Crystallization and preliminary X-ray analysis of human transglutaminase 3 from zymogen to active form.
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 beta-octylglucoside to overcome severe twinning problems. The crystal of the zymogen belongs to the triclinic space group P1 and diffracts to 2.2-A resolution, and the crystal of the active form belongs to the P2(1) space group at 2.7-A resolution.
Kim
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HC
Laboratory of Skin Biology, National Institute of Arthritis, Musculoskeletal, and Skin Diseases, Bethesda, Maryland 20892-2752, USA.
Nemes
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Z
Idler
W W
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Hyde
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CC
Steinert
P M
PM
Ahvazi
B
B
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D003451:Cryoprotective Agents / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D004279:DNA, Viral / Q000235:genetics; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000302:isolation & purification*; D005822:Genetic Vectors; D006801:Humans; D008081:Liposomes; D008970:Molecular Weight; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology / Q000821:virology*; D013997:Time Factors; D014162:Transfection; D011503:Transglutaminases / Q000737:chemistry*
D002135:Calcium-Binding Proteins; D003451:Cryoprotective Agents; D004279:DNA, Viral; D004792:Enzyme Precursors; D008081:Liposomes; D011994:Recombinant Proteins; C498712:TGM3 protein, human; D011503:Transglutaminases