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Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli

Velkov, Tony, Jones, Alun and Lim, Maria L. R. 2008, Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli, Preparative biochemistry and biotechnology, vol. 38, no. 4, pp. 422-441, doi: 10.1080/10826060802325725.

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Title Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli
Formatted title Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli
Author(s) Velkov, Tony
Jones, Alun
Lim, Maria L. R.
Journal name Preparative biochemistry and biotechnology
Volume number 38
Issue number 4
Start page 422
End page 441
Publisher Marcel Dekker
Place of publication New York, N.Y.
Publication date 2008-10
ISSN 1082-6068
1532-2297
Keyword(s) immobilized metal ion affinity chromatography
lac repressor
protein crystallization
protein purification
Summary A two-step chromatographic sequence is described for the purification of native lactose operon repressor protein from Escherichia coli cells. The first step involves Ni2+-based immobilized metal ion affinity chromatography of the soluble cytoplasmic extract. This method provides superior speed, resolution and yield than the established phosphocellulose cation-exchange chromatographic procedure. Anion-exchange chromatography is used for further purification to >95% purity. The identity and purity of the lactose repressor protein were demonstrated using sodium dodecylsulphate polyacrylamide electrophoresis, crystallization, tryptic finger-printing mass spectrometry, and inducer binding assays. The purified lac repressor exhibited inducer sensitivity for operator DNA binding and undergoes a conformational change upon inducer binding. By all these extensive biochemical criteria, the purified protein behaves exactly as that described for the Escherichia coli lactose operon repressor.
Language eng
DOI 10.1080/10826060802325725
Field of Research 060199 Biochemistry and Cell Biology not elsewhere classified
HERDC Research category C1.1 Refereed article in a scholarly journal
ERA Research output type C Journal article
HERDC collection year 2008
Copyright notice ©2008, Taylor & Francis Group, LLC
Persistent URL http://hdl.handle.net/10536/DRO/DU:30018681

Document type: Journal Article
Collection: School of Medicine
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Created: Fri, 11 Sep 2009, 09:56:30 EST by Rachael Wilson

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