Recombinant Cas3 Protein Production at Low Expression Temperature
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Solution Overview
Problem
Conventional methods struggle to produce Cas3 proteins in high purity and high yield with maintained activity, particularly for genome editing in various organisms, due to issues like low thermal stability and denaturation during expression.
Innovation Solution
Culturing insect cells at 20 to 28°C and using a phosphate buffer for purification to express and collect recombinant Cas3 proteins, followed by affinity purification and gel filtration chromatography to maintain activity and purity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Cas3 protein is produced using conventional expression methods, then the production process is simple, but the protein activity is lost due to denaturation and low thermal stability
Solution Approach 1:
The patent changes the expression temperature parameter to 20-28°C (lower than conventional temperatures) to maintain Cas3 protein thermal stability and prevent denaturation. This parameter modification directly addresses the low thermal stability issue while preserving protein activity throughout the production and purification process.
Solution Approach 2:
The patent introduces a phosphate buffer system as an intermediary medium during purification to maintain protein stability. The buffer acts as a protective environment that prevents denaturation during handling and purification steps, enabling the protein to maintain activity despite the complexity introduced by additional purification steps.
2Manufacturing precision
If Cas3 protein is purified to high purity, then the protein quality is improved, but the yield is reduced due to activity loss during purification
Solution Approach 1:
The patent modifies the purification buffer composition to include phosphate buffer at specific pH and ionic strength conditions optimized for Cas3 protein stability. This parameter optimization allows the protein to withstand purification steps without denaturation, achieving high purity while maintaining high yield by preventing activity loss during the purification process.
3Productivity
If Cas3 protein is expressed at higher temperature, then the production speed is increased, but the protein denatures and loses activity
Solution Approach 1:
The patent optimizes the expression temperature parameter to the specific range of 20-28°C, balancing production efficiency with protein stability. This temperature optimization allows sufficient production speed while preventing thermal denaturation, resolving the contradiction between productivity and protein activity maintenance.
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 recombinant Cas3 proteins in high purity and high yield, suitable for genome editing in various cells, including animal cells, with maintained activity even at 37°C for a short period.
Implementation Method 1
the purification of the Cas3 protein includes using a phosphate buffer
Implementation Method 2
The collection of the expressed Cas3 protein includes affinity purification and gel filtration chromatography
Data Source
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AI summary
The present inventors have found that by cultivating insect cells, into which the Cas3 gene has been introduced, at relatively low temperatures, it is possible to efficiently express recombinant Cas3 proteins with maintained activity, and by purifying the soluble fractions of these cells, it is possible to collect active forms of the recombinant Cas3 proteins in high purity and high yield.