Non-natural amino acid tRNA synthetases for para-methylazido-L-phenylalanine
AssigneeSutro Biopharma, Inc.
InventorsErik S. Zimmerman, Christopher Thanos
Life of this patent
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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.
Claims22
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.
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.
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.
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.
Dependencies are inferred from the claim text.