Exogenous peptide and protein expression levels using retroviral vectors in human cells

Citation
Tm. Sandrock et al., Exogenous peptide and protein expression levels using retroviral vectors in human cells, MOL THER, 4(5), 2001, pp. 398-406
Citations number
34
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Molecular Biology & Genetics
Journal title
MOLECULAR THERAPY
ISSN journal
1525-0016 → ACNP
Volume
4
Issue
5
Year of publication
2001
Pages
398 - 406
Database
ISI
SICI code
1525-0016(200111)4:5<398:EPAPEL>2.0.ZU;2-G
Abstract
Pseudotyped retroviral vectors combine the advantages of broad host range, high expression, stable chromosomal integration, and ease of preparation. T hese vectors greatly facilitate delivery into mammalian cells of sequences encoding individual peptide inhibitors-including those with therapeutic uti lity-and inhibitor libraries. However, retroviral vectors vary in behavior, particularly with respect to expression levels in different cell lines. Ex pression level is especially important in transdominant experiments because the concentration of an inhibitor (for example, an expressed peptide) is o ne of the key determinants in the degree of complex formation between the i nhibitor and its target. Thus, inhibitor concentration should have an impac t on the expressivity and/or penetrance of an induced phenotype. Here, we c ompare several retroviral vectors and human cell lines for relative express ion levels using a green fluorescent protein reporter. We show for a subset of these lines that cellular protein concentrations produced by single-cop y vectors range up to about 2 muM. We also examine other variables that con tribute to expression level, such as the nature of the expressed protein's carboxy terminus. Finally, we test the effect of increased concentration on phenotype with a nine-amino-acid peptide derived from the human papilloma virus protein E7 which overcomes E7-mediated cell growth.