Expression of Functional Recombinant Human Tissue Transglutaminase (TG2) Using the Bac-to-Bac Baculovirus Expression System

摘要

Purpose: Tissue transglutaminase (TG2) is a uniquemultifunctional enzyme. The enzyme possesses enzymatic activities such astransamidation/crosslinking and non-enzymatic functions such as cell migrationand signal transduction. TG2 has been shown to be involved in molecularmechanisms of cancers and several neurodegenerative diseases such asAlzheimer’s disease. The present study aimed at cloning and expression of fulllength human TG2 in Bac-to-Bac baculovirus expression system and evaluation ofits activity.Methods: pFastBac HTA donor vector containing coding sequenceof human TG2 was constructed. The construct was transformed to DH10Bac forgenerating recombinant bacmid. The verified bacmid was transfected to insectcell line (Sf9). Expression of recombinant TG2 was examined by RT-PCR, SDS-PAGEand western blot analysis. Functional analysis was evaluated by fluorometricassay and gel electrophoresis.Results: Recombinant bacmid was verified by amplification of aband near to 4500 bp. Expression analysis showed that the enzyme was expressedas a protein with a molecular weight near 80 kDa. Western blot confirmed thepresence of TG2 and the activity assays including flurometric assay indicatedthat the recombinant TG2 was functional. The electrophoresis assay conformedthat the expressed TG2 was the indeed capable of crosslinking in the presenceof physiological concentration calcium ions.Conclusion: Human TG2 was expressed efficiently in the activebiological form in the Bac-to-Bac baculovirus expression system. The expressedenzyme could be used for medical diagnostic, or studies which aim at findingnovel inhibitors of the enzymes . To best of our knowledge, this is probablythe first report of expression of full length human tissue transglutaminase(TG2) using the Bac-to-Bac expression system.