Abstract
To rigorously assess the dynamics and stability of plant microtubules, it is necessary to purify single tubulin isotypes on a large scale and analyze in vitro assembly properties. Expression system in E.coli is not applicable because biogenesis of assembly competent needs eukaryote-specific folding cofactors. To develop large scale tubulin expression and purification systems, we first utilized tobacco BY-2 cells.
Arabidopsis Α-tubulin was tagged with myc and hexahistidine at C-terminus, transformed to BY-2 by Agrobacterium, and overexpressed under CaMV35S promoter. We are currently purifying the recombinant tubulin by chelating affinity chromatography from crude extracts of an overproducing transgenic line.
We are also creating another expression system in budding yeast. Arabidopsis Α -tubulin and Β-tubulin were cloned in yeast expression vectors and will be co-expressed in yeast cells.
