USE OF A FUSION PROTEIN AS A MEANS OF LOCALISING OF TISSUE TRANSGLUTAMINASE

Transglutaminases are a family of Ca2+dépendait enzymes which post-translationally modify proteins leading to protein crosslinking via 8(y-glutamyl)lysine bridges. There is now increasing evidence to suggest that the most ubiquitous member of this family the tissue transglutaminase (tTG) may play an important role in the configuration and stabilisation of the extracellular matrix. However, there is no evidence to suggest how the enzyme is secreted from cells since it possesses no signal peptide sequence that would facilitate its secretion by conventional means. To study the passage of tTG from the intracellular to the extracellular environment we have engineered a tissue transglutaminase-βgalactosidase fiision protein and in order to specifically localise tTG in the intra and/or extracellular environment. The tissue transglutaminase cDNA has been subcloned into a galactosidase-expressing plasmid (pCHK), keeping the cDNA of each enzyme protein in frame with one another. This synthesised plasmid has then been successfully transfected into a number of mammalian cells whereby its subcellular localisation has been detected using immunofluorescence and immunogold microscopy.