Fig. 3 Site-specific labeling of the pol III clamp loader with FITC. (a) SrtA catalyzes the cleavage between T and
G in the LPXTG motif and subsequent peptide bond formation between T and the amino group of the
fluorescently labeled peptide that has to start with at least one G. The LPETG SrtA recognition sequence
and an MBP-tag were added to the C-terminus of the ψ subunit of the clamp loader by cloning. MBP is cleaved
off during the labeling reaction. (b) Nonreacted FITC-peptide, SrtA, and MBP can be separated from FITClabeled clamp loader by SEC. Elution was monitored using absorption at 280 nm (blue) and 494 nm (green).
Numbers 1–8 indicated in the elution profile represent the samples loaded on the SDS-PAGE in (c). (c)
Samples of the reaction mixture before (À) and after (+) addition of SrtA and FITC-peptide, and aliquots of SEC
fractions (1–8 as indicated in b) were analyzed by a 12% SDS-PAGE. The left panel shows the gel after
Coomassie staining; on the right panel, fluorescence was detected after excitation at 312 nm. For better
orientation, ψ-MBP, FITC-ψ, and FITC-peptide are marked with arrowheads
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Andrea Bogutzki and Ute Curth
G in the LPXTG motif and subsequent peptide bond formation between T and the amino group of the
fluorescently labeled peptide that has to start with at least one G. The LPETG SrtA recognition sequence
and an MBP-tag were added to the C-terminus of the ψ subunit of the clamp loader by cloning. MBP is cleaved
off during the labeling reaction. (b) Nonreacted FITC-peptide, SrtA, and MBP can be separated from FITClabeled clamp loader by SEC. Elution was monitored using absorption at 280 nm (blue) and 494 nm (green).
Numbers 1–8 indicated in the elution profile represent the samples loaded on the SDS-PAGE in (c). (c)
Samples of the reaction mixture before (À) and after (+) addition of SrtA and FITC-peptide, and aliquots of SEC
fractions (1–8 as indicated in b) were analyzed by a 12% SDS-PAGE. The left panel shows the gel after
Coomassie staining; on the right panel, fluorescence was detected after excitation at 312 nm. For better
orientation, ψ-MBP, FITC-ψ, and FITC-peptide are marked with arrowheads
404
Andrea Bogutzki and Ute Curth