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Titolo:
One-step purification, covalent immobilization, and additional stabilization of poly-His-tagged proteins using novel heterofunctional chelate-epoxy supports
Autore:
Mateo, C; Fernandez-Lorente, G; Cortes, E; 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, Ctr Invest Biol, Madrid, Spain CSIC Madrid SpainCSIC, Ctr Invest Biol, Madrid, Spain
Titolo Testata:
BIOTECHNOLOGY AND BIOENGINEERING
fascicolo: 3, volume: 76, anno: 2001,
pagine: 269 - 276
SICI:
0006-3592(200111)76:3<269:OPCIAA>2.0.ZU;2-R
Fonte:
ISI
Lingua:
ENG
Soggetto:
METAL AFFINITY-CHROMATOGRAPHY; SALT-INDUCED IMMOBILIZATION; BETA-GALACTOSIDASE; ACTIVATED SUPPORTS; ENZYMES; BIOCATALYSTS; ADSORPTION; RETENTION; CARRIER;
Keywords:
immobilized metal chelate affinity chromatography; epoxy supports; selective immobilization of enzymes; multipoint covalent attachment; glutaryl acylase;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Life Sciences
Citazioni:
32
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., "One-step purification, covalent immobilization, and additional stabilization of poly-His-tagged proteins using novel heterofunctional chelate-epoxy supports", BIOTECH BIO, 76(3), 2001, pp. 269-276

Abstract

Epoxy supports covalently immobilize proteins following a two-step mechanism; that is, the protein is physically adsorbed and then the covalent reaction takes place. This mechanism has been exploited to combine the selectivity of metal chelate affinity chromatography with the covalent immobilization capacity of epoxy supports. In this way, it has been possible to accomplish, in a simple manner, the purification, immobilization, and stabilizationof a poly-His-tagged protein. To fulfill this objective we developed a newkind of multifunctional epoxy support (chelate epoxy support [CES]), whichwas tested using a poly-His-tagged glutaryl acylase as a model protein (anup-heterodimeric enzyme of significant industrial interest). The selectivity of the immobilization in CES toward poly-His-tagged proteins was dependent to a large extent on the density and nature of the chelated metal. The highest selectivity was achieved by using low-density chelate groups (e.g., 5 mu mol/g) and metals with a low affinity (e.g., Co). However, the rate ofcovalent immobilization of the protein by its reaction with the epoxy groups on the support significantly increased at alkaline pH values. The multipoint attachment to the CES also depended on the reaction time. The immobilization of both glutaryl acylase subunits was achieved by incubation of the enzyme derivative at pH 10 for 24 h, with the best enzyme derivative 100-fold more stable than the soluble enzyme. By taking advantage of the selectivity properties of the novel support, we were able to immobilize up to 30 mgof protein per gram of modified Eupergit 250 using either pure enzyme or avery crude enzyme extract. (C) 2001 John Wiley & Sons, Inc.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 14/07/20 alle ore 02:58:13