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Case Studies Case 1: TEV Won't Cut Protein: 45 kDa enzyme Construct: His6-TEV site-Target Problem: No cleavage after 24 hours with excess TEV Diagnosis: Crystal structure of homolog showed N-terminus buried in a groove TEV site was inaccessible to protease Solution: Redesigned: His6-(GGGGS)-TEV site-(GGGGS)-Target Cleavage now complete in 4 hours Case 2: Thrombin Destroys the Protein Protein: 30 kDa signaling domain Construct: His6-Thrombin site-Target Problem: Multiple bands after cleavage