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Titolo:
Affinity chromatography of polyhistidine tagged enzymes - New dextran-coated immobilized metal ion affinity chromatography matrices for prevention ofundesired multipoint adsorptions
Autore:
Mateo, C; Fernandez-Lorente, G; Pessela, BCC; Vian, A; Carrascosa, AV; Garcia, JL; Fernandez-Lafuente, R; Guisan, JM;
Indirizzi:
CSIC, Inst Catalisis, Dept Biocatalisis, E-28049 Madrid, Spain CSIC Madrid Spain E-28049 isis, Dept Biocatalisis, E-28049 Madrid, Spain CSIC, Inst Fermentac Ind, Madrid 28006, Spain CSIC Madrid Spain 28006CSIC, Inst Fermentac Ind, Madrid 28006, Spain CSIC, Ctr Invest Biol, E-28006 Madrid, Spain CSIC Madrid Spain E-28006CSIC, Ctr Invest Biol, E-28006 Madrid, Spain
Titolo Testata:
JOURNAL OF CHROMATOGRAPHY A
fascicolo: 1-2, volume: 915, anno: 2001,
pagine: 97 - 106
Fonte:
ISI
Lingua:
ENG
Soggetto:
SELF-ASSEMBLED MONOLAYERS; ACTIVATED SUPPORTS; BETA-GALACTOSIDASE; EXTREME THERMOPHILE; SINGLE-STEP; PURIFICATION; PROTEINS; STABILIZATION; EXPRESSION; BINDING;
Keywords:
immobilized metal ion affinity chromatography; dextran-coated supports; enzymes; galactosidases;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Physical, Chemical & Earth Sciences
Citazioni:
41
Recensione:
Indirizzi per estratti:
Indirizzo: Guisan, JM CSIC, Inst Catalisis, Dept Biocatalisis, Campus Univ Autonoma, E-28049 Madrid, Spain CSIC Campus Univ Autonoma Madrid Spain E-28049 9 Madrid, Spain
Citazione:
C. Mateo et al., "Affinity chromatography of polyhistidine tagged enzymes - New dextran-coated immobilized metal ion affinity chromatography matrices for prevention ofundesired multipoint adsorptions", J CHROMAT A, 915(1-2), 2001, pp. 97-106

Abstract

New immobilized metal ion affinity chromatography (IMAC) matrices containing a high concentration of metal-chelate moieties and completely coated with inert flexible and hydrophilic dextrans are here proposed to improve the purification of polyhistidine (poly-His) tagged proteins. The purification of an interesting recombinant multimeric enzyme (a thermoresistant beta -galactosidase from Thermus sp. strain T2) has been used to check the performance of these new chromatographic media. IMAC supports with a high concentration (and surface density) of metal chelate groups promote a rapid adsorption of poly-His tagged proteins during IMAC. However, these supports also favor the promotion of undesirable multi-punctual adsorptions and problems may arise for the simple and effective purification of poly-His tagged proteins: (a) more than 30% of the natural proteins contained in crude extracts from E. coli become adsorbed, in addition to our target recombinant protein,on these IMAC supports via multipoint weak adsorptions; (b) the multimericpoly-His tagged enzyme may become adsorbed via several poly-His tags belonging to different subunits. In this way, desorption of the pure enzyme fromthe support may become quite difficult (e.g., it is not fully desorbed from the support even using 200 mM of imidazole). The coating of these IMAC supports with dextrans greatly reduces these undesired multi-point adsorptions: (i) less than 2% of natural proteins contained in crude extracts are nowadsorbed on these novel supports; and (ii) the target multimeric enzyme may be fully desorbed from the support using 60 mM imidazole. In spite of this dramatic reduction of multi-point interactions, this dextran coating hardly affects the rate of the one-point adsorption of poly-His tagged proteins(80% of the rate of adsorption compared to uncoated supports). Therefore, this dextran coating of chromatographic matrices seems to allow the formation of strong one-point adsorptions that involve small areas of the protein and support surface. However, the dextran coating seems to have dramatic effects for the prevention of weak or strong multipoint interactions that should involve a high geometrical congruence between the enzyme and the support surface. (C) 2001 Elsevier Science BN. All rights reserved.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 12/08/20 alle ore 05:55:01