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. Author manuscript; available in PMC: 2019 Jul 1.
Published in final edited form as: Cancer Immunol Res. 2018 May 23;6(7):870–880. doi: 10.1158/2326-6066.CIR-17-0661

Figure 1.

Figure 1

VHHCD11b-E749–57 conjugate design, purification, and validation. A) i. VHHCD11b (yellow) was cloned from the VHH genes of an immunized alpaca, expressed with a C-terminal sortase-recognition motif (LPETG), and purified via FPLC. ii. The E749–57 antigen (green) was synthesized with a G3 motif, and was site-specifically conjugated to VHHCD11b via a sortase A (purple)-mediated reaction. iii. The reaction mixture was purified in two steps. First, Ni-NTA beads were used to remove His-tag containing sortase A and unreacted VHHCD11b. Second, size exclusion was used to remove unreacted E749–57 antigen. B) Chromatograms showing the retention times of different species on a C8 HPLC column of (i) the purified sortase-ready VHH, (ii) the complete reaction mixture, and (iii) the final product following two-step purification. C i–iii) The calculated and observed mass(es) of the primary peaks (at 1.36, 1.43, and 1.43 min in the panels above, respectively) of the corresponding HPLC chromatograms.