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Titolo:
Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins
Autore:
Zhang, AH; Gonzalez, SM; Cantor, EJ; Chong, SR;
Indirizzi:
New England Biolabs Inc, Beverly, MA 01915 USA New England Biolabs Inc Beverly MA USA 01915 s Inc, Beverly, MA 01915 USA Univ Chihuahua, Dept Chem Sci, Chihuahua, Mexico Univ Chihuahua Chihuahua Mexico uahua, Dept Chem Sci, Chihuahua, Mexico
Titolo Testata:
GENE
fascicolo: 2, volume: 275, anno: 2001,
pagine: 241 - 252
SICI:
0378-1119(20010919)275:2<241:COAMFS>2.0.ZU;2-4
Fonte:
ISI
Lingua:
ENG
Soggetto:
GREEN-FLUORESCENT PROTEIN; CEREVISIAE VMA INTEIN; SACCHAROMYCES-CEREVISIAE; SPLICING ELEMENT; ADENOSINE-TRIPHOSPHATASE; RECOMBINANT PROTEINS; ESCHERICHIA-COLI; SPLIT INTEIN; DNAE GENE; CLEAVAGE;
Keywords:
protein splicing; cleavage; green fluorescent protein;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
34
Recensione:
Indirizzi per estratti:
Indirizzo: Chong, SR New England Biolabs Inc, 32 Tozer Rd, Beverly, MA 01915 USA New England Biolabs Inc 32 Tozer Rd Beverly MA USA 01915 915 USA
Citazione:
A.H. Zhang et al., "Construction of a mini-intein fusion system to allow both direct monitoring of soluble protein expression and rapid purification of target proteins", GENE, 275(2), 2001, pp. 241-252

Abstract

Affinity purification of recombinant proteins has been facilitated by fusion to a modified protein splicing element (intein). The fusion protein expression can be further improved by fusion to a mini-intein, i.e. an intein that lacks an endonuclease domain. We synthesized three rnini-inteins using overlapping oligonucleotides to incorporate Escherichia coli optimized codons and allow convenient insertion of an affinity tag between the intein (predicted) N- and C-terminal fragments. After examining the splicing and cleavage activities of the synthesized mini-inteins, we chose the mini-intein most efficient in thiol-induced N-terminal cleavage for constructing a novelintein fusion system. In this system, green fluorescent protein (GFP) was fused to the C-terminus of the affinity-tagged mini-intein whose. N-terminus was fused to a target protein. The design of the system allowed easy monitoring of soluble fusion protein expression by following GFP fluorescence, and rapid purification of the target protein through the intein-mediated cleavage reaction. A total of 17 target proteins were tested in this intein-GFP fusion system. Our data demonstrated that the fluorescence of the induced cells could be used to measure soluble expression of the intein fusion proteins and efficient intein cleavage activity. The final yield of the target proteins exhibited a linear relationship with whole cell fluorescence, The intein-GFP system may provide a simple route for monitoring real time. soluble protein expression, predicting final product yields, and screening the expression of a large number of recombinant proteins for rapid purification in high throughput applications. (C) 2001 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 21/09/20 alle ore 17:53:30