Compositions and Methods for Making Selenocysteine Containing Polypeptides
AssigneeYale University
InventorsDieter Soll, Caroline Aldag, Michael Hohn
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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.
Claims26
A non-naturally occurring tRNASec comprising a nucleic acid sequence at least 99% identical to SEQ ID NO:6, 7, 8, 9, 10, 11, 12, 13, 14, 15,1 6,17, 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, or a complement thereof.
An isolated nucleic acid comprising a nucleic acid sequence encoding a non-naturally occurring tRNASec, wherein the non-naturally occurring tRNASec can be recognized by SerRS and by EF-Tu, or variants thereof, and when aminoacylated with serine the Ser-tRNA can (1) be a substrate for Se1A or a variant thereof;
- or (2) be a substrate for PSTK and when aminoacylated with phosphorylated serine the Sep-tRNA can serve as a substrate for SepSecS or a variant thereof,
- wherein the anticodon of the non-naturally occurring tRNASec hybridizes to a stop codon.
A method of making a recombinant selenocysteine containing protein comprising co-expressing a non-naturally occurring tRNASec comprising a nucleic acid sequence 99% identical to a sequence selected from the group consisting of SEQ ID NO:6-56 in a host cell also expressing SerRS, EF-Tu, SelA, or PSTK and SepSecS, with a polynucleotide comprising a codon that hybridizes with the anticodon of the non-naturally occurring tRNASec.
Dependencies are inferred from the claim text.