Optimization of TEV protease cleavage conditions.

(A) A schematic model explaining the resistance of the GST-GB1 dual tagged wild type NS4A(1–48) peptide to TEV cleavage. Scissors illustrate the protease while the triangles represent the position of the cleavage sites. GST induced oligomerization of wild type NS4A(1 48) may block the protease cleavage site. (B) Analysis of the urea tolerance of TEV protease activity. GST-GB1 tag removal from NS4A(1 48; L6E, M10E) in the presence of different concentrations of urea (M). Fusion peptides were incubated with TEV protease at 20°C for 16 h at a fusion peptide to protease molar ratio of approximately 100. (C) Binding properties of GST-GB1-NS4A(1–48) to GSH sepharose in the presence of urea. Equal amounts of the fusion peptide were subjected to a mini-scale GSH-affinity chromatography in the presence of different concentrations of urea (M). Input, flow-through and elution fractions were analyzed by 15% SDS-PAGE. (D) TEV cleavage efficiency of wild type NS4A(1–48) peptide in the presence of urea. GST-GB1 tag removal from NS4A(1–48) in the presence of different concentrations of urea given in Molars. Fusion peptides were incubated in the absence (−) or presence (+) of TEV protease at 20°C for 16 h. The different “+” font sizes indicate the increasing amounts of TEV protease with fusion peptide to protease molar ratios of approximately 100, 50 and 10. The progress of the TEV digest is monitored by observing the decrease of the band of the dual tagged GST-GB1-NS4A(1–48) fusion protein and a parallel increase of the free GST-GB1 dual tag band (B, D).