# US 6,720,154 B1 · Purified stat proteins and methods of purifying thereof

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- Number: US 6,720,154 B1
- Document ID: US6720154B1
- Status: Granted
- Kind: B1
- Application number: 09/430,806
- Filed: 1999-11-02
- Granted: 2004-04-13
- Earliest claimed: 1997-10-15
- Assignees: The Rockefeller University
- Inventors: Uwe Vinkemeier; James E. Darnell, Jr.
- Web page: https://patentgrep.com/patent/US6720154B1

## CPC

- B01J 20/26: Synthetic macromolecular compounds
- B01D 15/3804: {Affinity chromatography}
- B01J 20/262: {obtained otherwise than by reactions only involving carbon to carbon unsaturated bonds, e.g. obtained by polycondensation (macromolecular compounds obtained otherwise than by reactions only involving unsaturated carbon-to-carbon bonds per se C08G)}
- B01J 20/265: {modified or post-treated polymers (polymer carriers or substrates subjected to further impregnating or coating B01J20/3208)}
- B01J 20/285: based on polymers
- B01J 20/3242: {Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group}
- C07K 14/4702: {Regulators; Modulating activity}
- C07K 14/4705: {stimulating, promoting or activating activity}
- G01N 33/6872: {Intracellular protein regulatory factors and their receptors, e.g. including ion channels}
- B01D 15/166: {Fluid composition conditioning, e.g. gradient}
- C12N 2799/026: where the vector is derived from a baculovirus
- G01N 2333/4706: stimulating, promoting or activating activity
- G01N 2500/02: Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention describes methods of producing milligram quantities of three forms of purified Stat1 protein from recombinant DNA constructs. In addition, the Stat proteins may be isolated in their phosphorylated or nonphosphorylated forms (Tyr 701). The proteins can be produced in baculovirus infected insect cells, or E. coli. A compact domain in the amino terminus of Stat1α was isolated and found to enhance DNA binding due to its ability to interact with a neighboring Stat protein. A relatively protease-resistant recombinant truncated form of the Stat protein was isolated in 40-50 mg quantities. Purification of the Stat proteins were performed after modifying specific cysteine residues of the Stat proteins to prevent aggregation. Activated EGF-receptor partially purified from membranes by immunoprecipitation was shown to be capable of in vitro catalysis of the phosphorylation of the tyrosine residue of Stat 1 known to be phosphorylated in vivo. Techniques are enclosed to separate the phosphorylated from the nonphosphorylated Stat proteins. The techniques disclosed are general for Stat proteins and may be used to isolate large quantities of purified Stat 2, 3, 4, 5A, 5B and 6. Methods for using purified Stat proteins, truncated Stat proteins, or Stat N-terminal fragments for drug discovery are also disclosed.

## Claims

### Claim 1

A method for identifying a drug that enhances the ability of adjacent STAT protein dimers to interact comprising measuring the ability of a test compound to enhance the association of a first STAT protein or a fragment of said first STAT protein with a second STAT protein or a fragment of said second STAT protein;
wherein said fragment of said first STAT protein comprises the N-terminal domain of said first STAT protein;
wherein said fragment of said second STAT protein comprises the N-terminal domain of said second STAT protein;
wherein the association is dependent upon the N-terminal domain of said first STAT protein, and the N-terminal domain of said second STAT protein; and
wherein a test compound which enhances the association is identified as a drug that enhances the interaction between adjacent activated STAT dimers.

### Claim 2 (depends on 1)

The method of claim 1 wherein said first STAT protein is selected from the group consisting of STAT 1, STAT 2, STAT 3, STAT 4, STAT 5A, STAT 5B, and STAT 6.

### Claim 3 (depends on 1)

The method of claim 1 wherein said second STAT protein is selected from the group consisting of STAT 1, STAT 2, STAT 3, STAT 4, STAT 5A, STAT 5B, and STAT 6.

### Claim 4 (depends on 1)

The method of claim 1 wherein said first STAT protein and said second STAT protein are the same STAT protein.

### Claim 5 (depends on 1)

The method of claim 1 wherein said first STAT protein is selected from the group consisting of STAT 1, STAT 2, STAT 4, and STAT 6.

### Claim 6 (depends on 1)

The method of claim 1 wherein said second STAT protein is selected from the group consisting of STAT 1, STAT 2, STAT 4, and STAT 6.