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Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation

Nature Protocols volume 2, pages 1225–1235 (2007)Cite this article

Abstract

Expressed protein ligation (EPL) allows semisynthesis of a target protein with site-specific incorporation of probes or unnatural amino acids at its N or C termini. Here, we describe the protocol that our lab has developed for incorporating fluorotyrosines (FnYs) at residue 356 of the small subunit of Escherichia coli ribonucleotide reductase using EPL. In this procedure, the majority of the protein (residues 1–353 out of 375) is fused to an intein domain and prepared by recombinant expression, yielding the protein in a thioester-activated, truncated form. The remainder of the protein, a 22-mer peptide, is prepared by solid-phase peptide synthesis and contains the FnY at the desired position. Ligation of the 22-mer peptide to the thioester-activated R2 and subsequent purification yield full-length R2 with the FnY at residue 356. The procedure to generate 100 mg quantities of Y356FnY-R2 takes 3–4 months.

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Figure 1: Proposed radical generation pathway in E. coli RNR based on the docking model of Uhlin and Eklund6.
Figure 2: Fluorotyrosine analogues.
Figure 3
Figure 4: Semisynthesis of R2 via EPL17.
Figure 5: Aspartimide formation during synthesis of FnY-22mer.
Figure 6: A typical analytical reverse-phase HPLC profile of pure 3,5-F2Y-22mer.
Figure 7: MonoQ elution profile after ligation of 3,5-F2Y-22mer to MESNA-activated truncated R2 (ref. 18).

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Acknowledgements

We thank Professor Robert Phillips for the generous gift of the plasmid pTZTPL used for expressing the enzyme TPL, Chia-Hung Wu for helpful comments on the manuscript and the National Institutes of Health for support (GM29595).

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Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139-4307, Massachusetts, USA

    Mohammad R Seyedsayamdost, Cyril S Yee & JoAnne Stubbe

  2. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139-4307, Massachusetts, USA

    JoAnne Stubbe

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Correspondence to JoAnne Stubbe.

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Seyedsayamdost, M., Yee, C. & Stubbe, J. Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation. Nat Protoc 2, 1225–1235 (2007). https://doi.org/10.1038/nprot.2007.159

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