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.
Claims19
A non-naturally occurring tRNASec comprising a nucleic acid sequence according to SEQ ID NO:
- 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 55, 56, 57, or 58.
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 and by EF-Tu, or variants thereof, and when aminoacylated with serine the Ser-tRNA (1) is a substrate for SelA or a variant thereof;
- or (2) is a substrate for PSTK and when aminoacylated with phosphorylated serine the Sep-tRNA serves as a substrate for SepSecS or a variant thereof,
- wherein the non-naturally occurring tRNA comprises a nucleic acid sequence according to SEQ ID NO:
- 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 55, 56, 57, or 58.
Dependencies are inferred from the claim text.