Abstract
Semisynthetic proteins engineered to contain non-coded elements such as post-translational modifications (PTMs) represent a powerful class of tools for interrogating biological processes. Here, we introduce a one-pot, chemoenzymatic method that allows broad access to chemically modified proteins. The approach involves a tandem transamidation reaction cascade that integrates intein-mediated protein splicing with enzyme-mediated peptide ligation. We show that this approach can be used to introduce PTMs and biochemical probes into a range of proteins including 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. We expect our approach will extend the scope of semisynthesis in protein engineering.
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Data availability
All relevant data are included in the Supplementary Information and are available from the authors. Plasmids for the following vectors have been deposited, along with maps and sequences, in Addgene: pET30-His6-Sumo-IntNtr-eSrt2A, Addgene plasmid #126015; pET30-His6-Sumo-MBP-CfaN, Addgene plasmid #126016; pET30-MBP-CfaN-His6, Addgene plasmid #126017; pET30-His6-Sumo-CfaC-MBP, Addgene plasmid #126018; pTXB1-His6-IntCtr-GyrA-His6, Addgene plasmid #126019. All other plasmids reported in this manuscript will be available upon request.
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Acknowledgements
The authors thank current members of the Muir laboratory (in particular G. Liszczak, M. Haugbro, A. Burton and J. Gramespacher) for discussions and comments. The authors also thank T. Srikumar of Princeton University Mass Spectrometry Facility. This work was supported by National Institutes of Health (NIH) grants R37 GM086868 and P01 CA196539. R.E.T. was supported by a Charles H. Revson Foundation postdoctoral fellowship and A.J.S. by a National Science Foundation graduate research fellowship (DGE-1148900). We dedicate this paper to R.T. Raines on the occasion of his 60th birthday.
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R.E.T. and T.W.M. conceived the project. R.E.T., A.J.S. and T.W.M. designed all experiments and analysed all data. R.E.T. performed all experiments. R.E.T. and T.W.M. wrote the manuscript.
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The Supplementary Information file contains methods, protein sequences and characterization, as well as supplementary figures and data.
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Thompson, R.E., Stevens, A.J. & Muir, T.W. Protein engineering through tandem transamidation. Nat. Chem. 11, 737–743 (2019). https://doi.org/10.1038/s41557-019-0281-2
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DOI: https://doi.org/10.1038/s41557-019-0281-2
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