# US 2021/0171584 A1 · Cell-free protein synthesis platform derived from cellular extracts of vibrio natriegens

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- Number: US 2021/0171584 A1
- Document ID: US20210171584A1
- Status: Published application
- Kind: A1
- Application number: 16/762,889
- Filed: 2018-11-12
- Published: 2021-06-10
- Earliest claimed: 2017-11-10
- Assignees: Northwestern University
- Applicants: Northwestern University
- Inventors: Michael Christopher Jewett; Benjamin James Des Soye; Samuel Ryan Davidson
- Web page: https://patentgrep.com/patent/US20210171584A1

## CPC

- C07K 14/28: from Vibrionaceae (F)
- C07K 1/00: General methods for the preparation of peptides {, i.e. processes for the organic chemical preparation of peptides or proteins of any length}
- C12N 1/20: Bacteria; Culture media therefor
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- 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)}

## Abstract

Disclosed are compositions, methods, and kits for performing cell-free RNA transcription and/or cell-free protein synthesis (CFPS). The disclosed compositions, methods, and kits include or utilize components prepared from Vibrio species such as cellular extracts from Vibrio natriegens.

## Claims

### Claim 1

A platform for in vitro transcription of mRNA and/or translation of peptides, polypeptides, or sequence defined polymers, the platform comprising a cellular extract prepared from a cell culture of a species of Vibrio.

### Claim 2 (depends on 1)

The platform of claim 1, wherein the species of Vibrio is Vibrio natriegens.

### Claim 3 (depends on 1)

The platform of claim 1, wherein the species of Vibrio is engineered to be deficient in a negative effector for cell-free protein synthesis (CFPS).

### Claim 4 (depends on 3)

The platform of claim 3, wherein the negative effector for CFPS is a selected from the group consisting of a homolog of E. coli endA, mazF, ma, mb, me, gor, lon, ompT, gdhA, gshA, sdaA, sdaB, speA, WaaL, tnaA, glpK, and any combination thereof.

### Claim 5 (depends on 1)

The platform of claim 1, wherein the species of Vibrio is engineered to express an upregulated gene product that is a positive effector for CFPS.

### Claim 6 (depends on 5)

The platform of claim 5, wherein the positive effector for CFPS is selected from the group consisting of a homolog of E. coli ackA, ndk, pykF, cdd, dsbC, dnaK, dnaJ, crpE, tig, groS, groL, infA, infB, fusA, efp, lepA, tufB, hs1R, ffr, and any combination thereof.

### Claim 7 (depends on 1)

The platform of claim 1, wherein the species of Vibrio is engineered to be deficient in a release factor 1.

### Claim 8 (depends on 1)

The platform of claim 1, wherein the species of Vibrio has been genomically-recoded to replace one or more stop codons with a different codon.

### Claim 9 (depends on 1)

The platform of claim 1, wherein the species of Vibrio has been engineered to express T7 RNA polymerase.

### Claim 10 (depends on 1)

The platform of claim 1, wherein the Vibrio cellular extract is prepared from cell cultures that are in stationary phase, optionally wherein stationary phase is defined as the cell culture having an OD600 of greater than about 3.0.

### Claim 11 (depends on 1)

The platform of claim 1, wherein the cellular extract comprises an S12 and/or S30 fraction of the cell culture.

### Claim 12 (depends on 1)

The platform of claim 1, further comprising one or more components selected from the group consisting of amino acids which optionally may include non-canonical amino acids, NTPs, salts, cofactors, an energy source and optionally an energy source comprising a phosphate group (such as phosphoenol pyruvate (PEP)) or non-phosphorylated energy group (e.g., glucose, pyruvate), a translation template, a transcription template, and any combination thereof.

### Claim 13 (depends on 1)

The platform of claim 1, further comprising an energy source and optionally an energy source comprising a phosphate group (such as phosphoenol pyruvate (PEP)), wherein the energy source is present at a concentration of greater than about 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, or 90 mM (preferably greater than about 67 mM), but less than about 100 mM, or within a concentration range bounded by of these values.

### Claim 14 (depends on 1)

The platform of claim 1, further comprising a source of potassium (K+), wherein the platform comprises potassium at a concentration greater than about 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, or 450 mM (preferably about 300 mM), but less than about 500 mM, or within a concentration range bounded by of these values; and/or further comprising a source of magnesium (Mg+), wherein the platform comprises magnesium at a concentration greater than about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 8 mM, 12 mM, 16 mM or 20 mM (preferably about 8 mM), but less than about 30 mM, or within a concentration range bounded by of these values.

### Claim 15 (depends on 1)

The platform of claim 1, wherein the platform or one or more components of the platform are preserved such as through freeze-drying.

### Claim 16 (depends on 1)

A method for in vitro transcription of mRNA and/or translation of a peptide, a polypeptide, or a sequence defined polymer, the method comprising transcribing a transcription templated encoding the mRNA and/or translating in vitro an mRNA encoding the sequence defined polymer, polypeptide, or peptide in the platform of claim 1.

### Claim 17 (depends on 16)

The method of claim 16, wherein the method comprises transcribing a DNA template in the platform to provide the translated mRNA.

### Claim 18 (depends on 16)

The method of claim 16, wherein the method is performed at a temperature between about 20-40° C.

### Claim 19

(canceled)

### Claim 20

(canceled)

### Claim 21

A kit comprising as components: (a) a cellular extract prepared from a cell culture of a species of Vibrio; and (b) a reaction mixture for translating an mRNA, optionally wherein the species of Vibrio is Vibrio natriegens.

### Claim 22

(canceled)

### Claim 23

A recombinant species of Vibrio, optionally Vibrio natriegens, wherein the species of Vibrio is engineered to be deficient in a negative effector for cell-free protein synthesis (CFPS).

### Claim at position 24

-29. (canceled)