Compositions and methods for making selenocysteine containing polypeptides
AssigneeYale University
InventorsDieter Soll, Caroline Aldag, Michael Hohn
Life of this patent
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Abstract
Non-naturally occurring tRNASec and methods of using them for recombinant expression of proteins engineered to include one or more selenocysteine residues are disclosed. The non-naturally occurring tRNASec can be used for recombinant manufacture of selenocysteine containing polypeptides encoded by mRNA without the requirement of an SECIS element. In some embodiments, selenocysteine containing polypeptides are manufactured by co-expressing a non-naturally occurring tRNASec a recombinant expression system, such as E. coli, with SerRS, EF-Tu, SelA, or PSTK and SepSecS, and an mRNA with at least one codon that recognizes the anticodon of the non-naturally occurring tRNASec.
Claims20
An isolated nucleic acid comprising a nucleic acid sequence encoding a non-naturally occurring tRNASec, wherein the non-naturally occurring tRNASec is recognized by SerRS, or a variant thereof, and when aminoacylated with serine the Ser-tRNASec is a substrate for SelA or a variant thereof,
- wherein the tRNASec is a variant of SEQ ID NO:1 wherein one or more base pairs selected from the group consisting of C7•G66, G49•U65, and C50•G64 are mutated,
- wherein when aminoacylated with seleocysteine the Sec-tRNASec is recognized by EF-Tu, and
- wherein the anticodon of the non-naturally occurring tRNASec hybridizes to a stop codon.
A tRNASec comprising at least 90% sequence identity to SEQ ID NO:1, wherein one or more nucleotides selected from group consisting of 8, 67, 68, 82, 83, and 84 are substituted relative to SEQ ID NO:1, wherein the anticodon of nucleotides 35-37 of SEQ ID NO:1 are CUA (amber), UCA (opal), or UUA (ochre), and wherein the tRNASec is a substrate for EF-Tu when aminoacylated.
A nucleic acid encoding a tRNASec comprising at least 90% sequence identity to SEQ ID NO:1, wherein one or more nucleotides selected from group consisting of 8, 67, 68, 82, 83, and 84 are substituted relative to SEQ ID NO:1, wherein the anticodon of nucleotides 35-37 of SEQ ID NO:1 are CUA (amber), UCA (opal), or UUA (ochre), and wherein the tRNASec is a substrate for EF-Tu when aminoacylated.
Dependencies are inferred from the claim text.