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, 38 4: 422-441. doi:10.1080/10826060802325725


Author Velkov, Tony
Jones, Alun
Lim, Maria L. R.
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
Journal name Preparative Biochemistry and Biotechnology   Check publisher's open access policy
ISSN 1082-6068
1532-2297
Publication date 2008
Sub-type Article (original research)
DOI 10.1080/10826060802325725
Volume 38
Issue 4
Start page 422
End page 441
Total pages 20
Place of publication Philadelphia, PA , United States
Publisher Taylor & Francis
Language eng
Formatted abstract
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.
Keyword Immobilized metal ion affinity chromatography
Lac repressor
Protein crystallization
Protein purification
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collection: Institute for Molecular Bioscience - Publications
 
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Created: Thu, 03 Sep 2009, 09:32:55 EST by Mr Andrew Martlew on behalf of Institute for Molecular Bioscience