# US 8,569,015 B2 · RPA optimization

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- Number: US 8,569,015 B2
- Document ID: US8569015B2
- Status: Granted
- Kind: B2
- Application number: 11/809,100
- Filed: 2007-05-30
- Published: 2008-03-06
- Granted: 2013-10-29
- Earliest claimed: 2006-05-30
- Assignees: Pfenex Inc.
- Applicants: Lada L. Rasochova; Philip P. Dao; Jamie P. Phelps
- Inventors: Lada L. Rasochova; Philip P. Dao; Jamie P. Phelps
- Web page: https://patentgrep.com/patent/US8569015B2

## CPC

- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C07K 14/32: from Bacillus (G)
- C12N 15/67: General methods for enhancing the expression
- C12N 15/78: for Pseudomonas
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- 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)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

An optimized synthetic polynucleotide encoding a Bacillus anthracis protective antigen and an anthrax vaccine based on the encoded protective antigen. Furthermore, heterologous expression in a host Pseudomonas fluorescens bacteria of an optimized polynucleotide sequence encoding a Bacillus anthracis protective antigen.

## Claims

### Claim 1

A method of producing a soluble recombinant Bacillus anthracis protective antigen protein (rPA) in a Pseudomonas fluorescens expression system comprising expressing the soluble rPA from a nucleotide sequence encoding the soluble rPA, wherein the nucleotide sequence is the soluble rPA encoding sequence of a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, or wherein the nucleotide sequence encoding the soluble rPA is a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.

### Claim 2 (depends on 1)

The method of claim 1, wherein the soluble rPA encoding nucleotide sequence lacks the sequence encoding the starting methionine.

### Claim 3 (depends on 1)

The method of claim 1, wherein the soluble rPA is expressed in the cytoplasm or periplasm of a host cell.

### Claim 4 (depends on 1)

The method according to claim 1, comprising expressing the soluble rPA to which a secretion signal has been fused, wherein said secretion signal co-translationally or post-translationally directs transfer of the soluble rPA to the periplasm of a host cell.