Intracellular Protein Extraction Using Water Miscible Alcohols
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Solution Overview
Problem
Current methods for extracting intracellular proteins from fermentation broths are inefficient and require cell wall disruption, followed by complex processing steps to isolate active proteins from a mixture of impurities, making them costly and difficult.
Innovation Solution
The use of water miscible or partially water miscible alcohols and glycol ethers to extract intracellular proteins from whole fermentation broth without disrupting cell walls, allowing for the formation of distinct phases that facilitate protein separation and recovery, maintaining enzyme activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If cell wall disruption techniques are used to extract intracellular proteins, then protein extraction efficiency is improved, but device complexity and processing difficulty increase due to the need for multiple separation and purification steps
Solution Approach 1:
The patent extracts only the necessary cellular components (intracellular proteins) directly into the aqueous phase using organic solvents, while leaving the cell walls intact. This selective extraction eliminates the need for mechanical cell disruption and subsequent complex separation steps, directly resolving the contradiction between extraction efficiency and processing complexity
Solution Approach 2:
The patent introduces organic solvents as intermediary substances that facilitate the transfer of intracellular proteins from the cellular compartment to the aqueous phase without requiring cell wall disruption. This intermediary mechanism simplifies the overall extraction process while maintaining high protein recovery efficiency
2Productivity
If cell wall disruption is performed to release intracellular proteins, then protein recovery is improved, but manufacturing precision deteriorates due to contamination with cellular impurities
Solution Approach 1:
The method selectively extracts intracellular proteins into the aqueous phase while leaving cellular impurities (DNA, host cell proteins, inclusion bodies) behind in the organic phase or cell debris. This selective partitioning achieves both high protein recovery and high purity simultaneously, resolving the contradiction between productivity and manufacturing precision
Solution Approach 2:
The patent segments the cellular components into two distinct phases: the aqueous phase containing the desired intracellular proteins and the organic phase containing cellular impurities. This phase separation enables independent optimization of protein recovery in one phase and impurity removal in the other, achieving both high recovery and high purity
3Productivity
If conventional extraction methods are used, then protein isolation is achieved, but loss of time increases due to multiple processing steps including filtration, centrifugation, and chromatography
Solution Approach 1:
The patent combines cell disruption, protein extraction, and initial purification into a single liquid-liquid extraction step. The organic solvent simultaneously disrupts cells, extracts proteins into the aqueous phase, and leaves impurities behind, eliminating the need for sequential filtration, centrifugation, and chromatography steps, thus dramatically reducing processing time while maintaining isolation efficiency
Solution Approach 2:
The organic solvent performs preliminary cell disruption and protein extraction in one step before any separation operations are needed. By pre-extracting proteins into the aqueous phase while leaving most impurities in the organic phase, the method eliminates subsequent time-consuming purification steps
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 method enables efficient extraction of proteins as a single liquid phase or two-phase system, reducing the need for cell disruption and subsequent processing steps, thereby improving the yield and maintaining enzyme activity, and is particularly effective for proteins expressed as inclusion bodies.
Implementation Method 1
The use of water miscible or partially water miscible alcohols and glycol ethers to extract intracellular proteins from whole fermentation broth without disrupting cell walls, allowing for the formation of distinct phases that facilitate protein separation and recovery
Data Source
AI summary
A method for extracting an intracellular peptide, protein or other polypeptide from a whole fermentation broth using a water miscible alcohol, or a water miscible or partially water miscible glycol ether.