Site-specific incorporation of phosphoserine into proteins in escherichia coli

Abstract

Nucleic acids encoding mutant elongation factor proteins (EF-Sep), phosphoseryl-tRNA synthetase (SepRS), and phosphoseryl-tRNA (tRNA Sep ) and methods of use in site specific incorporation of phosphoserine into & protein or polypeptide are described. Typically, SepRS preferentially aminoacylates tRNA Sep with O-phosphoserine and the tRNA Sep recognizes at least one codon such as a stop codon. Due to the negative charge of the phosphoserine, Sep-tRNA Sep does not bind elongation factor Tu (EF-Tu). However, mutant EF-Sep proteins are disclosed that bind Sep-tRNA Sep and protect Sep-tRNA Sep 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.

1 . A method of making a target protein, comprising expressing a messenger RNA (mRNA) encoding the target protein in a system comprising:
 an O-phosphoseryl-tRNA synthetase (SepRS) that preferentially aminoacylates a tRNA (tRNA Sep ) with phosphoserine;   a tRNA Sep  that can be aminoacylated with phosphoserine by SepRS to form a Sep-tRNA Sep  and recognize at least one codon in the mRNA encoding the target protein; and   a mutant elongation factor (EF-Sep) that binds Sep-tRNA Sep ;   wherein the SepRS preferentially aminoacylates the tRNA Sep  with phosphoserine and the resulting Sep-tRNA Sep  recognizes at least one codon such that phosphoserine is incorporated during translation to form the target protein.   
     
     
         2 . The method of  claim 1 , wherein the EF-Sep comprises an amino acid sequence at least 90% identical to any one of SEQ ID NOS:1-4, wherein
 (i) the amino acid sequence comprises the amino acids of any one of SEQ ID NOS:1-4 at positions corresponding to amino acid number 67, 216, 217, 219, 229, and 274 of any one of SEQ ID NOS:1-4; or   (ii) the amino acid sequence comprises the amino acids of any one of SEQ ID NOS:1-4 at positions corresponding to amino acid number 67, 217, 219, 229, and 274 of any one of SEQ ID NOS:1-4 and a substitution mutation corresponding to amino acid number 216 of any one of SEQ ID NOS:1-4.   
     
     
         3 . The method of  claim 1 , wherein the EF-Sep comprises an amino acid binding pocket for aminoacylated tRNA, wherein the binding pocket comprises
 (i) the binding pocket for aminoacylated tRNA of any one of SEQ ID NOS: 1-4, or any one of SEQ ID NO:1-4; or   (ii) the binding pocket for aminoacylated tRNA of SEQ ID NO:3 with a substitution at amino acid reside 216, optionally wherein the substitution is an asparagine-to-valine substitution (N216V).   
     
     
         4 . The method of  claim 1 , wherein the tRNA Sep  is cysteinyl-tRNA from  Methanocaldococcus jannaschii.    
     
     
         5 . The method of  claim 4 , wherein the tRNA Sep  is encoded by a the nucleic acid sequence SEQ ID NO:41. 
     
     
         6 . The method of  claim 1 , wherein the SepRS is the phosphoseryl-tRNA synthetase from  Methanococcus maripaludis  or  Methanocaldococcus jannaschii.    
     
     
         7 . The method of  claim 6 , wherein the SepRS is comprises an amino acid sequence at least 85% identical to SEQ ID NO:43 or 46. 
     
     
         8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein the nucleic acid encoding a gene with tRNA Sep  activity and the nucleic acid encoding a gene with SepRS activity are on one or more vectors. 
     
     
         10 . The method of  claim 9 , wherein the vector is an expression vector selected from the group consisting of a plasmid, a virus, a naked polynucleotide, and a conjugated polynucleotide. 
     
     
         11 . The method of  claim 9 , wherein the vector is expressed in cells selected from the group consisting of bacterial cells, archeaebacterial cells, and eukaryotic cells. 
     
     
         12 . The method of  claim 9 , wherein the vector is expressed in an in vitro transcription/translation system. 
     
     
         13 . The method of  claim 12 , wherein the vector is transcribed and translated prior to or along with nucleic acids encoding one or more proteins or polypeptides. 
     
     
         14 . The method of  claim 1 , wherein the nucleic acids are expressed in an organism. 
     
     
         15 . The method of  claim 1 , wherein the nucleic acids are under control of a promoter selected from the group consisting of constitutive, inducible and tissue-specific. 
     
     
         16 . A kit for producing a target protein containing phosphoserine, comprising a polynucleotide encoding EF-Sep, a polynucleotide encoding tRNA Sep , and a polynucleotide encoding SepRS. 
     
     
         17 . The kit of  claim 16 , wherein the kit further comprises phosphoserine. 
     
     
         18 . The kit of  claim 16 , further comprising a host system for expressing a polynucleotide encoding the protein, the polynucleotide encoding EF-Sep, the polynucleotide encoding tRNA Sep , and the polynucleotide encoding SepRS. 
     
     
         19 . A plurality of a target protein produced according to the method of  claim 1 . 
     
     
         20 . The plurality of the target protein of  claim 19  in a lysate of host cells used to produce the target protein. 
     
     
         21 . A method comprising screening candidate drugs for activity against a protein, wherein the protein is produced according to a process comprising the method of  claim 1 .