Crystallization and Preliminary X-Ray Analysis of Human Transglutaminase 3 from Zymogen to Active Form
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文摘
Transglutaminases(TGases; protein-glutamine-glutamyl-transferases) are a large family of calcium-dependent acyl-transfer enzymes that catalyze the formation of covalent cross links in proteins. Of these, the “epidermal” or “hair follicle” TGase 3 isoform is critically involved in barrier formation in epithelia. It is a zymogen, requiring proteolytic activation to achieve maximal specific activity. In order to understand its structure and function, we have devised methods for the rapid large-scale expression of the TGase 3 zymogen in the baculovirus system, and here we describe the purification of the zymogen and activated forms. We describe methods for the formation of high-quality, well-diffracting crystals within 3–5 days, using both dioxane and β-octylglucoside to overcome severe twinning problems. The crystal of the zymogen belongs to the triclinic space group P1 and diffracts to 2.2-Å resolution, and the crystal of the active form belongs to the P21 space group at 2.7-Å resolution.