Effect of substitution of Cys11 by Ser on the activity and dimerization of one of rice thioredoxin isoforms (OsTrx23) using site-directed mutagenesis

• Main Authors: Mitra Roudgar Nashta, Azar Shah Piri
• Format: Article
• Language: fas
• Published: Shahid Bahonar University of Kerman 2015-08-01
• Series: مجله بیوتکنولوژی کشاورزی
• Subjects: thioredoxin; site-directed mutagenesis; cysteine; protein dimerization
• Online Access: https://jab.uk.ac.ir/article_1362_e314e471d61bf2005aedf35576fcfcab.pdf

Thioredoxins (Trxs) are low-molecular-mass proteins with two cysteins in their active site WC(G/P)PC which are involved in reversible reduction of disulfide bonds. In contrast to animals and prokaryotes that typically possess one or a few genes encoding Trxs, higher plants contain eight different Trx types: <em>f</em>, <em>m</em>, <em>x</em>, <em>y</em>, <em>z</em>, <em>o</em>, <em>s</em>, and <em>h</em>. The cytoplasmic type Trxs (h- type) constitute a particularly large subgroup in higher plans. For instance, in the genome of rice (<em>Oryza sativa</em>), nine genes encoding putative Trx h were identified. Among these nine isoforms OsTrx20, OsTrx1, OsTrx18 and OsTrx23 have additional cys residue in N-terminal in comparison to the other OsTrxh isoforms. In order to study the critical role of this cysteine in OsTrx23, we replaced it with serine using site-directed mutagenesis. Both wild type and mutant proteins were heterologously expressed in Rosetta (DE3)- a strain of <em>Escherichia coli</em>-. The purification of these proteins enabled us to compare their activity in reaction with insulin as substrate. In addition, the dimerization of proteins were analysed under non- reducingand reducing condition with DTT. The results shows that whereas wild type OsTrx23 is present in monomer and dimer forms, the mutant OsTrx23_(C11S) is dominantly in monomer form. The activity of mutant was almost similar to wild type. These results suggest that Cys11 at the N-terminal of OsTrx23 has a key role in dimerization of this protein by creating disulfide bonds.