Expression ofNeurospora crassaβ-tubulin, target protein of benzimidazole fungicides, inEscherichia coli

Abstract

β-Tubulin of a wild-type Neurospora crassa strain was expressed using pET-16b, pET-29a(+), and pET-32a(+) expression vectors in Escherichia coli BL21 (DE3) strain. Yield of the expressed soluble protein was estimated to be about 0.1 mg ml−1 culture broth. The β-tubulins with S-Tag expressed by pET-29a(+) and pET-32a(+) bound to the S-protein Agarose by affinity binding but the thrombin and enterokinase treatments did not release β-tubulin, suggesting that the protease cleavage sites connecting S-Tag and β-tubulin were not exposed to approach of the proteases. The β-tubulin expressed by pET-16b did not bind to nickel resin, suggesting that its His-Tag was folded into the protein core. The protein expressed by pET-32a(+) was bound to the nickel resin and purified by the column chromatography. © 1999 Society of Chemical Industry