Expression, purification, crystallization and preliminary X-ray diffraction analysis of thioredoxin Trx1 from Saccharomyces cerevisiae

Abstract

Thioredoxins play key roles in the cellular response to oxidative stress. Three isoforms of thioredoxin have been identified in Saccharomyces cerevisiae: two that are cytosolic (Trx1 and Trx2) and one that is mitochondrial (Trx3). In the present work, the cytosolic form Trx1 was cloned, expressed, purified and crystallized. Crystals were obtained by the hanging-drop vapour-diffusion method. A data set was collected from a single crystal to 1.7 A resolution. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 32.29, b = 46.59, c = 64.20 A, alpha = beta = gamma = 90 degrees .

Figure 1

Alignment of the amino-acid sequences of S. cerevisiae Trx1, Trx2 and Trx3. Trx3 has an extra N-terminal signal peptide for mitochondial localization. The figure was produced with ESPript v. 2.2 (Gouet et al., 1999 ▶) based on an alignment using ClustalW2 at EBI.

Figure 2

15% SDS–PAGE analysis of Trx1. Lane 1, Trx1 after gel filtration. Lane 2, low-molecular-weight markers (kDa).

Figure 3

(a) Crystals of S. cerevisiae Trx1 obtained by the hanging-drop vapour-diffusion method. The average dimensions of the crystals were 0.1 × 0.08 × 0.7 mm. (b) X-­ray diffraction pattern at a resolution of 1.7 Å. The crystal-to-detector distance was 125 mm.