Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (6): 1084-1092.doi: 10.3864/j.issn.0578-1752.2012.06.006

• PLANT PROTECTION • Previous Articles     Next Articles

Soluble Expression and Purification of Fusarium graminearum β2-tubulin in Escherichia coli

 XU  Jian-Qiang, ZHOU  Yu-Jun, ZHANG  Cong, ZHOU  Ming-Guo   

  1. 1.南京农业大学植物保护学院/农业部作物病虫害监测及防控重点开发实验室，南京 210095
    2.河南科技大学林学院，河南洛阳 471003
  • Received:2011-09-07 Online:2012-03-15 Published:2011-11-29

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Abstract: 【Objective】 The objective of this study is to express the β2-tubulin of Fusarium graminearum in E. coli in soluble form and purify it by HisTrap™ HP columns.【Method】 β2-tubulin gene contained in the plasmid (pET32a+-β2-tubulin) was amplified, cloned to the vector pET30a+ , and then transformed into the hosts Rossatta (DE3) pLysS and BL21 (DE3). After the positive clones were screened by the colony PCR and double enzymatic digestion, the induced fusion proteins were obtained and verified by SDS-PAGE and Western blot. In order to express the fusion protein in soluble form, the inducing factors, including temperature, induction time, IPTG (Isopropyl β-D-Thiogalactoside) concentration, cell density, medium composition and hosts were screened. 【Result】The positive clones which could express more fusion protein after induced were screened, however, the fusion proteins formed inclusion bodies. The molecular weight of fusion proteins were confirmed to be 51.6 kD by SDS-PAGE, which also showed specific activity to anti-6×His monoclonal antibody. After the optimization of imidazole concentration in binding and washing buffer, the soluble fusion protein was purified and its structural integrity was preserved through the purification process by the verification of Western blot. 【Conclusion】 The methods described here can be used to express and purify other recombinant proteins in soluble form in E. coli. The purified fusion tubulin can be used in the studies of tubulin target drug resistant mechanisms as well as high throughout screening of new fungicide.

Key words: Fusarium graminearum, β2-tubulin, inclusion body, soluble expression, purification

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