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ACS Omega 9(1):817827

Fiorucci S, Biagioli M, Zampella A, Distrutti E

activity lost Diagnosis: Target protein contained internal thrombin-like site Both sites cleaved, fragmenting the protein Solution: Switched to TEV protease No internal TEV sites present Clean cleavage, full activity retained Case 3: Can't Separate Cleaved from Uncleaved Protein: 25 kDa structural protein Construct: His6-TEV site-Target Cleavage: 75% complete (couldn't improve further) Problem: Cleaved protein has native His-rich region, binds Ni-NTA Diagnosis: Subtractive purification impossible Both species bind column Solution: Switched to Strep-tag StrepTactin binding is tag-specific Cleaved protein flows through cleanly The Economics of Proper Design Getting It Wrong Clone construct: 1 week Express, purify: 1 week Attempt cleavage, fail: 1 week Troubleshoot: 2 weeks Redesign construct: 1 week Express, purify, cleave (success): 2 weeks Total: 8 weeks Getting It Right Analyze sequence for cleavage sites: 1 hour Design with flexible linker: 1 hour Clone construct: 1 week Express, purify, cleave (success): 2 weeks Total: 3 weeks The difference: 5 weeks of work, plus the frustration of failed experiments

Elevated soluble CD40 ligand (sCD40L) levels in the serum of patients with B cell lymphoma

IV nutrient therapy can also be used as a preventive measure to help boost your immune system and protect your body from illness

(1098) Bindels, L