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. Author manuscript; available in PMC: 2019 Sep 25.
Published in final edited form as: Nat Methods. 2019 Mar 25;16(4):319–322. doi: 10.1038/s41592-019-0357-3

Fig. 2. SNAC-tag cleavage in fusion proteins.

Fig. 2

a. TEV protease cleavage of HisTag-ENLYFQS-HB2225 construct, cleavage conditions: protein 1 mg/mL, 50 mM Tris, pH 8.0, 0.5 mM EDTA, 1 mM DTT, 0.25 M NaCl, 22 °C, 16 hours, TEV protease 0.04 mg/mL. b. Ni2+ cleavage of HisTag-GSHHW-HB2225 construct, cleavage conditions: protein 1 mg/mL, 1.0 mM NiCl2, 0.1 M CHES, 0.1 M acetone oxime, 0.1 M NaCl, pH 8.2, 22 °C, 16 hours. c. Cleavage compatibility test for Ni2+ cleavage of HisTag-GSHHW-HB2225. d. TEV protease cleavage of HisTag-T4L-ENLYFQS-3hbtmV2 construct, cleavage conditions: protein 1 mg/mL, 50 mM Tris, pH 8.0, 0.5 mM EDTA, 1mM DTT, 0.25 M NaCl, 22 °C, 5 mM DPC, 16 hours, TEV 0.04 mg/mL. e. Ni2+ cleavage of HisTag-T4L-GSHHW-3hbtmV2 construct, cleavage conditions: protein 1 mg/mL, 1.0 mM NiCl2 0.1 M CHES, 0.1 M acetone oxime, 0.1 M NaCl, pH 8.2, 22 °C, 5 mM DPC, 16 hours. f. Cleavage compatibility test for Ni2+ cleavage of HisTag-T4L-GSHHW-3hbtmV2. g. Ni2+ cleavage of HisTag-T4L-GSHHW-3hbtmV2 in different detergents. h. Cleavage of the membrane protein HisTag-T4L-XXXXXXXX-3hbtmV2. Lane 1 shows protein before cleavage, lanes 2 to 14 shows cleavage using difference sequences from phage selections, the sequence inserted for each lane is shown on top of the lane. i. Cleavage of the globular protein construct HisTag-XXXXXXXX-HB2225, lane 1 shows the position of the protein before cleavage, lane 2 to lane 13 shows cleavage using difference sequences from phage selection, the sequence inserted for each lane is shown on top of the lane. Cleavage percentages were quantified based on cleaved and uncleaved protein bands in each individual lane in Imagelab 5.2.1 and is summarized in Supplement Table 1, 2. Full gels are shown in Supplementary Fig. 6.