SepRS and EF-Tu Mutants for Site-Specific Phosphoserine Incorporation
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Solution Overview
Problem
Current methods for producing phosphorylated proteins, especially those phosphorylated at specific amino acids, face challenges in efficiency and scalability, particularly in bacterial systems where protein phosphorylation mechanisms are absent, limiting the production of serine-phosphorylated proteins to low yields.
Innovation Solution
The use of SepRS and EF-Tu mutants selected by molecular evolution, which incorporate phosphoserine into target proteins with high efficiency, enabling the production of phosphorylated proteins at specific sites, significantly increasing yields from micrograms to milligrams per liter.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional methods are used to produce phosphorylated proteins in bacterial systems, then the production process is simple, but the yield is very low (micrograms per liter)
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in SepRS (e.g., K347E, N352D, E412S, E414I, P495R, I496R, L512I) and EF-Tu (e.g., H67R, E216V, E216L, E216T, E216A, E216R, E216C, E216Y, D217G, F219Y, T229S, T229A, W274A, W274N) to alter their biochemical properties. These mutations enable the bacterial system to efficiently incorporate phosphoserine into target proteins, increasing yield from micrograms to milligrams per liter while maintaining relative system simplicity
Solution Approach 2:
The patent introduces phosphoserine as an intermediary substance that is incorporated into target proteins at specific positions through the mutated SepRS and EF-Tu system. This intermediary approach allows site-specific phosphorylation without requiring complex eukaryotic signaling machinery, thereby increasing productivity while avoiding excessive system complexity
2Productivity
If eukaryotic cells are used for protein phosphorylation, then the phosphorylation process is rapid and reversible, but it is very difficult to produce large amounts of uniformly phosphorylated proteins at specific amino acids
Solution Approach 1:
The patent applies local quality by using mutated SepRS and EF-Tu proteins that specifically recognize and incorporate phosphoserine at predetermined positions in the target protein sequence. The system achieves both high productivity (milligrams per liter) and high manufacturing precision (site-specific phosphorylation) by localizing the phosphorylation activity to specific amino acid positions through codon assignment and selective incorporation mechanisms
3Productivity
If bacterial systems are used for recombinant protein production, then large amounts of protein can be produced, but the natural protein phosphorylation mechanism does not exist
Solution Approach 1:
The patent enables bacterial systems to perform phosphorylation through self-service by introducing mutated SepRS and EF-Tu proteins that autonomously catalyze the incorporation of phosphoserine into target proteins. The bacterial machinery, enhanced by these engineered components, becomes self-sufficient for producing phosphorylated proteins without requiring external eukaryotic phosphorylation systems or complex additional machinery
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for the efficient and site-specific production of phosphorylated proteins, overcoming previous limitations in yield and scalability, facilitating research into cell signaling and potential therapeutic applications.
Implementation Method 1
A phosphate group (PO4) is reversibly incorporated into a protein by the action of kinase and phosphatase
Implementation Method 2
EF-Tu mutants selected by molecular evolution, which incorporate phosphoserine into target proteins with high efficiency
Data Source
AI summary
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.