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Titolo:
Systematic optimization of expression and refolding of the Plasmodium falciparum cysteine protease falcipain-2
Autore:
Sijwali, PS; Brinen, LS; Rosenthal, PJ;
Indirizzi:
Univ Calif San Francisco, Dept Pathol, San Francisco, CA 94143 USA Univ Calif San Francisco San Francisco CA USA 94143 ancisco, CA 94143 USA San Francisco Gen Hosp, Dept Med, San Francisco, CA 94143 USA San Francisco Gen Hosp San Francisco CA USA 94143 Francisco, CA 94143 USA
Titolo Testata:
PROTEIN EXPRESSION AND PURIFICATION
fascicolo: 1, volume: 22, anno: 2001,
pagine: 128 - 134
SICI:
1046-5928(200106)22:1<128:SOOEAR>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
DISULFIDE BOND FORMATION; EGG-WHITE LYSOZYME; ESCHERICHIA-COLI; INCLUSION-BODIES; RECOMBINANT PROTEINS; RENATURATION; STABILIZATION; INHIBITORS; PURIFICATION; AGGREGATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
44
Recensione:
Indirizzi per estratti:
Indirizzo: Rosenthal, PJ Univ Calif San Francisco, Dept Pathol, Box 0811, San Francisco, CA 94143 USA Univ Calif San Francisco Box 0811 San Francisco CA USA 94143
Citazione:
P.S. Sijwali et al., "Systematic optimization of expression and refolding of the Plasmodium falciparum cysteine protease falcipain-2", PROT EX PUR, 22(1), 2001, pp. 128-134

Abstract

The Plasmodium falciparum cysteine protease falcipain-2 is a potential newtarget for antimalarial chemotherapy. In order to obtain large quantities of active falcipain-2 for biochemical and structural analysis, a systematicassessment of optimal parameters for the expression and refolding of the protease was carried out. High-yield expression was achieved using M15(pREP4) Escherichia coli transformed with the pQE-30 plasmid containing a truncated profalcipain-2 construct. Recombinant falcipain-2 was expressed as inclusion bodies, solubilized, and purified by nickel affinity chromatography. Asystematic approach was then used to optimize refolding parameters. This approach utilized 100-fold dilutions of reduced and denatured falcipain-2 into 203 different buffers in a microtiter plate format. Refolding efficiencyvaried markedly. Optimal refolding was obtained in an alkaline buffer containing glycerol or sucrose and equal concentrations of reduced and oxidizedglutathione. After optimization of the expression and refolding protocols and additional purification with anion-exchange chromatography, 12 mg of falcipain-2 was obtained from 5 liters of E. coli, and crystals of the protease were grown. The systematic approach described here allowed the rapid evaluation of a large number of expression and refolding conditions and provided milligram quantities of recombinant falcipain-2. (C) 2001 Academic Press.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 05/04/20 alle ore 19:39:22