| 摘要 |
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. |
| 主权项 |
1. 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. |
