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

Mohammad R. Seyedsayamdost, Cyril S. Yee, Jo Anne Stubbe

Research output: Contribution to journalArticle

44 Scopus citations

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 Fn Y 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.

Original languageEnglish (US)
Pages (from-to)1225-1235
Number of pages11
JournalNature Protocols
Volume2
Issue number5
DOIs
StatePublished - May 1 2007
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)

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