Compositions and methods for making selenocysteine containing polypeptides
AssigneeYale University
InventorsDieter Soll, Takahito Mukai, Kyle Hoffman
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
A nucleic acid comprising a nucleic acid sequence encoding a non-naturally occurring tRNASec, wherein the tRNASec comprises the consensus sequence of
- i.
- (SEQ ID NO:
- 19)
- GGRGRRNRNNNNNNNNNGGYNNNNNNNNNNGNYU AANCNNNNNNNN
- NNNNNNNNNNNNNNNRNRGUYCRANYCYNYYNYYCYCCNCCA
- optionally, wherein an acceptor stem can be formed by base pairing between nucleotides 1-8 with nucleotides 87-80 respectively;
- a D-arm can be formed by base pairing between nucleotides 11-16 with nucleotides 26-21 respectively;
- an anti-codon arm can be formed by base pairing between nucleotides 27-32 with nucleotides 45-40 respectively;
- a V-arm can be formed by base pairing between nucleotides 47-53 with nucleotides 64-58 respectively;
- a T-arm can be formed by base pairing between nucleotides 65-68 with nucleotides 79-76 respectively;
- or a combination thereof;
- ii.
- (SEQ ID NO:
- 20)
- GGRRNNNNNNNNNNNNNYGGNNNNNNNNNNNRNYU AANYNNNNNNN
- NNNNNNNNNNNNNNNNNNNNRGGUUCRAYUCCYNNNNNYYCCRCCA
- optionally, an acceptor stem can be formed by base pairing between nucleotides 1-9 with nucleotides 91-83 respectively;
- a D-arm can be formed by base pairing between nucleotides 12-17 with nucleotides 27-22 respectively;
- an anti-codon arm can be formed by base pairing between nucleotides 28-33 with nucleotides 46-41 respectively;
- a V-arm can be formed by base pairing between nucleotides 48-56 with nucleotides 69-61 respectively;
- a T-arm can be formed by base pairing between nucleotides 70-72 with nucleotides 82-80 respectively;
- or a combination thereof;
- or
- iii.
- (SEQ ID NO:
- 21)
- GNNNNRYNANNNNNNNNGGYNNNNNNNNNNGNYY AANCNNNNNNNN
- NNNNNNNNNNNNNNNNNNGNUCRANNCNNNNNYNNNNCGCCA
- optionally, an acceptor stem can be formed by base pairing between nucleotides 1-8 with nucleotides 87-80 respectively;
- a D-arm can be formed by base pairing between nucleotides 11-16 with nucleotides 26-21 respectively;
- an anti-codon arm can be formed by base pairing between nucleotides 27-32 with nucleotides 45-40 respectively;
- a V-arm can be formed by base pairing between nucleotides 47-53 with nucleotides 64-58 respectively;
- a T-arm can be formed by base pairing between nucleotides 65-68 with nucleotides 79-76 respectively;
- or a combination thereof.
A non-naturally occurring tRNASec including sequence elements from a yeast tRNASer in combination with sequence elements from a non-yeast tRNA wherein the non-naturally occurring tRNASec is a substrate for a SerRS and a SelA and binds to eEF1α.
A variant of SelA comprising at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, or 99% sequence identity to any one of SEQ ID NO:79, 80, 82, or 84, wherein the variant does not have 100% sequence identity to SEQ ID NO:79, 80, 82, or 84.
Dependencies are inferred from the claim text.