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Titolo:
Characterization and use of green fluorescent proteins from Renilla mulleri and Ptilosarcus guernyi for the human cell display of functional peptides
Autore:
Peelle, B; Gururaja, TL; Payan, DG; Anderson, DC;
Indirizzi:
Rigel Pharmaceut Inc, Dept Prot Chem, S San Francisco, CA 94080 USA Rigel Pharmaceut Inc S San Francisco CA USA 94080 Francisco, CA 94080 USA
Titolo Testata:
JOURNAL OF PROTEIN CHEMISTRY
fascicolo: 6, volume: 20, anno: 2001,
pagine: 507 - 519
SICI:
0277-8033(200108)20:6<507:CAUOGF>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
SEQUENCE ALIGNMENT; GENETIC SELECTION; IMPORT SUBSTRATE; NUCLEOPORINS; SENSITIVITY; SCAFFOLD; APTAMERS; PATHWAY; CODON;
Keywords:
green fluorescent proteins; retroviral delivery; peptide libraries; fluorescence; circular dichroism;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
33
Recensione:
Indirizzi per estratti:
Indirizzo: Gururaja, TL Rigel Pharmaceut Inc, Dept Prot Chem, 240 E Grand Ave, S San Francisco, CA94080 USA Rigel Pharmaceut Inc 240 E Grand Ave S San FranciscoCA USA 94080
Citazione:
B. Peelle et al., "Characterization and use of green fluorescent proteins from Renilla mulleri and Ptilosarcus guernyi for the human cell display of functional peptides", J PROTEIN C, 20(6), 2001, pp. 507-519

Abstract

Green fluorescent protein (GFP) is useful as an intracellular scaffold forthe display of random peptide libraries in yeast. GFPs with a different sequence from Aequorea victoria have recently been identified from Renilla mulleri and Ptilosarcus gurneyi. To examine these proteins as intracellular scaffolds for peptide display in human cells, we have determined the expression level of retrovirally delivered human codon-optimized versions in Jurkat-E acute lymphoblastic leukemia cells using fluorescence activated cell sorting and Western blots. Each wild type protein is expressed at 40% hi-her levels than A. victoria mutants optimized for maximum fluorescence. We havecompared the secondary structure and stability of these GFPs with A. victoria GFP using circular dichroism (CD). All three GFPs essentially showed a perfect beta -strand conformation and their melting temperatures (T-m) are very similar, giving an experimental evidence of a similar overall structure. Folded Renilla GFP allows display of an influenza hemagglutinin epitope tag in several internal insertion sites, including one which is not permissive for such display in Aequorea GFP, giving greater flexibility in peptidedisplay options. To test display of a functional peptide, we show that theSV-40 derived nuclear localization sequence PPKKKRKV, when inserted into two different potential loops, results in the complete localization of Renilla GFP to the nucleus of human A549 cells.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 05/12/20 alle ore 13:32:21