Chimeric thermostable aminoacyl-tRNA synthetase for enhanced unnatural amino acid incorporation
AssigneeThe Trustees of Boston College
InventorsAbhishek Chatterjee, Katherine Grasso
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Abstract
The present invention describes methods to create chimeric aminoacyl-tRNA synthetases (aaRS) derived from bacteria which show optimal activity and high thermostability. These chimeric aaRSs can be more aggressively engineered to generate a wider assortment of Uaa-selective mutants that are stable at the physiological temperature. The invention further describes the composition of chimeric TyrRSs, generated from E. coli and G. stearothermophilus TyrRSs, which demonstrate enhanced stability relative to EcTyrRS and higher activity relative to both TyrRS enzymes.
Claims8
A composition comprising a polynucleotide encoding a biologically-active chimeric thermostable tyrosyl aminoacyl-tRNA synthetase, comprising an Escherichia coli (E. coli) tyrosyl aminoacyl-tRNA synthetase and a thermostable Geobacillus stearothermophilis (G. stearothermophilis) tyrosyl aminoacyl-tRNA synthetase constructed from a nucleic acid sequence of a mesophilic bacterial E. coli tyrosyl aminoacyl-tRNA synthetase and a nucleic acid sequence of a thermostable G. stearothermophilis tyrosyl aminoacyl-tRNA synthetase of a thermostable bacterial homolog, wherein the thermostable bacteria is selected from the group consisting of:
- Geobacillus stearothermophilis, Bacillus stearothermophilis, Thermus thermophilis or a Thermoanaerobacter species;
- wherein the mesophilic bacteria is selected from the group consisting of Escherichia coli, a Staphylococcus species, a Streptococcus species, or a Pseudomonas species;
- wherein the chimeric thermostable E. coli-G. stearothermophilis tyrosyl aminoacyl-tRNA synthetase aminoacylates an E. coli tRNAtyr with a tyrosine amino acid or a tyrosine amino acid analog in bacterial or mammalian cells;
- wherein the chimeric thermostable tyrosyl aminoacyl-tRNA synthetase has increased thermostability compared to the mesophilic E. coli wild-type tyrosyl aminoacyl-tRNA synthetase in bacterial or mammalian cells;
- and wherein the amino acid sequence of the chimera comprises an amino acid sequence selected from the group consisting of:
- SEQ ID NOs:
- 42, 43, 44, 46 or 47, or an amino acid sequence with at least 90% sequence identity to SEQ ID NOs:
- 42, 43, 44, 46 or 47.
Dependencies are inferred from the claim text.