Universal platform for genetic code expansion
AssigneeThe Trustees of Boston College
InventorsAbhishek Chatterjee, James Italia
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Abstract
Methods and compositions are described for selecting and identifying orthogonal aminoacyl synthetase-tRNA pairs and their use to incorporate unnatural amino acids in a site-specific manner in proteins. Specifically described is a novel E. coli tryptophanyl synthetase-tRNA pair that functions as both an opal and amber suppressor and that incorporates tryptophan analogs into proteins.
Claims3
A genetically engineered E. coli bacterial strain, wherein the endogenous E. coli tryptophanyl-tRNA synthetase (trpS) and the tryptophanyl-tRNA (trpT) genes are deleted and functionally replaced with an orthogonal eukaryotic tryptophanyl-tRNA synthetase-tRNA gene pair, wherein the E. coli strain is selected from the group consisting of:
- ATMW1 having the following genotype:
- EcNR1 pUltraG-ScW40CCA trpS::ZeoR trpT::GentR ΔgalK λRED::galK and BL21(De3) having the following genotype:
- BL21(DE3) pUltraG-ScW40CCA trpS::ZeoR trpT::GentR.
A cell comprising a variant E. coli tryptophanyl-tRNA synthetase (EcTrp-RS), wherein the variant EcTrp-RS preferentially aminoacylates an E. coli tryptophanyl tRNA (Ec-tRNATrp) with a tryptophan analog over the naturally-occurring tryptophan amino acid, wherein the variant EcTrp-RS comprises:
- the amino acid sequence of SEQ ID NO:
- 91, or an amino acid sequence with at least 90% sequence identity with the full-length SEQ ID NO:91, wherein the variant E. coli EcTrp-RS is mutated, relative to SEQ ID NO:91, such that the serine at position 8 is replaced with alanine;
- the valine at position 144 is replaced with either serine, glycine or alanine;
- and the valine at position 146 is replaced with either alanine, isoleucine or cysteine;
- and wherein the cell is E. coli and the E. coli strain is ATMW1 or BL21 (DE3) pUltraG-ScW40CCA trpS::ZeoR trpT::GentR.
Dependencies are inferred from the claim text.