首页|Prokaryotic expression analysis of an NBS-type PtDRGO1 gene isolated from Populus tomentosa Carr.
Prokaryotic expression analysis of an NBS-type PtDRGO1 gene isolated from Populus tomentosa Carr.
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In order to investigate the protein features of an NBS gene (PtDRGOl, EF157840) isolated from Populus tomentosa Carr., the full-length open reading frame was fused into a prokaryotic expression vector pGEX-KG. PCR analysis and double endonuclease digestion showed that the recombinant vector was successfully constructed and transferred into an expression host E. coli strain XA90. It was indicated by SDS-PAGE analysis that IPTG treatment successfully induced the expression of a fusion protein of about 79 kD, which was consistent with the predicted value. In addition, the prokaryotic expression system was also optimized. The result suggests that lmmol/L IPTG treatment for 4h at 37 C was most effective, and the product was predominately soluble and not extra-cellular secreting. Moreover, the fusion protein was purified with an affinity chromatography column using Glutathione Sepharose 4B. This work will lay a foundation for further studies on biological functions of the PtDRGOl gene.
Populus tomentosa CarrNBS geneprokaryotic expressionprotein purification
Yan LI、Qian ZHANG、Xing RAO、Haixia LI、Tingting LIU、Xinmin AN、Zhiyi ZHANG
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Assets and Laboratory Management Office, JiNan University,Guangzhou 510632, China
Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants of Ministry of Education, Beijing Forestry University,Beijing 100083, China
Ph.D. Programs Foundation of Ministry of Education of China