title: Expression, purification and kinetic characterisation of human tissue transglutaminase.
abstract: The expression of soluble recombinant transglutaminase (TGase) has proven to be a challenge for many research groups. Herein, we report a complementary method for the expression, in BL21(DE3) Escherichia coli, of recombinant human tissue transglutaminase (hTG2) whose solubility is enhanced through N-terminal fusion to glutathione S-transferase (GST). Moreover, we report the cleavage of the GST tag using PreScission™ Protease (PSP) and purification of hTG2 in its untagged form, distinctively suitable for subsequent studies of its remarkable conformational equilibrium. The effects of co-solvents and storage conditions on stability of purified hTG2 are also reported. Furthermore, we demonstrate for the first time the use of a convenient chromogenic assay to measure the activity of the human enzyme. The utility of this assay was demonstrated in the measurement of the kinetic parameters of a wide variety of substrates and inhibitors of both hTG2 and the extensively studied guinea pig liver TGase. Finally, comparison of these results provides further evidence for the functional similarity of the two enzymes.
authors.affiliation: Département de chimie, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal, PQ, Canada H3C 3J7.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Roy
authors.forename: Olivia
authors.initials: O
authors.lastname: Smith
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Clouthier
authors.forename: Jeffrey W
authors.initials: JW
authors.lastname: Keillor
mesh_terms: D000818:Animals; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli; D019204:GTP-Binding Proteins; D005982:Glutathione Transferase / Q000235:genetics; D006168:Guinea Pigs; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011503:Transglutaminases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins