# US 7,745,171 B2 · Cell-free protein synthesis method using insect cell extract solution

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 7,745,171 B2
- Document ID: US7745171B2
- Status: Granted
- Kind: B2
- Application number: 11/987,981
- Filed: 2007-12-06
- Published: 2008-09-11
- Granted: 2010-06-29
- Earliest claimed: 2002-12-27
- Assignees: Shimadzu Corporation
- Inventors: Toru Ezure; Shoken Higashide; Takashi Suzuki; Masaaki Ito; Koki Endo
- Web page: https://patentgrep.com/patent/US7745171B2

## CPC

- C12N 1/066: by physical processes
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

The present invention provides a preparation method of an insect cell extract solution for cell-free protein synthesis, the insect cell extract solution, a protein synthesis method in a cell-free system, which uses the insect cell extract solution, and a kit for cell-free protein synthesis containing the insect cell extract solution. The extract solution is easily prepared by the method of the present invention and can synthesize a higher amount of protein than by extract solutions prepared by conventional methods.

## Claims

### Claim 1

A method for cell-free protein synthesis, which comprises performing cell-free protein synthesis with an insect cell extract solution, wherein the insect cell extract solution is prepared by a process which comprises at least a step of rapid freezing at a temperature not higher than −80° C. an insect cell suspended in a solution for extraction, to allow for extraction from the insect cell, wherein the rapid freezing results in the insect cell being frozen for not longer than 10 seconds; wherein the cell-free protein synthesis is performed with an exogenous mRNA having a cap structure.

### Claim 2 (depends on 1)

The method of claim 1, wherein the process for preparing the insect cell extract solution further comprises thawing said insect cell after the rapid freezing and subjecting the cell to centrifugation.

### Claim 3 (depends on 2)

The method of claim 2, wherein the insect cell is thawed in a water bath or ice-water bath at −10° C. to 20° C.

### Claim 4 (depends on 1)

The method of claim 1, wherein the rapid freezing involves freezing the insect cell with liquid nitrogen.

### Claim 5 (depends on 1)

The method of claim 1, wherein the solution for extraction for the process for preparing the insect cell extract solution comprises a protease inhibitor.

### Claim 6 (depends on 1)

The method of claim 1, wherein the insect cell is a culture cell derived from Trichoplusia ni ovum or Spodoptera frugiperda ovary cell, or a combination of a cell derived from Trichoplusia ni ovum and a cell derived from Spodoptera frugiperda ovary cell.

### Claim 7 (depends on 1)

The method of claim 1, wherein the rapid freezing of the insect cell is at a temperature not higher than −150° C.

### Claim 8 (depends on 1)

The method of claim 1, wherein the process for preparing the insect cell extract solution further comprises subjecting the insect cell extract solution to a nuclease treatment before cell-free protein synthesis.

### Claim 9 (depends on 1)

The method of claim 1, wherein the cell-free protein synthesis is performed with a reaction solution comprising the insect cell extract solution and at least one of the following: potassium salt, magnesium salt, DTT, adenosine 5′-triphosphate, guanosine 5′-triphosphate, creatine phosphate, creatine kinase, an amino acid component, RNase inhibitor, tRNA, exogenous mRNA and a buffer.

### Claim 10 (depends on 9)

The method of claim 9, wherein the exogenous mRNA is an exogenous mRNA having a cap structure.