SH3-containing protein, DNA and uses thereof

Details SH3-containing protein, DNA and uses thereof SH3-containing protein, DNA and uses thereof

1999-07-01

2001-08-28

Ketter, James (Department: 1636)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of regulating cell metabolism or physiology

C530S300000, C530S350000, C530S358000, C530S400000

Reexamination Certificate

active

06281014

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention encompasses a protein designated TADG5 comprising an SH3 domain, a TADG5 DNA segment coding for the TADG5 protein, chimeric cells comprising the TADG5 DNA segment, vectors and plasmids comprising the TADG5 DNA segment and methods for producing the TADG5 protein as well as methods for using the TADG5 protein.
2. Description of the Related Art
Proteins containing SH3 domains have been previously identified. For example, chimeric protein tyrosine kinases comprising SH3 and SH2 domains are disclosed in U.S. Pat. No. 5,439,819. Proteins possessing SH2 and SH3 domains have been found to be important in cell cycle processes, especially in signal transduction pathways. Cyclin-dependent kinases possessing SH3 domains are well known participants in signal transduction processes. SH2 domains interact specifically with various proteins containing phosphotyrosine residues, whereas SH3 regions bind guanine nucleotide releasing factors, believed to be involved in important signaling pathways. The role in signal transduction of numerous molecules possessing SH3 and SH2 domains are discussed by Koch et al. (
Science
252:668-674 (1991)).
Interfering in the intracellular signal transduction pathways provides mechanisms for numerous therapeutic applications. While several proteins have been identified that interfere with various signal transduction mechanisms, novel proteins involved in signal transduction pathways are important to provide alternatives for therapy and drug development. The novel protein of the invention provides a heretofore unknown molecule which binds to the promoter region of a number of important genes and the Epstein-Barr virus.
A partial DNA sequence called HSU618 has 95% homology over approximately 560 nucleotides to the TADG5 gene (GenBank Accession No. U61837; submitted by F. Xu from the University of Southern California). The HSU618 sequence does not contain an open reading frame, and instead, contains stop codons in all reading frames. The sequence does, however, exhibit high homology with the TADG5 gene starting at nucleotide 87 of the TADG5 sequence through approximately base 654. HSU618 is indicated to have the capacity to bind cyclin G proteins. Because the HSU618 fragment lacks an open reading frame, it cannot be expressed to produce the corresponding protein fragment, nor could it be said to suggest the TADG5 protein amino acid sequence nor to disclose the isolated and purified DNA segment coding for the TADG5 protein.
The prior art is deficient in lack of the nucleotide and amino acid sequence of the SH3 domain-containing TADG5 protein. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The present invention encompasses a novel TADG5 protein having the amino acid sequence set out in SEQ ID No. 1, TADG5 DNA segments coding for the TADG5 protein, including a DNA segment isolated from human genetic material set out in SEQ ID No. 2, or a construct comprising the open reading frame found beginning at nucleotide 71 (with the methionine codon) to base 532, or optionally through nucleotide 535 (end of the in-frame stop codon downstream of the open reading frame). Preferably, the open reading frame segment from nucleotides 71 through 532 or 535 is coupled with a promoter segment and optionally coupled with additional DNA coding sequences useful in purification, such as a polyhistidine tail or an enzyme, such as GST.
In one embodiment of the present invention, there is provided an isolated TADG5 protein having the amino acid sequence of SEQ ID No. 1 or an allelic variant of SEQ ID No. 1 which retains the biological activity of the TADG5 protein. Further provided is an isolated DNA segment having the nucleotide sequence of SEQ ID No. 2 or an allelic variant thereof which encodes a protein having TADG5 biological activity. Additionally embodied are chimeric host cell comprising the above-described DNA segment, and antibodies directed towards a TADG5 protein, an allelic variant retaining TADG5 biological activity or functional fragments thereof.
In another embodiment of the present invention, there is provided an expression vector, comprising, in operable 5′ to 3′ linkage: (a) a promoter; and (b) a DNA segment encoding a TADG5 protein. Chimeric host cells comprising the expression vector are additional embodiments.
The above-referenced expression vector may further comprise: a signal sequence which is 3′ of the promoter and 5′ of the DNA segment encoding a TADG5 protein; a DNA sequence encoding a purification tag which is 5′ or 3′ of the DNA segment encoding a TADG5 protein; a linker sequence which is between the DNA sequence encoding a purification tag and the DNA segment encoding a TADG5 protein and encodes a cleavage site; transcriptional terminators and/or polyA sequences which are 3′ of the DNA segment encoding a TADG5 protein; and/or a gene encoding a selectable marker.
In yet another embodiment, there is provided a method of protein production, comprising the steps of: (a) introducing the above-described expression vector into a host cell; and (b) providing any necessary factors required by the promoter for expression of the gene encoding the TADG5 protein.
In still yet another embodiment, there is provided a peptide derived from the TADG5 protein shown in SEQ ID No. 1 or a biologically active allelic variant thereof, wherein the peptide is further characterized by binding to an oligonucleotide having the sequence selected from the group consisting of SEQ ID Nos. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or a strand complementary to one of the preceding sequences. Included in this embodiment is a method of regulating gene expression, comprising the steps of: binding the peptide(s) to a gene to increase or decrease expression of the gene.
In yet another embodiment, there is provided a method of identifying TADG5-binding DNA regulatory regions, comprising the steps of: (a) treating a DNA sample with a cleavage agent to produce DNA segments; and (b) binding the DNA segments to a TADG5 protein, allelic variants, or functional fragments thereof to produce TADG5-bound DNA segments. Generally, the TADG5-bound DNA segments comprise TADG5-binding DNA regulatory regions. Subsequently, the the TADG5-bound DNA segments can be isolated, and if so desired, released from the TADG5 protein.
In yet another embodiment of the present invention, there is provided a method of regulating gene expression, comprising the step of: binding to a regulatory region of a gene a molecule selected from the group consisting of: (a) a TADG5 protein, allelic variants retaining TADG5 biological activity or functional fragments thereof; (b) single-stranded homologous oligonucleotides which are homologous to sequences that bind a TADG5 protein, allelic variants retaining TADG5 biological activity or functional fragments thereof; (c) single-stranded complimentary oligonucleotides which are complimentary to sequences that bind a TADG5 protein, allelic variants retaining TADG5 biological activity or functional fragments thereof; (d) an antibody directed towards a TADG5 protein, allelic variants retaining TADG5 biological activity or functional fragments thereof; and (e) an antisense RNA molecule directed towards a TADG5 transcript, allelic variants retaining TADG5 biological activity or functional fragments thereof.


REFERENCES:
Clarke, ND and Berg, JM., “Zinc fingers in C. elegans . . . ”, Science 282, pp. 2018-2022, Dec. 1998.*
Desjarlais, J.R. et al., “Use of a zinc-finger consensus . . . ”. PNAS (USA) vol. 90, p2256-2260, Mar. 1993.

Affiliated with

Also associated with

Rating

SH3-containing protein, DNA and uses thereof does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with SH3-containing protein, DNA and uses thereof, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and SH3-containing protein, DNA and uses thereof will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2537935