# US 7,745,191 B2 · Method of producing heterodimer derivative of protein phosphatase type 2A enzyme

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- Number: US 7,745,191 B2
- Document ID: US7745191B2
- Status: Granted
- Kind: B2
- Application number: 12/094,067
- Filed: 2005-11-16
- Published: 2009-04-09
- Granted: 2010-06-29
- Earliest claimed: 2005-11-16
- Assignees: TROPICAL TECHNOLOGY CENTER Ltd.
- Inventors: Takeshi Yasumoto; Tsuyoshi Ikehara; Fukiko Shinjyo
- Web page: https://patentgrep.com/patent/US7745191B2

## CPC

- C12N 9/16: acting on ester bonds (3.1)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The purpose of the invention is to provide an activated protein phosphatase 2A (PP2A) in large quantities with high purity by a genetic engineering and to provide a method for producing a heterodimer derivative of PP2A which comprises infecting insect cultured cells with a baculovirus in which a cDNA encoding the catalytic subunit of PP2A carrying a first tag is integrated together with another baculovirus in which a cDNA encoding the A subunit of PP2A carrying a second tag is integrated, incubating the infected cells, disrupting the incubated cells to obtain a disrupted cell suspension, and then purifying the disrupted cell suspension with a solid phase carrying a substance capable of binding to the first tag and another solid phase carrying a substance capable of binding to the second tag, characterized in that the insect cells infected with the baculovirus are incubated at a temperature of from 18 to 22° C.

## Claims

### Claim 1

A method for producing a heterodimer derivative of protein phosphatase 2A (PP2A), comprising:
infecting cultured insect cells with baculovirus that express a polypeptide comprising the catalytic subunit of PP2A and a first tag and that express a polypeptide comprising the A subunit of PP2A and a second tag;
incubating the infected cells at a temperature ranging from 16° C. to 22° C.;
disrupting the incubated cells to obtain a disrupted cell suspension; and then
purifying the disrupted cell suspension with a solid phase carrying a substance capable of binding to the first tag and another solid phase carrying a substance capable of binding to the second tag, and recovering a heterodimer derivative of PP2A.

### Claim 2 (depends on 1)

The method of claim 1, wherein the catalytic subunit of PP2A is a catalytic α-subunit or a catalytic β-subunit.

### Claim 3 (depends on 1)

The method of claim 1, wherein
the first tag is a polyhistidine tag, and
the second tag is a FLAG tag.

### Claim 4 (depends on 1)

The method of claim 1, wherein the first tag binds to nickel (Ni) ions and
the second tag binds to an anti-FLAG M2 monoclonal antibody.

### Claim 5

A method for producing a catalytic subunit derivative of PP2A, comprising:
infecting insect cultured cells with a baculovirus containing cDNA encoding the catalytic subunit of PP2A carrying a tag;
incubating the infected cells at a temperature ranging from 16° C. to 22° C.;
disrupting the incubated cells to obtain a disrupted cell suspension; and then
purifying the disrupted cell suspension with a solid phase carrying a substance capable of binding to the tag, thus recovering a catalytic subunit derivative of PP2A.

### Claim 6 (depends on 5)

The method of claim 5, wherein the catalytic subunit of PP2A is a catalytic α-subunit or catalytic β-subunit.

### Claim 7 (depends on 5)

The method of claim 5, wherein the tag is a polyhistidine tag.

### Claim 8 (depends on 5)

The method of claim 5, wherein the substance capable of binding to the tag contains nickel (Ni) ions.

### Claim 9

A method for producing a phosphatase 2A (PP2A) protein heterodimer derivative that comprises the catalytic subunit of PP2A and the A subunit of PP2A, comprising:
infecting cultured insect cells with baculovirus encoding
a polypeptide comprising the catalytic subunit and a first tag and
a polypeptide comprising the A subunit of phosphatase 2A protein and a second tag;
incubating the infected cells at a temperature of from 16 to 22° C.; and
recovering a phosphatase 2A protein heterodimer derivative from said cells.

### Claim 10 (depends on 9)

The method of claim 9, wherein the polypeptide comprising the catalytic subunit and the polypeptide comprising the A subunit have different tags.

### Claim 11 (depends on 9)

The method of claim 9, wherein the polypeptide comprising the catalytic subunit comprises an α-isoform of the catalytic subunit.

### Claim 12 (depends on 9)

The method of claim 9, wherein the polypeptide comprising the catalytic subunit comprises an β-isoform of the catalytic subunit.

### Claim 13 (depends on 9)

The method of claim 9, wherein said PP2A heterodimer derivative is recovered by:
disrupting the incubated cells to obtain a disrupted cell suspension;
binding the polypeptide comprising the catalytic subunit and a first tag or, alternatively, binding the polypeptide comprising the A subunit and the second tag, to a first solid substrate,
washing the solid substrate to remove unbound materials,
eluting the bound materials by treating the solid substrate with a processing solvent that cleaves the linkage between the first or second tag and the first solid substrate,
applying the eluted material to a second solid substrate that binds to the tag not bound by the first solid substrate,
washing said second solid substrate to remove unbound material, and
recovering a heterodimer derivative of PP2A.

### Claim 14 (depends on 9)

The method of claim 9, wherein said first and second tag are selected from the group consisting of a polyhistidine tag, a FLAG tag, a GST tag, and an HA tag, provided the first and second tags are different.

### Claim 15 (depends on 9)

The method of claim 9, wherein said cultured insect cells are selected from the group consisting of armyworm and silkworm cells.

### Claim 16 (depends on 9)

The method of claim 9, wherein said cultured cells are selected from the group consisting of Sf9 cells, Sf21 cells, and High Five cells.

### Claim 17 (depends on 9)

The method of claim 9, further comprising cleaving the linkage between the catalytic subunit and its tag and/or cleaving the linkage between the A subunit and its tag.

### Claim 18 (depends on 9)

The method of claim 9, wherein the cultured insect cells are coinfected with a baculovirus encoding a polypeptide comprising the catalytic subunit and a first tag and a baculovirus encoding polypeptide comprising the A subunit of phosphatase 2A protein and a second tag.

### Claim 19 (depends on 18)

The method of claim 18, wherein the cultured insect cells are independently infected with a baculovirus encoding a polypeptide comprising the catalytic subunit and a first tag and a baculovirus encoding polypeptide comprising the A subunit of phosphatase 2A protein and a second tag.

### Claim 20 (depends on 9)

The method of claim 9, further comprising binding a PP2A beta subunit to said heterodimer derivative.