Yeast Cell Extract Nuclease Knockout for In Vitro Protein Synthesis
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Solution Overview
Problem
Current in vitro protein synthesis systems face challenges in maintaining the stability of nucleic acids due to the presence of nucleases, which affects the yield and efficiency of protein expression.
Innovation Solution
A method is developed using a yeast cell-free protein synthesis system that includes yeast cell extract with reduced EXN53 protein content, polyethylene glycol, optional sucrose, and an aqueous solvent, to stabilize nucleic acids and enhance protein synthesis stability and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If nuclease system is present in the in vitro protein synthesis system, then the system can degrade nucleic acids as part of natural cellular function, but the stability of nucleic acid substrates (mRNA and DNA) deteriorates, affecting protein yield
Solution Approach 1:
The patent extracts and removes the harmful nuclease components from the cell lysate through specific purification steps. The cell lysate is processed to selectively eliminate nucleases while retaining the essential protein synthesis machinery, thereby resolving the contradiction between maintaining natural cellular functions and protecting nucleic acid substrates from degradation
Solution Approach 2:
The patent utilizes the nuclease activity itself as a marker to guide the removal process. By exploiting the fact that nucleases degrade nucleic acids, the invention measures nucleic acid degradation as an indicator of nuclease activity and uses this information to optimize the purification conditions, ultimately converting the harmful effect into a useful monitoring tool
2Productivity
If standard cell lysate is used in the in vitro protein synthesis system, then the system maintains natural cellular composition, but protein production efficiency is reduced due to nuclease-mediated nucleic acid degradation
Solution Approach 1:
The patent extracts and removes the harmful nuclease components from the cell lysate through specific purification steps. The cell lysate is processed to selectively eliminate nucleases while retaining the essential protein synthesis machinery, thereby resolving the contradiction between maintaining natural cellular functions and protecting nucleic acid substrates from degradation
Solution Approach 2:
The patent changes the biochemical parameters of the cell lysate by adjusting pH, temperature, and ionic strength during purification to selectively precipitate or inactivate nucleases while preserving the activity of protein synthesis enzymes. This parameter optimization enables differential stabilization of functional components versus harmful nucleases
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
The method significantly improves the stability of nucleic acids and enhances the efficiency of protein production in the in vitro protein synthesis system, as evidenced by a doubling of luciferase activity in the modified yeast strain compared to the wild-type strain.
Implementation Method 1
Nuclease is a kind of protein that hydrolyzes the phosphodiester bond between nucleotides in the first step of nucleic acid degradation. Some nucleases which only act upon RNA are known as ribonuclease (RNase), and some nucleases which only act upon DNA are known as deoxyribonucleases (DNase).
Implementation Method 2
an in vitro transcription-translation system (abbreviated as IVTT system), which transcribes DNA template into mRNA intermediate through RNA polymerase
Implementation Method 3
completes one-step efficient translation of exogenous proteins by using components including amino acids, ATP, etc.
Data Source
AI summary
What provided is a method for regulating in vitro biosynthesis activity by knocking-out of a nuclease system, comprising: screening five nucleases among numerous nucleases, and performing down-regulation or knocking-out on one of the five nucleases (e.g., EXN53). The method can improve the stability of nucleic acid and the protein production efficiency of an in vitro protein synthesis system.