Recombinant Particle Protein Purification for Industrial Production
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Solution Overview
Problem
Existing methods for producing recombinant particle proteins face challenges such as low preparation purity, high industrial production cost, and difficulty in formation, necessitating a high-efficiency, stable, simple, and low-cost solution.
Innovation Solution
A method involving transfection of host cells with a plasmid vector, high-pressure homogenization, pH adjustment and heating of supernatants, addition of urea and sodium chloride, followed by anion exchange and hydrophobic chromatography to prepare recombinant particle proteins with a specific amino acid sequence (SEQ ID NO: 1).
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional preparation methods (salt precipitation, density gradient centrifugation, filtration, chromatography, dialysis) are used, then recombinant particle proteins can be prepared, but preparation purity is low and production cost is high
Solution Approach 1:
The patent applies preliminary action by performing high-pressure homogenization before chromatography to disrupt cell walls and release proteins, and by pre-treating with urea and sodium chloride to remove impurities. These preliminary steps prepare the sample for subsequent chromatography, improving overall purity while reducing the number of required chromatography steps and lowering production costs.
Solution Approach 2:
The patent utilizes parameter changes by adjusting pH to specific ranges (7.0-9.0 for first heating, 7.0-8.0 for second heating) and controlling temperature (80-100°C for first heating, 50-70°C for second heating) to optimize protein solubility and impurity removal. These parameter optimizations enable high purity preparation with reduced chromatography steps, addressing both purity and cost concerns.
2Manufacturing precision
If multiple chromatography steps are used to improve purity, then preparation purity increases, but production efficiency decreases and cost increases
Solution Approach 1:
The patent performs high-pressure homogenization and chemical treatment (urea and sodium chloride addition) as preliminary actions before chromatography. This pre-treatment removes many impurities in advance, allowing the subsequent chromatography process to achieve high purity with fewer steps, thereby maintaining production efficiency while improving purity.
Solution Approach 2:
By optimizing pH and temperature parameters during heating steps, the patent enhances the effectiveness of impurity removal. The first heating at pH 7.0-9.0 and 80-100°C, followed by second heating at pH 7.0-8.0 and 50-70°C, creates conditions where impurities are selectively removed, reducing the number of chromatography steps needed and improving production efficiency.
3Productivity
If high-pressure homogenization is used for cell disruption, then protein extraction efficiency improves, but formation difficulty increases and purity decreases
Solution Approach 1:
The patent addresses purity concerns after high-pressure homogenization by implementing specific pH adjustment (to 7.0-9.0) and temperature control (80-100°C for first heating, 50-70°C for second heating). These parameter changes create optimal conditions for protein solubility and impurity precipitation, ensuring high purity despite the aggressive cell disruption method.
Solution Approach 2:
The patent uses urea and sodium chloride as intermediary substances to facilitate impurity removal. Urea at specific concentrations (6-8 M) and sodium chloride are added as intermediaries between the high-pressure homogenization step and final purification, mediating the separation of proteins from impurities through solubility changes and precipitation.
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 achieves purity greater than 85% before chromatography, reduces chromatography steps, improves production efficiency, and lowers costs by minimizing impurities and solvent residues, ensuring uniform particle size and batch consistency.
Implementation Method 1
transfecting a host cell with a plasmid vector comprising a sequence encoding the recombinant particle protein for expression in the cell
Implementation Method 2
harvesting the bacterial cells, and disrupting the same through high-pressure homogenization
Implementation Method 3
adjusting the pH of a high-pressure homogenization supernatant to 7.0-9.0, heating the supernatant at 80-100° C. for more than 15 min
Implementation Method 4
performing centrifugation, discarding a pellet, collecting a supernatant
Implementation Method 5
adding urea and sodium chloride, wherein the urea is at a concentration of 6-8 M
Implementation Method 6
adding urea and sodium chloride
Implementation Method 7
performing anion exchange chromatography and hydrophobic chromatography
Implementation Method 8
performing anion exchange chromatography and hydrophobic chromatography
Data Source
AI summary
The present invention relates to a recombinant particle protein suitable for industrial production and a preparation method therefor. The product is prepared by the following method: transfecting a host cell with a plasmid vector comprising a sequence encoding the recombinant particle protein for expression in the cell, harvesting and disrupting the bacterial cells, heating, incubating with urea and sodium chloride, and performing chromatography. The particle size of the product is uniform, and the batch-to-batch consistency in particle size is good. The product prepared by the method can reduce the cost of large-scale industrial mass production, the method is simple to operate, and the product has low impurity residue and a good safety profile.