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High-recovery one-step purification of the DNA-binding protein Fur by mild guanidinium chloride treatment

Paper ID Volume ID Publish Year Pages File Format Full-Text
35038 45069 2010 5 PDF Available
Title
High-recovery one-step purification of the DNA-binding protein Fur by mild guanidinium chloride treatment
Abstract

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.

Keywords
GdnHCl, guanidinium chloride; PMSF, phenylmethylsulphonyl fluoride; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assays; Px, promoter of the gene X; TCA, tricholoracetic acid; TSP, 3-(trimethylsilyl)propionic acid-d4 sodium SALT; IMAC, Immobi
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High-recovery one-step purification of the DNA-binding protein Fur by mild guanidinium chloride treatment
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 45, Issue 2, February 2010, Pages 292–296
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us