Exogenous peptide and protein expression levels using retroviral vectors in human cells.
Pseudotyped retroviral vectors combine the advantages of broad host range, high expression, stable chromosomal integration, and ease of preparation. These vectors greatly facilitate delivery into mammalian cells of sequences encoding individual peptide inhibitors-including those with therapeutic utility-and inhibitor libraries. However, retroviral vectors vary in behavior, particularly with respect to expression levels in different cell lines. Expression level is especially important in transdominant experiments because the concentration of an inhibitor (for example, an expressed peptide) is one of the key determinants in the degree of complex formation between the inhibitor and its target. Thus, inhibitor concentration should have an impact on the expressivity and/or penetrance of an induced phenotype. Here, we compare several retroviral vectors and human cell lines for relative expression levels using a green fluorescent protein reporter. We show for a subset of these lines that cellular protein concentrations produced by single-copy vectors range up to about 2 microM. We also examine other variables that contribute 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.
Sandrock
T M
TM
Deltagen Proteomics, 615 Arapeen Dr., Suite 300, Salt Lake City, UT 84108, USA. Tsandrock@arcaris.com
Risley
B
B
Richards
B T
BT
Poritz
M A
MA
Austin
H A
HA
Yoo
S
S
Kim
M K
MK
Roth
B
B
Repetny
K
K
Hsu
F
F
Stump
M
M
Teng
D H
DH
Kamb
A
A
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D015723:Gene Library; D017930:Genes, Reporter / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D009052:Leukemia Virus, Murine / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D051379:Mice; D009856:Oncogene Proteins, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic
D019941:Cyclin-Dependent Kinase Inhibitor p16; D008164:Luminescent Proteins; D009856:Oncogene Proteins, Viral; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D016160:Retinoblastoma Protein; D049452:Green Fluorescent Proteins