Abstract
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. 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. 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. 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.
Highlights
Transglutaminases (TGs) are a protein family consisting of 9 members in mammals: factor XIII, protein band 4.2, and transglutaminases 1 to 7.1-3 They belong to a papainlike superfamily.[1,4] One of the most attractive members of this family is transglutaminase 2.Transglutaminase 2 (TG2) known as tissue transglutaminase is a monomeric protein with 76-80 kDa molecular mass which ubiquitously is expressed in many tissues
Western blot confirmed the presence of TG2 and the activity assays including flurometric assay indicated that the recombinant TG2 was functional
The results revealed that transcription of TG2 as a band near 2082 bp in the electrophoresis analysis (Figure 3)
Summary
Transglutaminases (TGs) are a protein family consisting of 9 members in mammals: factor XIII, protein band 4.2, and transglutaminases 1 to 7.1-3 They belong to a papainlike superfamily.[1,4] One of the most attractive members of this family is transglutaminase 2.Transglutaminase 2 (TG2) known as tissue transglutaminase (tTG) is a monomeric protein with 76-80 kDa molecular mass which ubiquitously is expressed in many tissues. TG2 interacts non-covalently with several cellular and extracellular matrix (ECM) proteins such as fibronectin,[4] integrin,[6] and heparan sulfates.[8] The enzyme has been involved (calcium independently) in multiple cellular processes for instance cellular proliferation and signal transduction.[5,9] TG2 has been implicated in some of diseases such as celiac disease,[10] certain types of cancer,[7] and several neurodegenerative diseases including Alzheimer’s disease (AD) (Table 1).[11,12] TG2 has been found as a novel pharmacological target in neurodegenerative diseases.[13] Despite of extensive studies that have been done on different aspects of TG2 specially in physiopathology of diseases,[2,7,14] its functions, activation, and novel inhibitors for the enzyme has not been completely worked out For this reason, we have aimed at the subcloning and expression of TG2 enzyme in this study
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