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Solutions: Use differently tagged protease: His-tagged protein + GST-tagged TEV Then: Ni-NTA (removes uncleaved protein) Glutathione (removes TEV) Use untagged protease: Commercially available Requires additional purification step to separate by size or charge Use immobilized protease: Protease covalently attached to resin Cleavage in batch, then filter Protease stays on resin Use biotinylated protease: Cleave in solution Remove protease with streptavidin beads Choosing the Right Protease TEV Protease Recognition site: ENLYFQS (or G) Advantages: Very high specificity Works at 4C (slowly) or room temperature (faster) Well-characterized Easy to produce in-house Disadvantages: Leaves scar (serine or glycine on target N-terminus) Can cleave non-canonical sites in some proteins Inhibited by zinc, oxidizing conditions Slow at 4C (overnight typical) Best for: General use, temperature-sensitive proteins, high-specificity requirements HRV3C (PreScission) Protease Recognition site: LEVLFQGP Advantages: Highly active at 4C (minutes to hours) Tolerates wider range of buffer additives than TEV Good specificity Disadvantages: Leaves GP scar on target Commercial sources can be expensive Somewhat less specific than TEV Best for: Cold-sensitive proteins, fast processing, high-throughput Thrombin Recognition site: LVPRGS Advantages: Very fast cleavage Works well at room temperature Cheap and widely available Disadvantages: Commercial preparations often contaminated Inhibited by reducing agents Requires calcium Best for: Quick pilot experiments, proteins without secondary thrombin sites Factor Xa Recognition site: IEGR Advantages: Leaves only arginine attached to target Fast cleavage Disadvantages: Non-specific cleavage common Requires calcium Sensitive to buffer conditions Best for: When minimal scar is important, robust proteins SUMO Protease Recognition site: SUMO domain fold (not sequence) Advantages: Recognizes structure, not sequence No scarnative N-terminus SUMO tag enhances expression and solubility Extremely high specificity Disadvantages: Requires SUMO tag (not universal) Commercial enzyme expensive SUMO tag is large (~11 kDa) Best for: When native N-terminus is essential, difficult-to-express proteins The Troubleshooting Decision Tree Cleavage failure

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