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Titolo:
Coexpression of proteins in bacteria using T7-based expression plasmids: Expression of heteromeric cell-cycle and transcriptional regulatory complexes
Autore:
Johnston, K; Clements, A; Venkataramani, RN; Trievel, RC; Marmorstein, R;
Indirizzi:
Univ Penn, Dept Biochem & Biophys, Philadelphia, PA 19104 USA Univ Penn Philadelphia PA USA 19104 & Biophys, Philadelphia, PA 19104 USA Univ Penn, Wistar Inst, Philadelphia, PA 19104 USA Univ Penn PhiladelphiaPA USA 19104 star Inst, Philadelphia, PA 19104 USA Univ Penn, Dept Chem, Philadelphia, PA 19104 USA Univ Penn Philadelphia PA USA 19104 Dept Chem, Philadelphia, PA 19104 USA
Titolo Testata:
PROTEIN EXPRESSION AND PURIFICATION
fascicolo: 3, volume: 20, anno: 2000,
pagine: 435 - 443
SICI:
1046-5928(200012)20:3<435:COPIBU>2.0.ZU;2-0
Fonte:
ISI
Lingua:
ENG
Soggetto:
HIGH-LEVEL EXPRESSION; YEAST TFIIA/TBP/DNA COMPLEX; CRYSTAL-STRUCTURE; ESCHERICHIA-COLI; INCLUSION-BODIES; T7 PROMOTER; VECTORS; DNA; RECEPTOR; GENES;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
38
Recensione:
Indirizzi per estratti:
Indirizzo: Marmorstein, R Univ Penn, Wistar Inst, 3601 Spruce St, Philadelphia, PA 19104 USA Univ Penn 3601 Spruce St Philadelphia PA USA 19104 9104 USA
Citazione:
K. Johnston et al., "Coexpression of proteins in bacteria using T7-based expression plasmids: Expression of heteromeric cell-cycle and transcriptional regulatory complexes", PROT EX PUR, 20(3), 2000, pp. 435-443

Abstract

This report describes the development and application of a dual vector coexpression system for the overproduction of heteromeric cell cycle and transcriptional regulatory protein complexes in bacteria. To facilitate these studies we constructed a T7-based expression plasmid, pRM1 that contains an origin of replication derived from p15A, and a gene encoding kanamycin resistance. This expression vector is compatible with ColE1-derived plasmids found in the pET family of T7 expression vectors, which encode ampicillin resistance. It also has the same multiple cloning sites as the pET- derived pRSET vector, allowing easy shuttling between the two expression vectors. Cotransformation of the pRM1 and pET-derived expression vectors into an Escherichia coli strain such as BL21(DE3) results in a significant level of coexpression of heteromeric protein complexes. We demonstrate the applicability of combining the pRM1 and pET-derived vectors for the coexpression of cell cycle regulatory components, pRB/E7 and pRB/E1a, and the transcriptional regulatory complexes, SRF/SAP-1 and SRF/Elk-1. We further use the pRB/E1a complex to demonstrate that these coexpressed complexes can be purified to homogeneity for further studies. Use of the pRM1 vector in combination with thepET-derived vectors should be generally applicable for the large-scale coexpression and purification of a wide variety of heteromeric protein complexes for biochemical, biophysical, and structural studies. (C) 2000 Academic Press.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 22/09/20 alle ore 14:01:25