Crystallization and preliminary X-ray analysis of tubulin-folding cofactor A from Arabidopsis thaliana

Abstract

Tubulin-folding cofactor A (TFC A) is a molecular post-chaperonin that is involved in the beta-tubulin-folding pathway. It has been identified in many organisms including yeasts, humans and plants. In this work, Arabidopsis thaliana TFC A was expressed in Escherichia coli and purified to homogeneity. After thrombin cleavage, a well diffracting crystal was obtained by the sitting-drop vapour-diffusion method at 289 K. The crystal diffracted to 1.6 A resolution using synchrotron radiation and belonged to space group I4(1), with unit-cell parameters a=55.0, b=55.0, c=67.4 A.

Figure 1

SDS–PAGE and Western blotting analyses of the proteins. (a) SDS–PAGE gel stained with Coomassie Brilliant Blue. Lane 1, molecular-weight markers (kDa); lane 2, His-tagged TFC A protein eluted from the Superdex 75 column; lane 3, dissolved TFC A crystals from protein that had been stored for three weeks at 277 K. (b) Western blotting analysis. The samples in lanes 4 and 5 correspond to those in lanes 2 and 3, respectively, but were diluted 100-fold.

Figure 2

Crystal of TFC A grown by the sitting-drop method. The dimensions of the crystal are approximately 0.2 × 0.2 × 0.1 mm.

Figure 3

Diffraction pattern of the TFC A crystal. (a) A typical diffraction image. (b) An enlarged view showing the highest resolution spots.