Highly efficient and tunable system for the incorporation of unnatural amino acids into proteins in Escherichia coli

Patent metadata

Field	Value
Publication number	US-12163173-B2
Application number	US-201716347880-A
Country	US
Kind code	B2
Filing date	Nov 9, 2017
Priority date	Nov 10, 2016
Publication date	Dec 10, 2024
Grant date	Dec 10, 2024

Abstract

The present invention provides a composition with a cell having an orthogonal translation system, and an expression vector. The invention also provides a method of producing a protein carrying an unnatural amino acid, and a kit for use in producing a protein carrying the unnatural amino acid.

First claim

The invention claimed is:

1. A cell, comprising:
a) an orthogonal translation system comprising an orthogonal tRNA, said orthogonal tRNA comprises an anticodon that corresponds to a stop codon (o-tRNA) and an unnatural amino acid (UAA)-specific orthogonal amino-acyl-tRNA synthetase (o-aaRS), and b) a vector comprising, a promoter region operably linked to an open reading frame, wherein said promoter region comprises a OR2-OR1-Pr promoter and said open reading frame comprises at least one of said stop codon located within: (i) the first 240 bases; or (ii) the first third of the open reading frame.

2. The cell of claim 1 , wherein said UAA is Propargyl-l-Lysine (PrK).

3. The cell of claim 1 , wherein said stop codon is a TAG stop codon.

4. The cell of claim 1 , wherein said cell is devoid of release factor 1 (RF1) expression.

5. The cell of claim 1 , wherein said cell further comprises an endogenous genome devoid of a TAG stop codon.

6. The cell of claim1 , wherein said cell is a genomically recoded E. coli (GRO) strain and said stop codon is a TAG stop codon.

7. The cell of claim 1 , wherein said OR2-OR1-Pr promoter region consists of, from 5′ to 3′, SEQ ID NO: 1, and SEQ ID NO: 2.

8. A method for producing a protein comprising an unnatural amino acid, comprising:
a) providing at least one cell of claim 1 ;
b) expressing in said cell the orthogonal translation system comprising the orthogonal tRNA, said orthogonal tRNA comprises the anticodon that corresponds to the stop codon (o-tRNA) and the UAA-specific orthogonal amino-acyl-tRNA synthetase (o-aaRS);
c) expressing in said cell the vector comprising, the promoter region operably linked to the open reading frame, wherein said promoter region comprises the OR2-OR1-Pr promoter and said open reading frame comprises at least one of: said stop codon within: (i) the first 240 bases; or (ii) the first third of the open reading frame; and
d) contacting said cell with said UAA, thereby producing said protein comprising said UAA.

9. The method of claim 8 , wherein said UAA is Proparagyl-l-Lysine.

10. The method of claim 8 , wherein said stop codon is a TAG stop codon.

11. The method of claim 8 , wherein said cell is devoid of RF1 expression.

12. The method of claim 8 , wherein said cell is devoid of said TAG stop codon.

13. The method of claim 8 , wherein said cell is a genomically recoded E. coli (GRO) strain.

14. The method of claims 8 , wherein said stop codon is a TAG stop codon.

15. The method of claim 8 , wherein said promoter region comprises an OR2-OR1-Pr promoter region.

16. The cell of claim 1 , wherein said vector further comprises a 5′ untranslated region (UTR) 3′ to said promoter region and wherein said 5′ UTR comprises SEQ ID NO: 3.

17. A kit comprising:
a) an orthogonal translation system comprising an orthogonal tRNA, said orthogonal tRNA comprises an anticodon that corresponds to a stop codon (o-tRNA) and an UAA-specific orthogonal amino-acyl-tRNA synthetase (o-aaRS);
b) a vector comprising a promoter region operably linked to a multiple cloning site (MCS), wherein said promoter region comprises a OR2-OR1-Pr promoter and has a transcriptional initiation rate between 1.0 and 10 mRNAs per second and a ribosome initiation rate between 1.0 and 4.5 ribosomes per second;
c) instructions for designing a nucleic acid molecule, wherein said nucleic acid molecule comprises at least one of said stop codon within: (i) the first 240 bases; or (ii) the first third of the open reading frame.

18. The kit of claim 17 , further comprising said UAA, a cell devoid of a TAG stop codon and devoid of RF1 expression or both.

19. The kit of claim 17 , wherein said stop codon is a TAG stop codon.

Assignees

• B G Negev Technologies And Applications Ltd At Ben Gurion Univ
• Univ Minnesota

Inventors

• Alfonta Lital
• Noireaux Vincent
• Schlesinger Orr
• Chemla Yonatan
• Ozer Eden

Classifications

• C12N2330/51

  Specially adapted vectors · CPC title

• C12N15/67

  General methods for enhancing the expression · CPC title

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title