Gene disruption and biochemical characterisation of 3-isopropylmalate dehydrogenase from Stagonospora nodorum
✍ Scribed by Cooley, R Neil; Monk, Tracy P; McLoughlin, Sheila B; Foster, Stephen G; Dancer, Jane E
- Publisher
- John Wiley and Sons
- Year
- 1999
- Tongue
- English
- Weight
- 200 KB
- Volume
- 55
- Category
- Article
- ISSN
- 1526-498X
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✦ Synopsis
4 CONCLUSION
We succeeded in expressing a fungal b-tubulin, which is contained in a very small amount in fungal cells and difficult to purify, in a large amount using the pET expression vector systems in E coli. The protein expressed using pET-32a(]) was puriüed by nickel resin column chromatography. This will open the way to obtaining b-tubulin for three-dimensional structure analysis of the complex with fungicide by NMR. Codon 198 of the b-tubulin cCDN has been altered from GAG (Glu) to GGG (Gly) by a sitedirected mutagenesis using PCR (unpublished data). This single base mutagenesis was reported to give a benzimidazole-resistant, diethofencarb-sensitivetype b-tubulin.4 We have started cloning of the mutated b-tubulin gene into an expression vector for a study to compare the binding mode of benzimidazole fungicides with that of diethofencarb to btubulin.
ACKNOWLEDGEMENT
We thank Dr M Fujimura of Sumitomo Chemical Co. Ltd. for supplying us with b-tubulin cCDNA of a wild-type N. crassa strain.