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Protein Expr Purif ; 78(2): 216-24, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21575725

RESUMO

Immobilized metal affinity chromatography (IMAC) is a widely used purification tool for the production of active, soluble recombinant proteins. Escherichia coli proteins that routinely contaminate IMAC purifications have been characterized to date. The work presented here narrows that focus to the most problematic host proteins, those retaining nickel affinity under elevated imidazole conditions, using a single bind-and-elute step. Two-dimensional difference gel electrophoresis, a favored technique for resolving complex protein mixtures and evaluating their expression, here discerns variation in the soluble extract pools that are loaded in IMAC and the remaining contaminants with respect to varied levels of recombinant protein expression. Peptidyl-prolyl isomerase SlyD and catabolite activator protein (CAP) are here shown to be the most persistent contaminants and have greater prevalence at low target protein expression.


Assuntos
Biotecnologia/normas , Cromatografia de Afinidade/métodos , Eletroforese em Gel Bidimensional/métodos , Proteínas de Escherichia coli/análise , Imidazóis/química , Proteínas Recombinantes/normas , Proteína Receptora de AMP Cíclico/análise , Proteína Receptora de AMP Cíclico/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Níquel/metabolismo , Peptidilprolil Isomerase/análise , Peptidilprolil Isomerase/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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