METHOD FOR PRODUCING PHOSPHOSERINE INCORPORATED PROTEINS BY USING SepRS MUTANTS AND EF-Tu MUTANTS

摘要

The present invention relates to a method of producing a phosphorylated protein using a SepRS (O-phosphoseryl-tRNA synthetase) mutant and an EF-Tu mutant, which have increased activity. More specifically, the invention relates to a method of producing a phosphorylated protein by incorporating phosphoserine into the specific position of a target protein or polypeptide using tRNASep serving to recognize at least one codon in the mRNA of the target protein or polypeptide, an O-phosphoseryl-tRNA synthetase (SepRS) mutant selected by a molecular evolution technique and serving to aminoacylate tRNASep with phosphoserine (Sep), and an EF-Tu mutant serving to bind and deliver Sep-tRNASep to the ribosome.;According to the invention, a phosphorylated protein can be produced in an amount of mg per liter using the SepRS and EF-Tu mutants. Thus, the invention is useful for the production of various phosphorylated proteins, including phosphorylated enzymes, the production of antibodies, the fabrication of protein chips, and cell-based screening for new drug development.

主权项

1. An EF-Tu mutant having at least one mutation selected from the group consisting of: H67R, E216V, E216L, E216T, E216A, E216R, E216C, E216Y, D217G, F219Y, T229S, T229A, W274A, and W274N in an EF-Tu having an amino acid sequence of SEQ ID NO:19.