Catalogo Articoli (Spogli Riviste)

OPAC HELP

Titolo:
MUTATIONS THAT SUPPRESS THE THERMOSENSITIVITY OF GREEN FLUORESCENT PROTEIN
Autore:
SIEMERING KR; GOLBIK R; SEVER R; HASELOFF J;
Indirizzi:
MRC,MOL BIOL LAB,HILLS RD CAMBRIDGE CB2 2QH ENGLAND MRC,MOL BIOL LAB CAMBRIDGE CB2 2QH ENGLAND MRC,UNIT PROT ENGN & DESIGN CAMBRIDGE CB2 2QH ENGLAND UNIV HALLE WITTENBERG,DEPT BIOCHEM,INST BIOCHEM BIOTECHNOL D-06124 HALLE GERMANY
Titolo Testata:
Current biology
fascicolo: 12, volume: 6, anno: 1996,
pagine: 1653 - 1663
SICI:
0960-9822(1996)6:12<1653:MTSTTO>2.0.ZU;2-V
Fonte:
ISI
Lingua:
ENG
Soggetto:
GENE-EXPRESSION; LOCALIZATION; CELLS; VISUALIZATION; GFP;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
35
Recensione:
Indirizzi per estratti:
Citazione:
K.R. Siemering et al., "MUTATIONS THAT SUPPRESS THE THERMOSENSITIVITY OF GREEN FLUORESCENT PROTEIN", Current biology, 6(12), 1996, pp. 1653-1663

Abstract

Background: The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has recently attracted great interest as the first example of a cloned reporter protein that is intrinsically fluorescent. Although successful in some organisms, heterologous expression of GFP hasnot always been straight forward. In particular, expression of GFP incells that require incubation temperatures around 37 degrees C has been problematic. Results: We have carried out a screen for mutant formsof GFP that fluoresce more intensely than the wild-type protein when expressed in E. coil at 37 degrees C. We have characterized a bright mutant (GFPA) with reduced sensitivity to temperature in both bacteria and yeast, and have shown that the amino acids substituted in GFPA actby preventing temperature-dependent misfolding of the GFP apoprotein. We have shown that the excitation and emission spectra of GFPA can bemanipulated by site-directed mutagenesis without disturbing its improved folding characteristics, and have produced a thermostable folding mutant (GFP5) that can be efficiently excited using either long-wavelength ultraviolet or blue light. Expression of GFP5 results in greatly improved levels of fluorescence in both microbial and mammalian cells cultured at 37 degrees C. Conclusions: The thermotolerant mutants of GFP greatly improve the sensitivity of the protein as a visible reporter molecule in bacterial, yeast and mammalian cells, The fluorescence spectra of these mutants can be manipulated by further mutagenesis without deleteriously affecting their improved folding characteristics, soit may be possible to engineer a range of spectral variants with improved tolerance to temperature. Such a range of sensitive reporter proteins will greatly improve the prospects for GFP-based applications in cells that require relatively high incubation temperatures.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 01/12/20 alle ore 07:27:18