Prokaryotic expression analysis of an NBS-typePtDRG01gene isolated fromPopulus tomentosaCarr.

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Abstract

In order to investigate the protein features of an NBS gene (PtDRG01, 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 1 mmol/L IPTG treatment for 4 h 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 PtDRG01 gene.

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