Expression, purification and kinetic characterisation of human tissue transglutaminase

Protein Expr Purif. 2013 Jan;87(1):41-6. doi: 10.1016/j.pep.2012.10.002. Epub 2012 Oct 22.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromatography, Affinity
  • Cloning, Molecular
  • Enzyme Inhibitors / chemistry
  • Enzyme Stability
  • Escherichia coli
  • GTP-Binding Proteins
  • Glutathione Transferase / genetics
  • Guinea Pigs
  • Humans
  • Kinetics
  • Liver / enzymology
  • Proteolysis
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification
  • Transglutaminases / antagonists & inhibitors
  • Transglutaminases / biosynthesis*
  • Transglutaminases / genetics
  • Transglutaminases / isolation & purification

Substances

  • Enzyme Inhibitors
  • Recombinant Fusion Proteins
  • transglutaminase 2
  • Transglutaminases
  • Glutathione Transferase
  • GTP-Binding Proteins