Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
2000-05-23
2001-08-07
McKelvey, Terry (Department: 1636)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C530S324000, C530S350000
Reexamination Certificate
active
06271341
ABSTRACT:
BACKGROUND OF THE INVENTION
The ability to regulate gene expression is desirable in a variety of situations, including in the production of recombinant proteins, in gene therapy, and in analyses of cell development and differentiation. A wide variety of gene regulation systems have been described, some of which stimulate gene expression in a constitutive manner and some of which stimulate gene expression in an inducible manner. A popular approach to regulating gene expression is to create a transcriptional activator fusion protein (also referred to herein as a “transactivator”) which is composed of a DNA binding domain, which has specificity for a particular target DNA binding site, and a transcriptional activation domain. To regulate expression of a gene of interest, the gene is operatively linked to the target DNA binding site and then both the gene and an expression vector encoding the transactivator fusion protein are coexpressed in a host cell. Upon binding of the transactivator fusion protein to the target DNA binding site, expression of the gene of interest is stimulated.
A constitutive transcriptional activator is created in cases where the DNA binding domain binds to its target site constitutively (i.e., without the need for an inducing agent to regulate DNA binding). One example of such a constitutive transactivator is GAL4-VP16 (Sadowski, I. et al.(1988)
Nature
335:563-564), composed of the yeast GAL4 DNA binding domain linked to the C-terminal region of herpes simplex virus virion protein 16 (Triezenberg, S. J. et al. (1988)
Genes Dev.
2:718-729). In contrast, when the DNA binding domain only binds to its target site in the presence or absence of an inducing agent, an inducible transcriptional activator is created. Examples of such inducible transcriptional activators are TetR-VP16, composed of a bacterial Tet repressor linked to VP16 (which binds to tetO sequences in the absence, but not the presence of tetracycline) (Gossen, M., and Bujard, H. (1992)
Proc. Natl. Acad. Sci. U.S.A
89, 5547-5551) and rTetR-VP16, composed of a mutated Tet repressor linked to VP16 (which binds to tetO sequences in the presence but not the absence of tetracycline) (Gossen, M., et al. (1995)
Science
268, 1766-1769).
The C-terminal transcriptional activation domain of HSV VP16 has been used frequently as the activator component of transactivator fusion proteins because of its strong capacity to stimulate transcription in eukaryotic cells. It has been shown, however, that overexpression of transcription factors can result in “squelching” (Gill, G., and Ptashne, M. (1988)
Nature
334, 721-724), which is seen as a consequence of titrating components of the transcriptional machinery from their respective intracellular pools. For VP16, which is one of the most potent transactivators known, it has been demonstrated that its overexpression, e.g. as a fusion protein with GAL4, is not tolerated by cells (Berger, S. L., et al. (1992)
Cell
70, 251-265, Kelleher, R. J., et al. (1990)
Cell
61, 1209-1215). Considering that VP16 interacts with a variety of essential components of the transcriptional machinery, including the adaptor/coactivator protein ADA2 in
S. cerevisiae
(Silverman, N., et al. (1994)
Proc. Natl. Acad Sci. U.S.A
91, 11665-11668) and its human homologue (Candau, R., et al. (1996)
Mol. Cell Biol.
16, 593-602), with TFIIB (Lin, Y. S., et al. (1991)
Nature
353, 569-571), TFIID (Stringer, K. F., et al. (1990)
Nature
345, 783-786), TFIIH (Xiao, H., et al. (1994)
Mol. Cell Biol.
14, 7013-7024) and dTAFII40 (Goodrich, J. A., et al. (1993)
Cell
75, 519-530), this is not surprising. Gilbert and coworkers (Gilbert, D. M., et al. (1993)
Mol. Cell. Biol
13, 462-472) have found a correlation between squelching and growth arrest which indicates that toxicity through squelching is a quantitative problem where the intracellular concentration and the strength of activation domains are crucial parameters.
Thus, while the potent transcriptional activation ability of VP16 makes it an attractive component for use in transactivator fusion proteins, in certain instances it may be desirable to have a fusion protein with a lower transcriptional activation potential than that provided by wild type VP16. Alternatively, in other situations, it may be desirable to have a fusion protein with an even higher transcriptional activation potential than that provided by wild type VP16. Accordingly, additional transactivator fusion proteins with graded transactivation potentials are needed.
SUMMARY OF THE INVENTION
This invention provides a panel of fusion protein transactivators which contain VP16-derived minimal activation domains and which possess a graded transactivation potential spanning more than 3 orders of magnitude. These transactivators have the advantage that they are tolerated in cells at high concentrations provide the ability to regulate levels of gene expression in a very precise manner.
One aspect of the invention pertains to nucleic acid molecules that encode the transcriptional activator fusion proteins of the invention. In one embodiment, the nucleic acid molecule encodes a fusion protein which activates transcription, the fusion protein comprising a first polypeptide comprising a DNA binding domain operatively linked to a second polypeptide comprising a transcriptional activation domain, wherein the transcriptional activation domain comprises at least one copy of a mutated acidic region of herpes simplex virus virion protein 16 (HSV VP16), the mutated acidic region consisting of amino acid positions 436 to 447 of HSV VP16 and having an amino acid substitution at position 442 as compared to wild type HSV VP16. The mutated acidic region of HSV VP16 can have, for example, the amino acid sequence of SEQ ID NO: 2 (in which the phenylalanine at position 442 of wild type VP16 has been mutated to glycine, referred to herein as VP16[G]). Alternatively, the mutated acidic region of HSV VP16 can have, for example, the amino acid sequence of SEQ ID NO: 3 (in which the phenylalanine at position 442 of wild type VP16 has been mutated to tyrosine, referred to herein as VP16[Y]).
In other embodiments, of the invention the transcriptional activation domain of the fusion protein is composed of two or more copies of the minimal activation domain of VP16, at least one of which has a mutation at position 442. For example, in one embodiment, the transcriptional activation domain comprises two copies of VP16[G] (having the amino acid sequence of SEQ ID NO: 4). In another embodiment, the transcriptional activation domain comprises, in the N-terminal to C-terminal direction, one copy of the wild type VP16 minimal activation domain (referred to as VP16[F]) and one copy of VP16[G]) (having the amino acid sequence of SEQ ID NO: 5). In yet another embodiment, the transcriptional activation domain comprises, in the N-terminal to C-terminal direction, one copy of VP16[G] and one copy of VP16[F] (having the amino acid sequence of SEQ ID NO: 6). In yet another embodiment, the transcriptional activation domain comprises, in the N-terminal to C-terminal direction, one copy of VP16[F], one copy of VP16[G] and one copy of VP16[Y] (having the amino acid sequence of SEQ ID NO: 7). In still another embodiment, the transcriptional activation domain comprises, in the N-terminal to C-terminal direction, one copy of VP16[G], one copy of VP16[F] and one copy of VP16[Y] (having the amino acid sequence of SEQ ID NO: 8).
In another embodiment, a nucleic acid molecule of the invention encodes a fusion protein which activates transcription, the fusion protein comprising a first polypeptide comprising a DNA binding domain operatively linked to a second polypeptide comprising a transcriptional activation domain, wherein the transcriptional activation domain consists of three copies of an acidic region of herpes simplex virus virion protein 16 (HSV VP16), the acidic region consisting of amino aci
Baron Udo
Bujard Hermann
Gossen Manfred
BASF - Aktiengesellschaft
DeConti, Jr. Esq. Giulio A.
Lahive & Cockfield LLP
McKelvey Terry
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