom W. Muir 1
  1. Department of Chemistry, Princeton University, Princeton, NJ 08544, USA
  2. Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94158, USA

Proteins engineered to contain non-coded elements such as post-translational modifications (PTMs) represent a powerful class of tools for interrogating biological processes (1,2). Access to these complex macromolecules, whether through the ribosomal incorporation of unnatural amino acids or chemical tailoring of proteins in vitro, has benefited from the amalgamation of chemical synthesis and recombinant DNA technologies, allowing access to molecular space beyond the reach of either field in isolation. Ligation-based methods of protein semisynthesis, where targets are assembled from building blocks comprising synthetic peptides and recombinant proteins (3,4), are widely used for the generation of proteins bearing non-coded elements. Despite impressive progress made in the protein ligation field as a whole, many large and complex protein targets remain beyond the reach of existing technologies. We have developed a chemoenzymatic method of protein semisynthesis that allows broad access to chemically modified proteins in high yields, from native peptides and recombinant proteins at low substrate concentrations. The approach involves a tandem transamidation reaction cascade that integrates intein-mediated protein splicing with enzyme-mediated peptide ligation – Transpeptidase-Assisted Intein Ligation (TAIL) (5). We show that TAIL can be used to introduce PTMs and biochemical probes into a range of proteins including linker histone H1, Cas9 nuclease and the transcriptional regulator MeCP2, which causes Rett Syndrome when mutated. The versatility of the approach is further illustrated through the chemical tailoring of histone proteins within a native chromatin setting. 

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  4. Muir, T. W., Sondhi, D. & Cole, P. A. Expressed protein ligation: A general method for protein engineering. Proc. Natl. Acad. Sci. U. S. A. 95, 6705-6710 (1998).
  5. Thompson, R. E., Stevens, A. J., & Muir, T. W. Protein Engineering through Tandem Transamidation. (Submitted)