Investigation of structural features of mutant Metallothioneins I: Hingeless T. durum metallothionein (Mutant metallotionin proteininin yapısal özellikleri: köprüsüz T. durum metallotionini)
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Aktürk, Anıl and Yeşilırmak, Filiz and Sayers, Zehra (2009) Investigation of structural features of mutant Metallothioneins I: Hingeless T. durum metallothionein (Mutant metallotionin proteininin yapısal özellikleri: köprüsüz T. durum metallotionini). In: 14th Biomedical Engineering Conference (BIYOMUT 2009), Izmir, Turkey
Metallothioneins (MTs) are classified as cysteine (Cys) rich, metal binding, low molecular weight , ubiquitously expressed  and non-enzymatic  proteins. First MT to be identified was a Cd-binding protein isolated from horse kidney. Initially, MT protein family was thought to be specific to animals; however, further studies showed that MTs are synthesized also by plant and prokaryotic cells . An MT gene, dmt, was identified in durum wheat and was cloned in E. coli . The protein coded by this gene, dMT which binds Cd2+, has a three-domain structure: the metal binding N- (1-19th residues) and C-termini (61-75th residues) and a long hinge region (20-60th residues). Cys are distributed equally in N- and C-termini with a “Cys-X-Cys” motif, where X is any amino acid other than Cys. Hinge region possesses no Cys. According to these features, dMT has been classified as a Type I plant MT . dMT was expressed in E.coli as a GST (glutathion-S-transferase) fusion protein (GSTdMT)  and GSTdMT and the protein cleaved from GST, dMT, were purified and investigated by both biochemical and biophysical methods . In this poster investigations on a mutant dMT, in which the hinge region connecting the two metal binding domains is removed will be presented. Removal of the hinge region and re-linking of the sequences coding for the terminal domains were achieved by PCR and the “chimeric” dMT is inserted into the vector pEGX4T-2 for over expression in E. coli as a GST fusion. Results of cloning and expression studies will be presented and discussed in terms of stability and metal binding properties of dMT.
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