Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli
Data(s) |
01/10/2008
|
---|---|
Resumo |
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. <br /> |
Identificador | |
Idioma(s) |
eng |
Publicador |
Marcel Dekker |
Relação |
http://dro.deakin.edu.au/eserv/DU:30018681/velkov-ni2basedimmobilized-2008.pdf http://dx.doi.org/10.1080/10826060802325725 |
Direitos |
2008, Taylor & Francis Group, LLC |
Palavras-Chave | #immobilized metal ion affinity chromatography #lac repressor #protein crystallization #protein purification |
Tipo |
Journal Article |