title: Site-specific incorporation of fluorotyrosines into the R2 subunit of E. coli ribonucleotide reductase by expressed protein ligation.
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 (F(n)Ys) 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 F(n)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 F(n)Y at residue 356. The procedure to generate 100 mg quantities of Y356F(n)Y-R2 takes 3-4 months.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139-4307, USA.
authors.forename: Mohammad R
authors.initials: MR
authors.lastname: Seyedsayamdost
authors.forename: Cyril S
authors.initials: CS
authors.lastname: Yee
authors.forename: Joanne
authors.initials: J
authors.lastname: Stubbe
mesh_terms: D004926:Escherichia coli; D015335:Molecular Probes / Q000096:biosynthesis*; D015394:Molecular Structure; D015202:Protein Engineering / Q000379:methods*; D019154:Protein Splicing / Q000235:genetics*; D012264:Ribonucleotide Reductases / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D015335:Molecular Probes; C001245:3-fluorotyrosine; D014443:Tyrosine; D012264:Ribonucleotide Reductases