# US 10,501,734 B2 · Compositions and methods of use thereof for making polypeptides with many instances of nonstandard amino acids

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,501,734 B2
- Document ID: US10501734B2
- Status: Granted
- Kind: B2
- Application number: 15/117,406
- Filed: 2015-02-06
- Published: 2016-12-08
- Granted: 2019-12-10
- Earliest claimed: 2014-02-06
- Assignees: Yale University
- Applicants: Yale University
- Inventors: Farren J. Isaacs; Miriam Amiram; Adrian Haimovich; Dieter Söll
- Web page: https://patentgrep.com/patent/US10501734B2

## CPC

- C12N 9/93: {Ligases (6)}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/1058: {Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms}
- C12Y 601/01: Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
- 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

Compositions, systems, and methods for preparation of polypeptides having multiple iterations of non-standard amino acids are provided. The compositions and method can be used to produce recombinant proteins at a greater yield than the same or similar polypeptides made using conventional compositions, systems, and methods. Accordingly, in some embodiments, the polypeptides are ones that could not be made using conventional methods and reagents, or could not be made a sufficient yield or purity to serve a practical purpose using conventional methods and reagents. Polypeptides made using the disclosed compositions, systems, and methods are also provided.

## Claims

### Claim 1

A composition comprising
a plurality of a translated peptide or polypeptide comprising at least one instance of a non-standard amino acid,
mRNA encoding the peptide or polypeptide, and
a non-auxotrophic translation system comprising a heterologous, multiplex automated genome evolution (MAGE) evolved aminoacyl tRNA synthetase (AARS) and a cognate tRNA that can be charged with the non-standard amino acid by the AARS and express an elastin-like (ELP) green fluorescent protein (GFP) fusion protein of SEQ ID NO:33 comprising thirty instances of the non-standard amino acid at an average yield of more than 18% of the corresponding ELP-GFP of SEQ ID NO:34 with no non-standard amino acids,
wherein the translated peptide or polypeptide is prepared by translation of the mRNA encoding the peptide or polypeptide in genomically recoded organism (GRO) E. coli cells expressing the translation system.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the MAGE comprises
carrying out one or more rounds of mutagenesis on host organism cells comprising an orthogonal translation system comprising a precursor heterologous AARS integrated into the host's genome and a cognate tRNA to form a library of variants of the precursor heterologous AARS,
carrying out one or more rounds of selection on the library of variants to identify host cells comprising variant AARS with improved or altered specificity and/or activity for the non-standard amino and/or the cognate tRNA, and
selecting the heterologous AARS from the library of variant AARS.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the mutagenesis is targeted to the AARS alone or in combination with one or more of tRNA, EF-Tu, RNA components of the ribosome, and protein components of the ribosome.

### Claim 4 (depends on 2)

The composition of claim 2, wherein the site(s) for introducing an amino acid sequence variation in the AARS are predetermined, but the mutation(s) per se are random, semi-random, degenerate, or specific and introduce mismatch(es), deletion(s), and/or insertion(s).

### Claim 5 (depends on 4)

The composition of claim 4, wherein the mutagenesis comprises one or more rounds of transforming or transfecting host cells using transformation medium or transfection medium including at least one nucleic acid oligomer containing one or more mutations, replacing the transformation medium or transfection medium with growth medium, incubating the cells in the growth medium, and optionally repeating the steps if necessary or desired until the sequence(s) of the one or more nucleic acid oligomer is introduced into the host cells' genome or vector within the host cells.

### Claim 6 (depends on 5)

The composition of claim 5 wherein, the one or more nucleic acid oligomers is a pool of oligomers having a diversity of different random or non-random mutations at the location(s) of desired mutagenesis.

### Claim 7 (depends on 2)

The composition of claim 2 wherein selection comprises negative selection comprising removal of host cells comprising undesirable AARS variants from the library of AARS variants.

### Claim 8 (depends on 7)

The composition of claim 7 wherein negative selection comprises TolC-based selection wherein mutated AARS variants capable of mischarging the cognate tRNA with natural amino acids permit read-through of a tolC construct in the host genome, rendering the organism sensitive to colicin E1.

### Claim 9 (depends on 2)

The composition of claim 2 wherein selection comprises positive selection comprising choosing desirable AARS variants from the library of AARS variants.

### Claim 10 (depends on 2)

The composition of claim 2 wherein positive selection comprises selecting host cells that have improved yield of a protein of interest encoded by an mRNA comprising at least one codon recognized by the orthogonal translation system tRNA anticodon and expressed in the host cell.

### Claim 11 (depends on 2)

The composition of claim 2 wherein the selection comprises Rec negative selection in the presence of one or more of the undesirable non-standard amino acids to establish orthogonality toward the undesirable non-standard amino acid(s) in addition to the twenty canonical amino acids, followed by positive selection for increased activity for one or more desired non-standard amino acids.

### Claim 12 (depends on 1)

The composition of claim 1, wherein the AARS comprises at least 90% sequence identity, but not 100% sequence identity, to the non-standard amino acid (amino acid ligand) binding pocket of any of SEQ ID NOS:2-15, the tRNA anticodon recognition interface of any of SEQ ID NO:2-15, or a combination thereof, or SEQ ID NO:1.

### Claim 13 (depends on 12)

The composition of claim 12, wherein the AARS comprises amino acids 65, 107, 108, 109, 158, 159, 162, 167, 257, and 261 of any of SEQ ID NOS:2-15.

### Claim 14 (depends on 1)

The composition of claim 1, wherein the AARS comprises the non-standard amino acid (amino acid ligand) binding pocket of any of SEQ ID NOS:2-15, the tRNA anticodon recognition interface of any of SEQ ID NO:2-15, or a combination thereof.

### Claim 15 (depends on 14)

The composition of claim 14, wherein the AARS comprises the amino acid sequence of any of SEQ ID NOS:2-15.

### Claim 16 (depends on 1)

The composition of claim 1, wherein the AARS and its cognate tRNA are operably linked to expression control sequences and transformed, transfected, or integrated into the GRO,
wherein the heterologous mRNA comprises a nucleic acid sequence comprising at least one instance of a codon absent from the GRO,
and wherein the tRNA comprises an anticodon that can bind to the codon absent from the GRO and introduce the non-standard amino acid into the nascent peptide or polypeptide during translation of the heterologous mRNA.

### Claim 17 (depends on 16)

The composition of claim 16 wherein the GRO wherein the codon absent from the GRO is a non-sense codon recoded as a sense codon, and wherein expression of the corresponding non-sense codon release factor is reduced or absent.

### Claim 18 (depends on 17)

The composition of claim 17 wherein the non-sense codon is TAG.

### Claim 19 (depends on 1)

The composition of claim 1 wherein the non-standard amino acid is selected is pAcF, pAzF, StyA, 4IF, 4BrF, 4ClF, 4MeF, 4Cf3F, MeY, 4NO2F, 4BuF, BuY, 2NaA, PhF, or 3,4-dihydroxyphenylalanine.

### Claim 20 (depends on 1)

The composition of claim 1 comprising at least 20 instances of the non-standard amino acid.

### Claim 21 (depends on 1)

The composition of claim 1 wherein the E. coli is C321.Δ A.

### Claim 22 (depends on 1)

The composition of claim 1 comprising one or more instances of pAzF.

### Claim 23 (depends on 22)

The composition of claim 22, wherein the side chain of pAzF is conjugated to another molecule.

### Claim 24 (depends on 23)

The composition of claim 23, wherein the molecule is a drug or an imaging molecule.

### Claim 25 (depends on 1)

The composition of claim 1, wherein the translation system can express the ELP-GFP fusion protein of SEQ ID NO:33 comprising thirty instances of the non-standard amino acid at an average yield of more than 66.5% of the corresponding ELP-GFP of SEQ ID NO:34 with no non-standard amino acids.

### Claim 26 (depends on 1)

The composition of claim 1, wherein the plurality of the peptide or polypeptide has at least 95% correct non-standard amino acid incorporated at the desired residue(s) relative to an undesired amino acid at the same residue.

### Claim 27 (depends on 1)

The composition of claim 1, wherein the peptide or polypeptide comprises the sequence (VPGXG)n (SEQ ID NO:19), wherein “X” is the non-standard amino acid and wherein “n” is an integer from 1 to 500.

### Claim 28 (depends on 1)

The composition of claim 1, wherein the peptide or polypeptide is an Elastin-like Protein (ELP).

### Claim 29 (depends on 28)

The composition of claim 28, wherein the ELP comprises the amino acid sequence of any of SEQ ID NOS:20-27.

### Claim 30 (depends on 29)

The composition of claim 29 wherein “X” in SEQ ID NO:20-27 is pAcF, pAzF, StyA, 4IF, 4BrF, 4ClF, 4MeF, 4Cf3F, MeY, 4NO2F, 4BuF, BuY, 2NaA, PhF, or 3,4-dihydroxyphenylalanine.

### Claim 31 (depends on 28)

The composition of claim 28, wherein the ELP comprises the amino acid sequence of any of SEQ ID NOS:19-27, wherein “X” is 3,4-dihydroxyphenylalanine.

### Claim 32 (depends on 28)

The composition of claim 28, wherein the ELP comprises the amino acid sequence of SEQ ID NOS:19-27 wherein “X” is pAzF and the side chain of pAzF is conjugated to a molecule.

### Claim 33 (depends on 32)

The composition of claim 32, wherein molecule is a fatty acid.

### Claim 34 (depends on 33)

The composition of claim 33, wherein the fatty acid is palmitic acid.

### Claim 35 (depends on 1)

The composition of claim 1, wherein the peptide or polypeptide is a protein polymer.

### Claim 36 (depends on 18)

The composition of claim 18, wherein the release factor is release factor 1 (RF1).

### Claim 37

A composition comprising
a plurality of a translated peptide or polypeptide comprising at least four instances of a non-standard amino acid,
mRNA encoding the peptide or polypeptide, and
a translation system comprising a heterologous aminoacyl tRNA synthetase (AARS) and a cognate tRNA that can be charged with the non-standard amino acid by the AARS express a superfolder green fluorescent protein (GFP) comprising three instances of the non-standard amino acid at more than 33% yield of the corresponding wildtype superfolder green fluorescent protein (GFP) with no non-standard amino acids,
wherein the translated peptide or polypeptide is prepared by translation of the mRNA encoding the peptide or polypeptide in genomically recoded organism (GRO) E. coli cells expressing the translation system, and
wherein the plurality of the peptide has at least 95% correct non-standard amino acid incorporated at the desired residue(s) relative to an undesired amino acid at the same residue.

### Claim 38 (depends on 37)

The composition of claim 37 wherein the E. coli is C321.Δ A.