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Titolo:
Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports
Autore:
Armisen, P; Mateo, C; Cortes, E; Barredo, JL; Salto, F; Diez, B; Rodes, L; Garcia, JL; Fernandez-Lafuente, R; Guisan, JM;
Indirizzi:
CSIC, Inst Catalisis & Petroleoquim, Dept Biocatalisis, Madrid, Spain CSIC Madrid Spain isis & Petroleoquim, Dept Biocatalisis, Madrid, Spain Hispanagar SA, Burgos, Spain Hispanagar SA Burgos SpainHispanagar SA, Burgos, Spain CSIC, Ctr Invest Biol, Dept Mol Microbiol, Madrid, Spain CSIC Madrid Spain C, Ctr Invest Biol, Dept Mol Microbiol, Madrid, Spain Antibiot SA, Lab Ingn Genet, Leon, Spain Antibiot SA Leon SpainAntibiot SA, Lab Ingn Genet, Leon, Spain Ctr Ingn Genet & Biotecnol, La Habana, Cuba Ctr Ingn Genet & Biotecnol LaHabana Cuba & Biotecnol, La Habana, Cuba
Titolo Testata:
JOURNAL OF CHROMATOGRAPHY A
fascicolo: 1-2, volume: 848, anno: 1999,
pagine: 61 - 70
Fonte:
ISI
Lingua:
ENG
Soggetto:
AFFINITY-CHROMATOGRAPHY; MOLECULAR-BIOLOGY; ESCHERICHIA-COLI; IMMOBILIZATION; PENICILLIN; PURIFICATION; EQUILIBRIUM; EXPRESSION; BINDING;
Keywords:
immobilized metal-ion affinity chromatography; affinity supports; glutaryl acylase; enzymes;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Physical, Chemical & Earth Sciences
Citazioni:
23
Recensione:
Indirizzi per estratti:
Indirizzo: Guisan, JM CSIC, Inst Catalisis & Petroleoquim, Dept Biocatalisis, Madrid,Spain CSIC Madrid Spain oleoquim, Dept Biocatalisis, Madrid, Spain
Citazione:
P. Armisen et al., "Selective adsorption of poly-His tagged glutaryl acylase on tailor-made metal chelate supports", J CHROMAT A, 848(1-2), 1999, pp. 61-70

Abstract

A poly-His tag was fused in the glutaryl acylase (GA) from Acinetobacter sp. strain YS114 cloned in E. coli yielding a fully active enzyme. Biochemical analyses showed that the tag did not alter the maturation of the chimeric GA (poly-His GA) that undergoes a complex post-translational processing from an inactive monomeric precursor to the active heterodimeric enzyme. This enzyme has been used as a model to develop a novel and very simple procedure for one-step purification of poly-His proteins via immobilized metal-ion affinity chromatography on tailor-made supports. It was intended to improve thr selectivity of adsorption of the target protein on tailor-made chelate supports instead of performing a selective desorption. The rate and extent of the adsorption of proteins from a crude extract from E. coli and of pure poly-His tagged GA on different metal chelate supports was studied. Up to 90% of proteins from E. coli were adsorbed on commercial chelate supports having a high density of ligands attached to the support through long spacer arms, while this adsorption becomes almost negligible when using low ligand densities, short spacer arms and Zn2+ or Co2+ as cations. On the contrary, poly-His GA adsorbs strongly enough on all supports. A strong affinityinteraction between the poly-His tail and a single chelate moiety seems tobe the responsible for the adsorption of poly-His GA. By contrast, multipoint weak interactions involving a number of chelate moieties seem to be mainly responsible for adsorption of natural proteins. By using tailor-made affinity supports, a very simple procedure for one-step purification of GA with minimal adsorption of host proteins could be performed. Up to 20 mg of GA were adsorbed on each mi of chelate support while most of accompanying proteins were hardly adsorbed on such supports. Following few washing steps, the target enzyme was finally recovered (80% yield) by elution with 50 mM imidazole with a very high increment of specific activity (up to a 120 purification factor). (C) 1999 Elsevier Science B.V. All rights reserved.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 07/07/20 alle ore 14:37:50