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US 9,090,928 B2Granted
Site-specific incorporation of phosphoserine into proteins in Escherichia coli
AssigneeYale University
InventorsHee-Sung Park, Dieter Soll
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AssigneeYale University
InventorsHee-Sung Park, Dieter Soll
Estimating the term…
Nucleic acids encoding mutant elongation factor proteins (EF-Sep), phosphoseryl-tRNA synthetase (SepRS), and phosphoseryl-tRNA (tRNASep) and methods of use in site specific incorporation of phosphoserine into a protein or polypeptide are described. Typically, SepRS preferentially aminoacylates tRNASep with O-phosphoserine and the tRNASep recognizes at least one codon such as a stop codon. Due to the negative charge of the phosphoserine, Sept-tRNASep does not bind elongation factor Tu (EF-Tu). However, mutant EF-Sep proteins are disclosed that bind Sep-tRNASep and protect Sep-tRNASep from 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.
A method for site specific incorporation of phosphoserine into a target protein, comprising expressing a messenger RNA (mRNA) encoding the target protein in a system comprising:
A kit for producing a target protein containing phosphoserine, comprising a polynucleotide encoding an elongation factor (EF-Sep) comprising an amino acid sequence at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, wherein the amino acid sequence comprises the amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 at positions corresponding to amino acid numbers 67, 216, 217, 219, 229, and 274 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, and wherein the EF-Sep can bind to a Sep-tRNASep and can catalyze the covalent transfer of the phosphoserine amino acid of the Sep-tRNASep onto a polypeptide, a polynucleotide encoding tRNASep, and a polynucleotide encoding SepRS.
An elongation factor (EF-Sep) comprising an amino acid sequence at least 90% identical to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, wherein the amino acid sequence comprises the amino acids of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 at positions corresponding to amino acid number 67, 216, 217, 219, 229, and 274 of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, and
An EF-Sep comprising the amino acid sequence of the amino acid binding pocket for aminoacylated tRNA of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4,
Dependencies are inferred from the claim text.