# US 7,410,788 B2 · Compositions and methods for production of disulfide bond containing proteins in host cells

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- Number: US 7,410,788 B2
- Document ID: US7410788B2
- Status: Granted
- Kind: B2
- Application number: 11/058,926
- Filed: 2005-02-16
- Published: 2006-02-09
- Granted: 2008-08-12
- Earliest claimed: 1999-10-05
- Assignees: President and Fellows of Harvard College; Board of Regents, The University of Texas System
- Inventors: Jonathan Beckwith; Fredrik Aslund; Paul H. Bessette; George Georgiou; Daniel Ritz; Jackie Eun-ah Lim
- Web page: https://patentgrep.com/patent/US7410788B2

## CPC

- C12N 9/0004: {Oxidoreductases (1.)}
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- 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 invention provides composition and methods for producing proteins of interest which comprise at least one disulfide bond, include proteins which in their mature form do not contain disulfide bonds, but whose precursor molecule contained at least one disulfide bond. The methods employ a host cell modified to more efficiently produce properly folded disulfide bond containing proteins. The host cells generally contain a mutation in one or more reductase genes, and can be further genetically modified to increase their growth rate, and are further optionally modified to increase the expression of a catalyst of disulfide bond formation. Host cells, methods for using such to produce proteins of interest, proteins of interest produced by these methods are within the scope of the invention.

## Claims

### Claim 1

A prokaryotic cell that is genetically modified to shift the redox status of the cytoplasm to a more oxidative state, which cell further expresses a heterologous polypeptide selected from the group consisting of catalysts of disulfide bond formation, disulfide bond isomerization and the combination of disulfide bond formation and disulfide bond isomerization, and which cell is further modified to increase its ability to proliferate, wherein the modification to increase its ability to proliferate is a mutation in an AhpC gene.

### Claim 2 (depends on 1)

The prokaryotic cell of claim 1, wherein the expression or activity of a reductase is decreased relative to that in the corresponding wild type cell.

### Claim 3 (depends on 2)

The prokaryotic cell of claim 2, wherein the reductase is selected from the group consisting of thioredoxin reductase, glutathione reductase, and glutathione.

### Claim 4 (depends on 3)

The prokaryotic cell of claim 3, in which the expression or activity of a second reductase is decreased relative to that in the corresponding wild type cell.

### Claim 5 (depends on 4)

The prokaryotic cell of claim 4, wherein the second reductase is selected from the group consisting of thioredoxin reductase, glutathione reductase, and glutathione.

### Claim 6 (depends on 2)

The prokaryotic cell of claim 2, wherein the gene encoding the reductase is mutated.

### Claim 7 (depends on 6)

The prokaryotic cell of claim 6, wherein the gene encoding the reductase contains a null mutation.

### Claim 8 (depends on 5)

The prokaryotic cell of claim 5, wherein the genes encoding the first and the second reductases contain a null mutation.

### Claim 9 (depends on 2)

The prokaryotic cell of claim 2, wherein the activity of the reductase is inhibited.

### Claim 10 (depends on 9)

The prokaryotic cell of claim 9, wherein the activity of the reductase is inhibited by contacting the prokaryotic cell with an agent.

### Claim 11 (depends on 1)

The prokaryotic cell of claim 1, wherein the modification to increase the ability of the cell to proliferate is a mutation in the endogenous ahpC gene.

### Claim 12 (depends on 1)

The prokaryotic cell of claim 1, wherein the mutation is located in a region containing four triplet repeats.

### Claim 13 (depends on 12)

The prokaryotic cell of claim 12, wherein the mutated ahpC protein comprises the amino acid sequence set forth in SEQ ID NO: 24.

### Claim 14 (depends on 12)

The prokaryotic cell of claim 12, wherein the mutation in the ahpC gene comprises an insertion of three nucleotides in the TCT triplet repeat region
located at about codons 36-39 of an ahpC gene.

### Claim 15 (depends on 14)

The prokaryotic cell of claim 14, wherein the triplet rich region of the mutated ahpC gene encodes a stretch of four phenylalanines.

### Claim 16 (depends on 14)

The prokaryotic cell of claim 14, wherein the three nucleotides are TCT.

### Claim 17 (depends on 15)

The prokaryotic cell of claim 15, wherein the mutated ahpC gene encodes a protein comprising SEQ ID NO: 11.

### Claim 18 (depends on 17)

The prokaryotic cell of claim 17, wherein the TCT triplet rich region has the nucleotide sequence set forth in SEQ ID NO: 10.

### Claim 19 (depends on 1)

The prokaryotic cell of claim 1, which is a prokaryotic cell having ATCC Designation No. PTA-938 (FA112) and further expresses a heterologous polypeptide selected from the group consisting of catalysts of disulfide bond formation, disulfide bond isomerization and the combination of disulfide bond formation and disulfide bond isomerization.

### Claim 20 (depends on 1)

The prokaryotic cell of claim 1, which is a prokaryotic cell having ATCC Designation No. PTA-939 (FA113) and further expresses a heterologous polypeptide selected from the group consisting of catalysts of disulfide bond formation, disulfide bond isomerization and the combination of disulfide bond formation and disulfide bond isomerization.

### Claim 21 (depends on 1)

A prokaryotic cell of claim 1, further comprising a heterologous nucleic acid encoding a protein comprising at least one cysteine that forms a disulfide bond with another cysteine.

### Claim 22 (depends on 1)

The prokaryotic cell of claim 1, wherein the heterologous polypeptide is a catalyst of disulfide bond isomerization.

### Claim 23 (depends on 22)

The prokaryotic cell of claim 22, wherein the reductase is selected from the group consisting of thioredoxin reductase, glutathione reductase, and glutathione.

### Claim 24 (depends on 23)

The prokaryotic cell of claim 23, in which the expression or activity of a second reductase is decreased relative to that in the corresponding wild type cell.

### Claim 25 (depends on 24)

The prokaryotic cell of claim 24, wherein the second reductase is selected from the group consisting of thioredoxin reductase, glutathione reductase, and glutathione.

### Claim 26 (depends on 23)

The prokaryotic cell of claim 23, wherein the gene encoding the reductase is mutated.

### Claim 27 (depends on 26)

The prokaryotic cell of claim 26, wherein the gene encoding the reductase contains a null mutation.

### Claim 28 (depends on 24)

The prokaryotic cell of claim 24, wherein the genes encoding the first and the second reductases contain a null mutation.

### Claim 29 (depends on 22)

The prokaryotic cell of claim 22, wherein the modification to increase the ability of the cell to proliferate is a mutation in the endogenous ahpC gene.

### Claim 30 (depends on 29)

The prokaryotic cell of claim 29, wherein the mutation in the ahpC gene comprises an insertion of three nucleotides in the TCT triplet repeat region located at about codons 36-39 of an ahpC gene.

### Claim 31 (depends on 30)

The prokaryotic cell of claim 30, wherein the three nucleotides are TCT.

### Claim 32 (depends on 31)

The prokaryotic cell of claim 31, wherein the mutated ahpC protein comprises the amino acid sequence set forth in SEQ ID NO: 24.

### Claim 33 (depends on 22)

A prokaryotic cell of claim 22, further comprising a heterologous nucleic acid encoding a protein comprising at least one cysteine that forms a disulfide bond with another cysteine.

### Claim 34 (depends on 22)

A method for producing a protein having at least one cysteine, which forms a disulfide bond with another cysteine, comprising isolating the protein from a cell of claim 22 in which has been introduced a nucleic acid encoding the protein, which protein has at least one cysteine that forms a disulfide bond with another cysteine, and which cell has been grown under conditions in which the protein is produced.

### Claim 35 (depends on 1)

The prokaryotic cell of claim 1, wherein the catalyst is a DsbC protein without a signal sequence or a variant thereof that is encoded by a nucleic acid that hybridizes to a nucleic acid encoding a DsbC protein under conditions including a wash in 0.2×SSC at 50° C., wherein the variant acts as a catalyst selected from the group consisting of catalysts of disulfide bond formation, disulfide bond isomerization and the combination of disulfide bond formation and disulfide bond isomerization.

### Claim 36 (depends on 1)

The prokaryotic cell of claim 1, wherein the catalyst is a variant of a protein of the thioredoxin superfamily having a redox potential that is higher than that of its wild type counterpart.

### Claim 37 (depends on 36)

The prokaryotic cell of claim 36, wherein the variant is a “Grx” variant of thioredoxin A.

### Claim 38 (depends on 35)

The prokaryotic cell of claim 35, wherein the catalyst is a DsbC protein without a signal sequence.

### Claim 39 (depends on 1)

A method for producing a protein having at least one cysteine, which forms a disulfide bond with another cysteine, comprising isolating the protein from a cell of claim 1 in which has been introduced a nucleic acid encoding the protein, which protein has at least one cysteine that forms a disulfide bond with another cysteine, and which cell has been grown under conditions in which the protein is produced.