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. Author manuscript; available in PMC: 2016 Jan 7.
Published in final edited form as: Angew Chem Int Ed Engl. 2014 Oct 31;54(2):441–445. doi: 10.1002/anie.201408126

Figure 1.

Figure 1

Sortase A catalyzes protein-small molecule isopeptide ligation. (a) Overview of the reaction used to conjugate biotin to the pilin domain in the Fn3-PLN3-ELP fusion protein, along with the expected tryptic peptides of the product (boxed). SrtA-ELP and biotin-LPETGRAGG were used at 2- and 10-fold molar excess to Fn3-PLN3-ELP, respectively. (b) SDS-PAGE of biotinylation reactions with Fn3-ELP (36 kDa) and Fn3-PLN3-ELP (43 kDa) and Western blot with streptavidin-Cy5 indicate that only the reaction containing both the pilin domain and biotin-modified LPETG peptide yields biotinylated target protein. (c) MALDI-TOF spectrum of the unpurified Fn3-PLN3-ELP biotinylation reaction product, corresponding to lane 2 of the Western blot in panel (b), after trypsinization. Biotin is installed specifically at the pilin domain lysine (ions 1 and 2), with all other ions corresponding to unmodified peptides from the Fn3-PLN3-ELP and SrtA-ELP fusion proteins. (d) MALDI-TOF spectrum of the tryptic digest of SrtA-ELP reacted with Fn3-PLN3-ELP without biotin-LPETGRAGG. All peaks corresponding to panel (c) are present except those corresponding to biotinylated ions 1 and 2.