Compositions and Methods Of Use Thereof For Making Polypeptides With Many Instances Of Nonstandard Amino Acids

Patent metadata

Field	Value
Publication number	US-2016355802-A1
Application number	US-201515117406-A
Country	US
Kind code	A1
Filing date	Feb 6, 2015
Priority date	Feb 6, 2014
Publication date	Dec 8, 2016
Grant date	—

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.

First claim

1 . A method of making aminoacyl tRNA synthetases (AARS) variants comprising
carrying out one or more rounds of mutagenesis on host organism cells comprising an orthogonal translation system comprising a precursor heterologous non-standard amino acid anaminoacyl tRNA synthetase (AARS) integrated into the host's genome and a cognate tRNA to form a library of variant 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.

2 . The method of claim 1 , wherein the mutagenesis is targeted to the AARS, tRNA, EF-Tu, RNA, components of the ribosome, protein components of the ribosome, or a combination thereof.

3 . The method of claim 1 , wherein the site(s) for introducing an amino acid sequence variation 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).

4 . The method of claim 3 , wherein the mutagenesis comprises one or more rounds of multiplex automated genome evolution (MAGE) comprising 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 one nucleic acid oligomer is introduced into the host cells' genome or vector within the host cells.

5 . The method of claim 4 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.

6 . The method of claim 1 wherein selection comprises negative selection comprising removal of host cells comprising undesirable AARS variants from the library of AARS variants.

7 . The method of claim 6 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.

8 . The method of claim 1 wherein selection comprises positive selection comprising choosing desirable AARS variants from the library of AARS variants.

9 . The method of claim 1 wherein positive selection comprises selecting host cells that have improved yield relative to the of a protein of interest encoded by an mRNA comprising at least one codon recognized by the OTS tRNA anticodon and expressed in the host cell.

10 . The method of claim 1 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 undesireable 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.

11 . The isolated nucleic acid encoding an AARS variant identified according to claim 1 .

12 . An isolated AARS variant encoded by the nucleic acid of claim 11 .

13 . An isolated AARS variant comprising 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.

14 . The isolated AARS variant of claim 13 comprising amino acids 65, 107, 108, 109, 158, 159, 162, 167, 257, and 261 of any of SEQ ID NOS:2-15.

15 . An isolated variant AARS comprising 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.

16 . The isolated variant AARS of claim 15 comprising the amino acid sequence of any of SEQ ID NOS:2-15.

17 . A method of making a polypeptide comprising one or more iterations of a non-standard amino acid comprising expressing a messenger RNA (mRNA) encoding the target protein in a system comprising:
orthogonal translation system (OTS) comprising the nucleic acid sequence encoding the variant AARS of claim 12 and its cognate tRNA operably linked to expression control sequences and transformed, transfected, or integreated into a genomically recoded organism (GRO) with at least one codon reduced or absent from its genome, and a plurality of a non-standard amino acids, wherein the mRNA comprises a nucleic acid sequence comprising at least one iteration of the codon deleted from the GRO, and wherein the tRNA comprises and anticodon that can bind to the codon reduced or absent from the GRO.

18 . The method of claim 17 wherein the GRO is E. coli.

19 . The method of claim 18 wherein the codon reduced or absent from the GRO is TAG.

20 . The method of claim 19 wherein the non-standard amino acid is selected is pAcF, pAzF, StyA, 41F, 4BrF, 4ClF, 4MeF, 4Cf3F, MeY, 4NO 2 F, 4BuF, BuY, 2NaA, PhF, or 3,4-dihydroxyphenylalanine.

21 . A polypeptide translated by the method of claim 17 .

22 . A polypeptide comprising the amino acid sequence of any of SEQ ID NOS:20-27.

23 . A polypeptide comprising the amino acid sequence of any of SEQ ID NOS:19-27, wherein the polypeptide was made according to the method of claim 17 .

24 . The polypeptide of claim 22 wherein “X” is pAcF, pAzF, StyA, 4IF, 4BrF, 4ClF, 4MeF, 4Cf3F, MeY, 4NO 2 F, 4BuF, BuY, 2NaA, PhF, or 3,4-dihydroxyphenylalanine.

25 . The polypeptide of claim 21 comprising at least 20 iterations of a non-standard amino acid.

26 . A pharmaceutical composition comprising the polypeptide of claim 21 .

27 . A device coated with the polypeptide of claim 21 .

28 . A host cell comprising the variant AARS of claim 12 .

29 . The host cell of claim 28 , wherein the variant AARS is integrated into the host cell's genome.

30 . The host cell of claim 29 , wherein the host cell is a genomically recoded E. coli.

31 . A polypeptide comprising the amino acid sequence of any of SEQ ID NOS:19-27, wherein “X” is 3,4-dihydroxyphenylalanine.

32 . A polypeptide comprising 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.

34 . The polypeptide of claim 33 , wherein molecule is a fatty acid.

35 . The polypeptide of claim 34 , wherein the fatty acid is fatty acids palmitic acid.

36 . A silver nanoparticle formulation comprising silver nanoparticles and the polypeptide of claim 31 .

37 . A polypeptide translated by the method of claim 17 comprising one or more iterations of pAzF.

38 . The polypeptide of claim 37 , wherein the side chain of pAzF is conjugated to another molecule.

39 . The polypeptide of claim 38 , wherein the molecule is a drug or an imaging molecule.

40 . The method of claim 1 wherein the host organism is a genetically recoded organism (GRO).

Assignees

• Univ Yale

Inventors

• Isaacs Farren J
• Amiram Miriam
• Haimovich Adrian
• Söll Dieter

Classifications

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N15/1058

  Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms · CPC title

• C12Y601/01

  Ligases forming aminoacyl-tRNA and related compounds (6.1.1) · CPC title

• C12N9/93 Primary

  Ligases (6) · CPC title