# US 2017/0283469 A1 · Proteolytic inactivation of select proteins in bacterial extracts for improved expression

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- Number: US 2017/0283469 A1
- Document ID: US20170283469A1
- Status: Published application
- Kind: A1
- Application number: 15/483,766
- Filed: 2017-04-10
- Published: 2017-10-05
- Earliest claimed: 2012-10-12
- Assignees: Sutro Biopharma, Inc.
- Applicants: Sutro Biopharma, Inc.
- Inventors: Christopher D. THANOS; Christopher J. MURRAY; Junhao YANG; Heather STEPHENSON
- Web page: https://patentgrep.com/patent/US20170283469A1

## CPC

- C07K 14/245: Escherichia (G)
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/065: {Purification, fragmentation}
- C07K 16/32: against translation products of oncogenes
- C12N 9/52: derived from bacteria {or Archaea}
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/689: for bacteria
- G01N 30/02: Column chromatography
- G01N 30/06: Preparation
- C07K 2319/50: containing protease site
- C12Y 304/00: Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y 304/21: Serine endopeptidases (3.4.21)
- 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)
- 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)}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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 disclosure provides modified proteins that are capable of being cleaved by the protease OmpT1. The proteins can be modified in an exposed surface motif to incorporate OmpT1 cleavage sites. Also provided are nucleic acids encoding the modified proteins, bacterial cells that express the modified proteins, and cell free synthesis systems containing modified RF1. The disclosure further provides methods for reducing the deleterious activity of a modified protein in a cell free synthesis system by contacting the modified protein with OmpT1. Also provided are methods for reducing RF1 competition at an amber codon in the cell free synthesis system, and methods for expressing a protein in the cell free synthesis system. The modified proteins of the invention can be used to increase the yield of proteins having non-natural amino acids incorporated at an amber codon.

## Claims

### Claim at position 1

-31. (canceled)

### Claim 32

A mutant Releasing Factor 1 protein (RF1) comprising a cleavage site for an Outer Membrane Protein T1 (OmpT1) located within a switch loop region corresponding to amino acids 287-304 of SEQ ID NO:1.

### Claim 33 (depends on 32)

The mutant RF1 protein of claim 32, wherein the mutant RF1 comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1, and has activity to recognize a stop codon in an mRNA sequence and terminate translation.

### Claim 34 (depends on 32)

A bacterial cell comprising both an OmpT1 and the RF1 protein of claim 32.

### Claim 35 (depends on 32)

A nucleic acid encoding the mutant RF1 protein of claim 32.

### Claim 36 (depends on 35)

A bacterial cell comprising the nucleic acid of claim 35, wherein the nucleic acid is incorporated into the genome of the bacterial cell.

### Claim 37 (depends on 32)

A bacterial cell extract comprising the mutant RF1 protein of claim 32.

### Claim 38 (depends on 32)

A cell free synthesis system comprising in a single reaction mixture:
i) components from a bacterial lysate sufficient to translate a nucleic acid template encoding a protein;
ii) a nucleic acid template encoding a protein of interest and having at least one amber codon;
iii) tRNA complementary to the amber codon; and
iv) the mutant RF1 protein of claim 32.

### Claim 39 (depends on 32)

The mutant RF1 protein of claim 32, wherein the cleavage activity by OmpT1 is greater than 50% of the wild type RF1 (SEQ ID NO:1) after 30 minutes at 30° C. when the modified and wild-type proteins are present at a similar concentration in a cell-free extract from bacteria expressing OmpT1.

### Claim 40 (depends on 32)

The mutant RF1 protein of claim 32, where the switch loop region comprises an amino acid sequence selected from the group of sequences consisting of AAR, AARG (SEQ ID NO:47), WLAARRGRG (SEQ ID NO:48), and WGGRWARKKGTI (SEQ ID NO:49).

### Claim 41

A mutant Releasing Factor 1 protein (RF1) comprising a cleavage site for an Outer Membrane Protein T1 (OmpT1) located within a switch loop region corresponding to amino acids 287-304 of SEQ ID NO:1, where the switch loop region comprises an amino acid sequence selected from the group of sequences consisting of AAR, AARG (SEQ ID NO:47), WLAARRGRG (SEQ ID NO:48), and WGGRWARKKGTI (SEQ ID NO:49).

### Claim 42 (depends on 41)

The mutant RF1 protein of claim 41, wherein the amino acid sequence corresponding to amino acids 287-304 of SEQ ID NO:1 comprises an amino acid sequence selected from the group of sequences consisting of:
QARRGSTRRNLLGSGDRS;
(SEQ ID NO: 26)
QQARRGTRRNLLGSGDRS;
(SEQ ID NO: 27)
QQAARRGRRNLLGSGDRS;
(SEQ ID NO: 28)
QQAEARRGRNLLGSGDRS;
(SEQ ID NO: 29)
QQAEAARRGNLLGSGDRS;
(SEQ ID NO: 30)
QQAEASARRGLLGSGDRS;
(SEQ ID NO: 31)
QQAEASTARRGLGSGDRS;
(SEQ ID NO: 32)
QQAEASTRARRGGSGDRS;
(SEQ ID NO: 33)
QQAEASTRRARRGSGDRS;
(SEQ ID NO: 34)
QQAEASTRRNARRGGDRS;
(SEQ ID NO: 35)
QQAEASTRRNLARRGDRS;
(SEQ ID NO: 36)
QQAEASTRRNLLARRGRS;
(SEQ ID NO: 37)
QQAEASTRRNLLGARRGS;
(SEQ ID NO: 38)
QQAEASTRRNLLGSARRG;
(SEQ ID NO: 39)
QQAEASTRRNLLGSGARR
(SEQ ID NO: 40)
QQAWLAARRGRGGSGDRS;
(SEQ ID NO: 41)
QQAEWLAARRGRGSGDRS;
(SEQ ID NO: 42)
QQAEAWLAARRGRGGDRS
(SEQ ID NO: 43)
QQWGGRWARKKGTIGDRS;
(SEQ ID NO: 44)
QQAWGGRWARKKGTIDRS;
(SEQ ID NO: 45)
and
QQAEWGGRWARKKGTIRS.
(SEQ ID NO: 46)

### Claim 43 (depends on 41)

The mutant RF1 protein of claim 41, wherein the mutant RF1 comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1, and has activity to recognize a stop codon in an mRNA sequence and terminate translation.

### Claim 44 (depends on 41)

The mutant RF1 protein of claim 41, wherein the cleavage activity by OmpT1 is greater than 50% of the wild type RF1 (SEQ ID NO:1) after 30 minutes at 30° C. when the modified and wild-type proteins are present at a similar concentration in a cell-free extract from bacteria expressing OmpT1.

### Claim 45 (depends on 41)

A bacterial cell comprising both an OmpT1 and the RF1 protein of claim 41.

### Claim 46 (depends on 41)

A nucleic acid encoding the mutant RF1 protein of claim 41.

### Claim 47 (depends on 46)

A bacterial cell comprising the nucleic acid of claim 46, wherein the nucleic acid is incorporated into the genome of the bacterial cell.

### Claim 48 (depends on 41)

A bacterial cell extract comprising the mutant RF1 protein of claim 41.

### Claim 49 (depends on 41)

A cell free synthesis system comprising in a single reaction mixture:
i) components from a bacterial lysate sufficient to translate a nucleic acid template encoding a protein;
ii) a nucleic acid template encoding a protein of interest and having at least one amber codon;
iii) tRNA complementary to the amber codon; and,
iv) the mutant RF 1 protein of claim 41.

### Claim 50 (depends on 38)

The cell free synthesis system of claim 38, wherein the reaction mixture further comprises a non-natural amino acid and corresponding amino acid synthetase, the synthetase able to charge the tRNA complimentary to the amber codon with the non-natural amino acid.

### Claim 51 (depends on 50)

The cell free synthesis system of claim 50, wherein the system generates ATP via an active oxidative phosphorylation system.

### Claim 52 (depends on 32)

The mutant RF1 protein of claim 32, wherein the OmpT1 is wild-type OmpT1 or a functional homolog thereof having OmpT1 enzyme activity, or comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:3, or specifically binds polyclonal antibodies that bind to OmpT1.

### Claim 53 (depends on 32)

A method for expressing protein in a cell-free synthesis system, comprising:
i. combining a nucleic acid template with a bacterial extract comprising an RF1 protein of claim 32 that has been cleaved by OmpT1; and
ii. expressing a protein from the nucleic acid template.

### Claim 54 (depends on 35)

A method for preparing a cell free synthesis extract, the method comprising the steps of:
i) culturing an OmpT1 positive bacteria comprising the nucleic acid of claim 35 to express the mutant RF1 protein; and
ii) lysing the bacteria to create a cell free synthesis extract.

### Claim 55 (depends on 54)

The method of claim 54, wherein the OmpT1 positive bacteria is Escherichia coli.

### Claim 56 (depends on 32)

A method for expressing a protein of interest in a bacterial cell-free synthesis system, comprising:
i. combining a nucleic acid template encoding a protein of interest with a bacterial cell free synthesis extract to produce a bacterial cell-free synthesis system, wherein the bacterial cell free synthesis extract comprises the mutant RF1 protein of claim 32 and an OmpT1;
ii. allowing the mutant RF1 protein to be cleaved by the OmpT1; and
iii. expressing the protein of interest from the nucleic acid template.