Yale University

Dieter Soll

Hee-Sung Park

Caroline Aldag

Michael Hohn

Jesse Rinehart

Takahito Mukai

Corwin Miller

Kyle Hoffman

Non-naturally occurring tRNAand methods of using them for recombinant expression of proteins engineered to include one or more selenocysteine residues are disclosed. The non-naturally occurring tRNAcan 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 tRNAa recombinant expression system, such as, 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 tRNA.

Compositions and methods for making selenocysteine containing polypeptides

Nucleic acids encoding mutant elongation factor proteins (EF-Sep), phosphoseryl-tRNA synthetase (SepRS), and phosphoseryl-tRNA (tRNA) and methods of use in site specific incorporation of phosphoserine into a protein or polypeptide are described. Mutant EF-Sep proteins are disclosed that bind Sep-tRNAand protect Sep-tRNAfrom deacylation. In a preferred embodiment the nucleic acids are on vectors and are expressed in cells such as bacterial cells, archeaebacterial cells, and eukaryotic cells. Proteins or polypeptides containing phosphoserine produced by the methods described herein can be used for a variety of applications such as research, antibody production, protein array manufacture and development of cell-based screens for new drug discovery.

Site-specific incorporation of phosphoserine into proteins in

Nucleic acids encoding mutant elongation factor proteins (EF-Sep), phosphoseryl-tRNA synthetase (SepRS), and phosphoseryl-tRNA (tRNA) and methods of use in site specific incorporation of phosphoserine into a protein or polypeptide are described. Typically, SepRS preferentially aminoacylates tRNAwith O-phosphoserine and the tRNArecognizes at least one codon such as a stop codon. Due to the negative charge of the phosphoserine, Sept-tRNAdoes not bind elongation factor Tu (EF-Tu). However, mutant EF-Sep proteins are disclosed that bind Sep-tRNAand protect Sep-tRNAfrom deacylation. In a preferred embodiment the nucleic acids are on vectors and are expressed in cells such as bacterial cells, archeaebacterial cells, and eukaryotic cells. Proteins or polypeptides containing phosphoserine produced by the methods described herein can be used for a variety of applications such as research, antibody production, protein array manufacture and development of cell-based screens for new drug discovery.

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Nucleic acids encoding genes with SepRS and tRNASep activity for site specific incorporation of phosphoserine into a protein or polypeptide and methods of use thereof are described. Typically, SepRS preferentially aminoacylates tRNASep with O-phosphoserine and the tRNASep recognizes at least one codon such as a stop codon. In a preferred embodiment the nucleic acids are on vectors. In one embodiment, the vectors are expressed in cells such as bacterial cells, archeaebacterial cells, and eukaryotic cells. In an alternative embodiment, the vectors are expressed in an in vitro transcription/translation system. Proteins or polypeptides containing phosphoserine produced by the methods described herein can be used for a variety of applications such as research, antibody production, protein array manufacture and development of cell-based screens for new drug discovery.

Growing community of inventors

Guilford, CT, United States of America

Dieter Soll