Cell-Free Protein Synthesis With Cold Shock Proteins at Low Temperature
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Solution Overview
Problem
Existing cell-free protein synthesis systems face challenges in producing active proteins efficiently at low temperatures, often resulting in protein precipitation and reduced yields.
Innovation Solution
Incorporating cold shock proteins (Csp) such as CspA, CspB, CspC, CspE, CspG, or CspI into the reaction solution of a cell-free protein synthesis system, along with a nucleic acid encoding their amino acid sequence, allows for high-efficiency protein synthesis at temperatures below 30°C.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If the reaction temperature is lowered to suppress protein precipitation, then protein solubility is improved, but protein synthesis yield significantly decreases
Solution Approach 1:
The invention changes the chemical parameter of adding cold shock proteins to the reaction system. This allows the reaction to proceed efficiently at lower temperatures (4-30°C) by stabilizing RNA and protecting it from degradation, thereby maintaining both protein solubility and synthesis yield that would otherwise be contradictory
Solution Approach 2:
Cold shock proteins act as intermediary substances that mediate between the low temperature environment and the protein synthesis process. These proteins protect RNA from cold-induced degradation and maintain the functionality of the transcription-translation system, enabling efficient protein production at temperatures that would otherwise suppress synthesis activity
2Productivity
If the reaction temperature is set at 30 to 37°C to increase protein synthesis yield, then productivity is improved, but protein precipitation occurs and active protein production decreases
Solution Approach 1:
The invention changes the temperature parameter from the conventional 30-37°C range to a lower range of 4-30°C, and simultaneously changes the chemical parameter by adding cold shock proteins. This combination allows maintaining high synthesis yields while preventing precipitation, effectively resolving the contradiction between productivity and protein stability
3Productivity
If cold shock protein is added to enable high-efficiency synthesis at low temperature, then protein synthesis activity is improved, but reaction system complexity increases
Solution Approach 1:
The cold shock proteins perform multiple functions within the reaction system: they protect RNA from degradation, maintain transcription-translation efficiency at low temperatures, and prevent protein aggregation. This self-service capability of the added protein reduces the need for other complex control mechanisms, thereby limiting the increase in overall system complexity
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 enables the production of active proteins with high efficiency and solubility even at low temperatures, significantly enhancing yield and reducing precipitation.
Implementation Method 1
it has been found that the use of a cold shock protein enables the protein synthesis to be implemented by a cell-free protein synthesis system with high protein synthesis activity even at a low temperature of lower than 30° C.
Implementation Method 2
The cell-free protein synthesis system is a technique to synthesize a protein in vitro by adding amino acids as substrates, an energy source such as adenosine triphosphate (ATP), an energy regeneration system, and a salt such as magnesium ions, as well as a gene encoding a target protein, to a cell extract containing factors necessary for protein synthesis.
Data Source
AI summary
The object of the present invention is to provide a protein production method capable of producing an active protein with high efficiency even at a low temperature, and a cell-free protein synthesis kit. A protein production method including producing a protein with a reaction solution of a cell-free protein synthesis system containing either one or both of a cold shock protein and a nucleic acid containing a coding region encoding an amino acid sequence of the cold shock protein. A cell-free protein synthesis kit including one or both of a cold shock protein and a nucleic acid containing a coding region encoding an amino acid sequence of the cold shock protein, and a reaction solution of a cell-free protein synthesis system.