# US 9,040,253 B2 · Method for enhancing recombinant protein production by cell-free protein expression system

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- Number: US 9,040,253 B2
- Document ID: US9040253B2
- Status: Granted
- Kind: B2
- Application number: 13/087,075
- Filed: 2011-04-14
- Published: 2011-10-27
- Granted: 2015-05-26
- Earliest claimed: 2010-04-14
- Assignees: Sutro Biopharma, Inc.
- Applicants: Sushmita Mimi Roy; Sunil Bajad; Evan Green; Henry Heinsohn
- Inventors: Sushmita Mimi Roy; Sunil Bajad; Evan Green; Henry Heinsohn
- Web page: https://patentgrep.com/patent/US9040253B2

## CPC

- C12P 21/06: produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00)
- C07K 14/535: Granulocyte CSF; Granulocyte-macrophage CSF
- C12N 1/06: Lysis of microorganisms
- C12N 1/20: Bacteria; Culture media therefor
- C12N 9/1247: {DNA-directed RNA polymerase (2.7.7.6)}
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- G01N 33/6848: {Methods of protein analysis involving mass spectrometry}
- C12Y 207/07006: DNA-directed RNA polymerase (2.7.7.6)
- 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)}
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- C12Y: ENZYMES

## Abstract

The present invention provides a method for monitoring of profile changes of components in a dynamic system such as a cell-free in vitro protein synthesis system by using liquid chromatography (LC) combined with mass spectrometry (MS). In an additional aspect, this invention provides a method for enhancing the yield and/or reproducibility in a cell-free protein synthesis system by modulating the level and/or activity of a protein component that has regulatory effects on the system.

## Claims

### Claim 1

A method for enhancing recombinant protein production in a cell-free protein synthesis system comprising:
contacting a cell lysate prepared from prokaryotic cells comprising a recombinant genomic modification that abolishes CspE expression, and an exogenous nucleic acid template encoding a protein; and
producing the protein encoded by the exogenous nucleic acid template in the cell-lysate.

### Claim 2 (depends on 1)

The method of claim 1, wherein the recombinant genomic modification comprises at least one nucleotide substitution or deletion in the genomic sequence encoding for the CspE.

### Claim 3 (depends on 1)

The method of claim 1, wherein the prokaryotic cells further comprise a recombinant genomic modification that abolishes CspA expression.

### Claim 4 (depends on 3)

The method of claim 3, wherein the recombinant genomic modification comprises at least one nucleotide substitution or deletion in the genomic sequence encoding for the CspA gene.