Non-natural amino acid tRNA synthetases for para-methylazido-L-phenylalanine

Patent metadata

Field	Value
Publication number	US-9938516-B2
Application number	US-201415028396-A
Country	US
Kind code	B2
Filing date	Oct 10, 2014
Priority date	Oct 11, 2013
Publication date	Apr 10, 2018
Grant date	Apr 10, 2018

Abstract

The invention provides methods and compositions for a mutein aminoacyl-tRNA synthetase that preferentially charges a tRNA with a non-natural amino acid. Also provided are methods for incorporating the non-natural amino acid, para-methylazido-L-phenylalanine into a protein and further conjugating a biologically active adduct to the para-methylazido-L-phenylalanine.

First claim

What is claimed is:

1. A composition comprising an aminoacyl-tRNA synthetase (RS) wherein the RS:
i) preferentially aminoacylates to a degree of greater than 90% a tRNA with para-methylazido-L-phenylalanine (pAMF) compared to the 20 common naturally occurring amino acids;
ii) has a sequence identity of over 90% to Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS) having SEQ ID NO:1; and
iii) using SEQ ID NO: 1 as a reference sequence, has:
a) at position L65 amino acid: A or V;
b) at position F108 amino acid: Y or W;
c) at position D158 amino acid: A;
d) at position Y32 amino acid: T or V or A; and
e) at position I159 amino acid: S or G or V.

2. The composition of claim 1 , having an amino acid substitution at position Q109 selected from the group of amino acids consisting of L or M or I.

3. The composition of claim 1 , wherein the RS has a sequence identity of at least 90% of SEQ ID NO:1 and has amino acid substitutions selected from the group consisting of:
i) Y32T, L65A, F108Y, Q109L and I159S;
ii) Y32V, L65A, F108W, Q109M and I159G;
iii) Y32A, L65V, F108W and I159G;
iv) Y32V, L65V, F108Y and I159V;
v) Y32T, L65V, F108W and I159S; and
vi) Y32V, L65V, F108W, G109I and I159S.

4. The composition of claim 1 , wherein the RS using SEQ ID NO:1 as a reference sequence, has
a) at position L65 amino acid: V;
b) at position F108 amino acid: W;
c) at position D158 amino acid: A;
d) at position Y32 amino acid: A; and
e) at position I159 amino acid: G.

5. A polynucleotide encoding an aminoacyl-tRNA synthetase (RS) wherein the RS:
i) preferentially aminoacylates to a degree of greater than 90% a tRNA with para-methylazido-L-phenylalanine (pAMF) compared to the 20 common naturally occurring amino acids;
ii) has a sequence identity of over 90% to Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS) having SEQ ID NO: 1; and
iii) using SEQ ID NO: 1 as a reference sequence, has:
a) at position L65 amino acid: A or V;
b) at position F108 amino acid: Y or W;
c) at position D158 amino acid: A;
d) at position Y32 amino acid: T or V or A; and
e) at position I159 amino acid: S or G or V.

6. The polynucleotide of claim 5 , having an amino acid substitution at position Q109 selected from the group of amino acids consisting of L or M or I.

7. The polynucleotide of claim 5 , wherein the RS has a sequence identity of at least 90% of SEQ ID NO: 1 and has amino acid substitutions selected from the group consisting of:
i) Y32T, L65A, F108Y, Q109L and I159S;
ii) Y32V, L65A, F108W, Q109M and I159G;
iii) Y32A, L65V, F108W and I159G;
iv) Y32V, L65V, F108Y and I159V;
v) Y32T, L65V, F108W and I159S; and
vi) Y32V, L65V, F108W, G109I and I159S.

8. The polynucleotide of claim 5 , wherein the RS using SEQ ID NO:1 as a reference sequence, has
a) at position L65 amino acid: V;
b) at position F108 amino acid: W;
c) at position D158 amino acid: A;
d) at position Y32 amino acid: A; and
e) at position I159 amino acid: G.

9. A cell free protein synthesis system for selectively incorporating para-methylazido-L-phenylalanine (pAMF) into a protein of interest, the system comprising:
a) a cell free extract of bacteria having biologically functioning tRNA, amino acids and ribosomes necessary for cell free protein synthesis;
b) a polynucleotide having a coding region encoding the protein of interest and including a suppression codon selectively positioned within its coding region;
c) methylazido-L-phenylalanine in a concentration sufficient to permit selective incorporation of pAMF into the protein of interest;
d) a tRNA able to be charged with pAMF and complementary to the suppression codon of the protein of interest; and
e) an aminoacyl-tRNA synthetase (RS) wherein the RS:
i) preferentially aminoacylates to a degree of greater than 90% a tRNA with pAMF compared to the 20 common naturally occurring amino acids;
ii) has a sequence identity of over 90% to Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS) having SEQ ID NO:1; and
iii) using SEQ ID NO:1 as a reference sequence, has:
a) at position L65 amino acid: A or V;
b) at position F108 amino acid: Y or W;
c) at position D158 amino acid: A;
d) at position Y32 amino acid: T or V or A; and
e) at position I159 amino acid: S or G or V.

10. The cell free protein synthesis system of claim 9 , wherein the RS has an amino acid substitution at position Q109 selected from the group of amino acids consisting of L or M or I.

11. The cell free protein synthesis system of claim 9 , wherein the RS has a sequence identity of at least 90% of SEQ ID NO: 1 and has amino acid substitutions selected from the group consisting of:
i) Y32T, L65A, F108Y, Q109L and I159S;
ii) Y32V, L65A, F108W, Q109M and I159G;
iii) Y32A, L65V, F108W and I159G;
iv) Y32V, L65V, F108Y and I159V;
v) Y32T, L65V, F108W and I159S; and
vi) Y32V, L65V, F108W, G109I and I159S.

12. The cell free protein synthesis system of claim 9 , wherein the RS using SEQ ID NO:1 as a reference sequence, has
a) at position L65 amino acid: V;
b) at position F108 amino acid: W;
c) at position D158 amino acid: A;
d) at position Y32 amino acid: A; and
e) at position I159 amino acid: G.

13. The cell free protein synthesis system of claim 9 , wherein the cell free extract has an active oxidative phosphorylation system.

14. The cell free protein synthesis system of claim 9 , wherein the protein of interest is an antibody or antibody fragment.

15. A method for selectively incorporating para-methylazido-L-phenylalanine (pAMF) into a protein of interest, the method comprising the steps of:
a) combining a cell free extract of bacteria having containing biologically functioning tRNA, amino acids and ribosomes necessary for cell free protein synthesis with the following reagents:
i) a polynucleotide having a coding region encoding the protein of interest and including a suppression codon selectively positioned within its coding region;
ii) methylazido-L-phenylalanine in a concentration sufficient to permit selective incorporation of pAMF into the protein of interest;
iii) a tRNA able to be charged with pAMF and complementary to the suppression codon of the protein of interest; and
iv) an aminoacyl-tRNA synthetase (RS) wherein the RS:
preferentially aminoacylates to a degree of greater than 90%, a tRNA with pAMF compared to the 20 common naturally occurring amino acids;
has a sequence identity of over 90% to Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS) having SEQ ID NO: 1; and
using SEQ ID NO: 1 as a reference sequence, has:
a) at position L65 amino acid: A or V;
b) at position F108 amino acid: Y or W;
c) at position D158 amino acid: A;
d) at position Y32 amino acid: T or V or A; and
e) at position I159 amino acid: S or G or V; and

b) incubating the combination of step (a) under conditions permitting selective incorporation of pAMF into the protein of interest.

16. The method of claim 15 , wherein the RS further has an amino acid substitution at position Q109 selected from the group of amino acids consisting of L or M or I.

17. The method of claim 15 , wherein the RS has sequence identity of at least 90% of SEQ ID NO:1 and further has an amino acid substitution selected from the group of substitutions consisting of:
i) Y32T, L65A, F108Y, Q109L, and I159S;
ii) Y32V, L65A, F108W, Q109M, and I159G;
iii) Y32A, L65V, F108W, and I159G;
iv) Y32V, L65V, F108Y, and I159V;
v) Y32T, L65V, F108W, and I159S; and
vi) Y32V, L65V, F108W, G109I, and I159S.

18. The method o

Assignees

• Sutro Biopharma Inc

Inventors

• Zimmerman Erik S
• Thanos Christopher

Classifications

• C12Y601/01

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

• A61K31/40

  having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil · CPC title

• C12Y601/01001

  Tyrosine-tRNA ligase (6.1.1.1) · CPC title

• C12P21/02

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

• C12N9/93 Primary

  Ligases (6) · CPC title