Site-specific incorporation of phosphoserine into proteins in Escherichia coli

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:

• an O-phosphoseryl-tRNA synthetase (SepRS) that aminoacylates a tRNA (tRNASep) with phosphoserine;
• a tRNASep that can be aminoacylated with phosphoserine by SepRS to form a Sep-tRNASep and recognize at least one codon in the mRNA encoding the target protein;
• and
• an elongation factor (EF-Sep) comprising:
• (i) 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, 217, 219, 229, and 274 of SEQ ID NO:
• 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, and a substitution mutation corresponding to amino acid number 216 of SEQ ID NO:
• 1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4;
• or
• (ii) an amino acid binding pocket for aminoacylated tRNA,
• wherein the binding pocket comprises the binding pocket for aminoacylated tRNA of SEQ ID NO:3 with an asparagine-to-valine substitution at amino acid residue 216 (N216V) of SEQ ID NO:3;
• wherein the EF-Sep that binds the Sep-tRNASep and catalyzes the covalent transfer of the phosphoserine amino acid of the Sep-tRNASep onto a polypeptide;
• wherein the SepRS aminoacylates tRNASep with phosphoserine and the tRNASep recognizes at least one codon such that phosphoserine is incorporated into a protein or polypeptide, and
• wherein the EF-Sep binds and protects Sep-tRNASep from deacylation.