# US 8,771,990 B2 · Method of producing lipidated polypeptides

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- Number: US 8,771,990 B2
- Document ID: US8771990B2
- Status: Granted
- Kind: B2
- Application number: 13/296,558
- Filed: 2011-11-15
- Published: 2012-05-17
- Granted: 2014-07-08
- Earliest claimed: 2010-11-15
- Assignees: National Health Research Institutes
- Applicants: Chih-Hsiang Leng; Chi-Ling Tseng; Shih-Jen Liu; Hsin-Wei Chen; Pele Choi-Sing Chong
- Inventors: Chih-Hsiang Leng; Chi-Ling Tseng; Shih-Jen Liu; Hsin-Wei Chen; Pele Choi-Sing Chong
- Web page: https://patentgrep.com/patent/US8771990B2

## CPC

- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C07K 2319/90: containing a motif for post-translational modification
- 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)

## Abstract

A method of producing a recombinant lipidated polypeptide in E. coli. The method includes providing an E. coli host cell adapted to express a recombinant lipidated polypeptide; and culturing the E. coli host cell in a minimal medium under conditions that allow expression of the polypeptide in lipidated form.

## Claims

### Claim 1

A method of producing a recombinant lipidated polypeptide in E. coli, the method comprising:
providing an E. coli host cell adapted to express a recombinant lipidated polypeptide, wherein the E. coli host cell is C41 (DE3), C43 (DE3), DK8 (DE3)S, or C2014 (DE3) and has a nucleic acid sequence encoding the recombinant lipidated polypeptide, which contains a bacterial lipoprotein signal peptide; and
culturing the E. coli host cell in a M9 medium under conditions that allow expression of the polypeptide in lipidated form, wherein the ratio of immature to mature lipoprotein (I/M ratio) of the recombinant lipidated polypeptide thus expressed is 0.01 or lower, the M9 medium lacking an organic nitrogen source and a ferrous source, containing 100 mM or less of phosphate and glucose as the sole carbon source, and having a pH of 7.0-8.5.

### Claim 2 (depends on 1)

The method of claim 1, wherein the minimal medium contains 70 mM or less of phosphate.

### Claim 3 (depends on 1)

The method of claim 1, wherein the I/M ratio is 0.001 or lower.

### Claim 4 (depends on 1)

The method of claim 1, wherein the lipoprotein signal peptide includes the D1 sequence of Ag473.

### Claim 5 (depends on 1)

The method of claim 1, wherein the lipoprotein signal peptide comprises the P3 sequence of the acriflavine-resistance protein E precursor.

### Claim 6 (depends on 1)

The method of claim 1, wherein the nucleic acid sequence is operably linked to an inducible promoter.

### Claim 7 (depends on 6)

The method of claim 6, wherein the promoter is inducible by isopropylthio-β-D-galactoside (IPTG).

### Claim 8 (depends on 1)

The method of claim 1, wherein the recombinant polypeptide includes an antigen of a pathogen.

### Claim 9 (depends on 1)

The method of claim 1, wherein the E. coli host cell is C43 (DE3).

### Claim 10 (depends on 8)

The method of claim 8, wherein the antigen is E3 or a pneumolysin.

### Claim 11 (depends on 10)

The method of claim 10, wherein the lipoprotein signal peptide includes the D1 sequence of Ag473.

### Claim 12 (depends on 10)

The method of claim 10, wherein the lipoprotein signal peptide comprises the P3 sequence of the acriflavine-resistance protein E precursor.