Cloning and Expression of Human Recombinant Transglutaminase 6

Abstract

Transglutaminases (TGases) are an enzyme group that catalyses different post-translational modifications in the glutamine amino acid in proteins and peptides. Nine different types of TGase have been characterised, and three of them are closely linked to gluten-related autoimmune disorders. When consuming gluten, patients with celiac disease have autoantibodies against transglutaminase 2 (TG2), while patients with dermatitis herpetiformis have autoantibodies against both TG2 and transglutaminase 3 (TG3). Studies have also shown that patients with neurological manifestations of gluten consumption have antibodies against transglutaminase 6 (TG6).

The immune response that occurs in celiac disease and dermatitis herpetiformis has been well studied. This has been achieved, among other methods, by creating human recombinant TG2 and TG3. The immune response that occurs in patients with neurological manifestations has been less studied. To investigate this immune response, access to recombinant TG6 is necessary. Therefore, the overall aim of this project is to establish a system to produce biotinylated human recombinant TG6.

Recombinant biotinylated TG6 was attempted to be produced in both prokaryotic and eukaryotic expression systems. E. coli was used for prokaryotic expression, while a baculovirus expression system was used for eukaryotic expression in insect cells. Histidine-tag (His-tag) was used for the purification of produced recombinant protein. Several attempts to produce TG6 were conducted, both with an N-terminal His-tag and a C-terminal His-tag. Protein detection was done by SDS-PAGE and Western blot (WB) after expression.

All attempts to produce recombinant TG6 yield a low protein concentration. The results from SDS-PAGE showed that the protein eluate consisted of many proteins, but throughout all production attempts, two protein bands were stronger than the rest. The production with N-terminal His-tag exhibited one band on WB-analysis with anti-His, while the production with C-terminal His-tag exhibited two bands on the same analysis. Production of TG6 in E. coli was not analysed by WB. Mass spectrometry analysis of proteins produced with a C-terminal his- tag verified TG6 in a protein band at approximately 75 kDa. The production of TG6 cannot be achieved in the same quantity as TG2 and TG3. Previous studies have also shown low yield when TG6 has been produced.