کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
35038 | 45069 | 2010 | 5 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: High-recovery one-step purification of the DNA-binding protein Fur by mild guanidinium chloride treatment High-recovery one-step purification of the DNA-binding protein Fur by mild guanidinium chloride treatment](/preview/png/35038.png)
Engineering of DNA-binding domains of regulatory proteins aimed to control gene expression requires a deep knowledge of protein–DNA interactions acquired from structural data on purified species. Most DNA-binding proteins work as dimers establishing multiple protein–protein contacts mainly driven by hydrophobic interactions, being its cleansing a difficult task because of solubility problems. One-step purification of soluble, functional recombinant FurA from the cyanobacterium Anabaena sp. PCC 7120 has been achieved using mild chaotropic conditions. FurA was isolated using a Zn-iminodiacetate chromatography of the crude extract obtained after sonication of Escherichia coli in the presence of 2 M guanidium chloride. CD and 1D NMR spectroscopies demonstrate that FurA conserves the native tertiary structure. Functional analysis reveals FurA ability to recognise and bind target DNAs. We propose that the use of chaotropic agents under mild denaturating conditions might have general application in the purification of DNA-binding proteins and other proteins prone to aggregation.
Journal: Process Biochemistry - Volume 45, Issue 2, February 2010, Pages 292–296