# US 2014/0154743 A1 · Methods and materials for enhancing functional protein expression in bacteria

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- Number: US 2014/0154743 A1
- Document ID: US20140154743A1
- Status: Published application
- Kind: A1
- Application number: 13/982,669
- Filed: 2012-02-03
- Published: 2014-06-05
- Earliest claimed: 2011-02-03
- Assignees: Xoma Technology Ltd.
- Inventors: Raphael D. Levy; Chung-Leung Chan; Kiranjit K. Ahluwalia; Toshihiko Takeuchi
- Web page: https://patentgrep.com/patent/US20140154743A1

## CPC

- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 14/005: from viruses
- C07K 14/245: Escherichia (G)
- C12N 9/90: Isomerases (5.)
- C12N 15/67: General methods for enhancing the expression
- C12Y 502/01008: Peptidylprolyl isomerase (5.2.1.8), i.e. cyclophilin
- C07K 2319/00: Fusion polypeptide
- C07K 2319/35: containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
- 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)
- C12Y: ENZYMES

## Abstract

Novel materials and methods useful for expressing heterologous proteins in prokaryotic cells are provided, including prokaryotic cells expressing FkpA and/or Skp.

## Claims

### Claim 1

A prokaryotic host cell that expresses a heterologous protein for recombinant production comprising:
(a) a polynucleotide encoding (i) FkpA that is not linked to a functional signal sequence, or a functional fragment thereof, or (ii) Skp that is not linked to a functional signal sequence, or a functional fragment thereof, and
(b) a polynucleotide encoding the heterologous protein linked to a functional signal sequence.

### Claim 2 (depends on 1)

The host cell of claim 1 wherein the host cell comprises a polynucleotide encoding FkpA that is not linked to a functional signal sequence, or a functional fragment thereof, optionally wherein the host cell further comprises a polynucleotide encoding Skp that is not linked to a functional signal sequence, or a functional fragment thereof.

### Claim 3 (depends on 1)

The host cell of claim 1 wherein the host cell comprises a polynucleotide encoding Skp that is not linked to a functional signal sequence, or a functional fragment thereof.

### Claim 4

(canceled)

### Claim 5 (depends on 1)

The host cell of claim 1 wherein the prokaryotic host cell is selected from the group consisting of Escherichia coli, Salmonella typhimurium, Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus brevi, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ralstonia eutropha, Staphylococcus carnosus and Serratia marcescans.

### Claim 6 (depends on 1)

The host cell of claim 1 wherein the heterologous protein is associated with mis-folding or a slow folding rate in the absence of FkpA and Skp.

### Claim 7

(canceled)

### Claim 8 (depends on 1)

The host cell of claim 1 wherein the heterologous protein is fused to a filamentous phage coat protein or fragment thereof.

### Claim 9 (depends on 1)

The host cell of claim 1 wherein the heterologous protein is an antibody.

### Claim 10

(canceled)

### Claim 11 (depends on 1)

The host cell of claim 1 wherein the functional fragment of FkpA is a chaperone domain fragment.

### Claim 12 (depends on 11)

The host cell of claim 11 wherein the chaperone domain fragment comprises amino acids 26-140 of SEQ ID NO: 1 or wherein the chaperone domain fragment comprises an amino acid sequence at least 75% identical to at least 100 amino acids of amino acids 26 through 140 of SEQ ID NO: 1.

### Claim 13

(canceled)

### Claim 14 (depends on 1)

The host cell of claim 1 wherein the functional fragment of FkpA is a peptidylprolyl isomerase domain fragment.

### Claim 15 (depends on 14)

The host cell of claim 14 wherein the peptidylprolyl isomerase domain fragment comprises amino acids 141-270 of SEQ ID NO: 1 or wherein the peptidylprolyl isomerase domain fragment comprises an amino acid sequence at least 75% identical to a fragment of at least 100 amino acids of amino acids 141 through 270 of SEQ ID NO: 1.

### Claim 16

(canceled)

### Claim 17 (depends on 3)

The host cell of claim 3 wherein the functional fragment of Skp comprises amino acids 21-161 of SEQ ID NO: 2.

### Claim 18 (depends on 1)

A plurality of cells comprising at least 103 different prokaryotic host cells according to claim 1, each such host cell expressing a different heterologous protein.

### Claim 19

(canceled)

### Claim 20

A method for increasing recombinant production of a functional heterologous protein in a prokaryotic host cell, comprising co-expressing:
(a) a polynucleotide encoding (i) FkpA that is not linked to a functional signal sequence, or a functional fragment thereof, or (ii) Skp that is not linked to a functional signal sequence, or a functional fragment thereof, and
(b) a polynucleotide encoding a heterologous protein that is linked to a functional signal sequence,
whereby the amount of functional heterologous protein produced is increased compared to expressing (b) in the absence of (a).

### Claim 21 (depends on 20)

The method of claim 20 wherein the functional signal sequence linked to the heterologous protein directs secretion to the periplasm.

### Claim 22 (depends on 20)

The method of claim 20 wherein the host cell comprises (a) a polynucleotide encoding FkpA that is not linked to a functional signal sequence, or a functional fragment thereof, and (b) a polynucleotide encoding Skp that is not linked to a functional signal sequence, or a functional fragment thereof.

### Claim 23 (depends on 20)

The method of claim 20 wherein the prokaryotic host cell is selected from the group consisting of Escherichia coli, Salmonella typhimurium, Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus brevi, Pseudomonas aeruginosa, Pseudomonas fluorescens, Ralstonia eutropha, Staphylococcus carnosus, and Serratia marcescans.

### Claim 24 (depends on 20)

The method of claim 20 wherein the heterologous protein is associated with mis-folding or a slow folding rate in the absence of FkpA and Skp.

### Claim 25

(canceled)

### Claim 26 (depends on 20)

The method of claim 20 wherein the heterologous protein is fused to a filamentous phage coat protein or fragment thereof.

### Claim 27 (depends on 20)

The method of claim 20 wherein the heterologous protein is an antibody.

### Claim 28

(canceled)

### Claim 29 (depends on 20)

The method of claim 20 wherein the functional fragment of FkpA is a chaperone domain fragment.

### Claim 30 (depends on 29)

The method of claim 29 wherein the chaperone domain fragment comprises amino acids 26-140 of SEQ ID NO: 1 or wherein the chaperone domain fragment comprises an amino acid sequence at least 75% identical to at least 100 amino acids of amino acids 26 through 140 of SEQ ID NO: 1.

### Claim 31

(canceled)

### Claim 32 (depends on 20)

The method of claim 20 wherein the functional fragment of FkpA is a peptidylprolyl isomerase domain fragment.

### Claim 33 (depends on 32)

The method of claim 32 wherein the peptidylprolyl isomerase domain fragment comprises amino acids 141-270 of SEQ ID NO: 1 or wherein the peptidylprolyl isomerase domain fragment comprises an amino acid sequence at least 75% identical to a fragment of at least 100 amino acids of amino acids 141 through 270 of SEQ ID NO: 1.

### Claim 34

(canceled)

### Claim 35 (depends on 20)

The method of claim 20 wherein the functional fragment of Skp comprises amino acids 21-161 of SEQ ID NO: 2.

### Claim 36 (depends on 1, 20)

The host cell of claim 1 or the method of claim 20, wherein the host cell has a leaky phenotype.

### Claim 37 (depends on 1, 20)

The host cell of claim 1 or the method of claim 20, wherein the polynucleotide of (a) and the polynucleotide of (b) are operatively linked to the same regulatory control sequence.

### Claim 38 (depends on 1, 20)

The host cell of claim 1 or the method of claim 20, wherein the polynucleotide of (a) and the polynucleotide of (b) are on the same plasmid or wherein the polynucleotide of (a) and the polynucleotide of (b) are on different plasmids.

### Claim 39

(canceled)