Yaghoub Yazdani
1,2, Shahram Azari
2, Hamid Reza Kalhor
3*1 Infectious Diseases Research Center and Laboratory Sciences Research Center, Golestan University of Medical Sciences, Gorgan, Iran.
2 Department of Molecular Medicine, Faculty of Advanced Medical Science Technologies, Golestan University of Medical Sciences, Gorgan, Iran.
3 Biochemistry Research Laboratory, Department of Chemistry, Sharif University of Technology, Tehran, Iran.
Abstract
Purpose: Tissue transglutaminase (TG2) is a unique
multifunctional enzyme. The enzyme possesses enzymatic activities such as
transamidation/crosslinking and non-enzymatic functions such as cell migration
and signal transduction. TG2 has been shown to be involved in molecular
mechanisms of cancers and several neurodegenerative diseases such as
Alzheimer’s disease. The present study aimed at cloning and expression of full
length human TG2 in Bac-to-Bac baculovirus expression system and evaluation of
its activity.
Methods: pFastBac HTA donor vector containing coding sequence
of human TG2 was constructed. The construct was transformed to DH10Bac for
generating recombinant bacmid. The verified bacmid was transfected to insect
cell line (Sf9). Expression of recombinant TG2 was examined by RT-PCR, SDS-PAGE
and western blot analysis. Functional analysis was evaluated by fluorometric
assay and gel electrophoresis.
Results: Recombinant bacmid was verified by amplification of a
band near to 4500 bp. Expression analysis showed that the enzyme was expressed
as a protein with a molecular weight near 80 kDa. Western blot confirmed the
presence of TG2 and the activity assays including flurometric assay indicated
that the recombinant TG2 was functional. The electrophoresis assay conformed
that the expressed TG2 was the indeed capable of crosslinking in the presence
of physiological concentration calcium ions.
Conclusion: Human TG2 was expressed efficiently in the active
biological form in the Bac-to-Bac baculovirus expression system. The expressed
enzyme could be used for medical diagnostic, or studies which aim at finding
novel inhibitors of the enzymes . To best of our knowledge, this is probably
the first report of expression of full length human tissue transglutaminase
(TG2) using the Bac-to-Bac expression system.