Brevibacillus Host for Protein A Hyperexpression
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Solution Overview
Problem
Current methods for producing protein A, such as using Escherichia coli or Bacillus subtilis, face challenges with low productivity and degradation issues, making the purification process complex and costly.
Innovation Solution
Employing a Brevibacillus genus bacterium as a host for the hyperexpression and secretion of protein A, utilizing genetic recombination techniques to achieve high-yield production and prevent degradation, allowing for easy purification and immobilization on a matrix for antibody purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If Escherichia coli or Bacillus subtilis is used as host for protein A production, then production cost is reduced, but productivity is low and protein degradation occurs
Solution Approach 1:
The patent changes the host organism parameter from conventional E. coli or B. subtilis to Brevibacillus brevis, which naturally possesses lower protease activity. This parameter change resolves the contradiction by providing both high productivity and reduced degradation while maintaining cost-effectiveness through a single organism system.
2Manufacturing precision
If multiple chromatography techniques are used for antibody purification, then purity is improved, but process complexity and time increase
Solution Approach 1:
The patent extracts and eliminates the need for multiple complex chromatography steps by using a single affinity chromatography step with protein A. The high purity of secreted protein A allows direct use as affinity medium, removing the need for additional purification steps while maintaining high antibody purity.
Solution Approach 2:
The patent makes protein A serve multiple functions: it is both the target protein to be produced and the affinity ligand for antibody purification. This multi-functionality simplifies the overall process by eliminating the need for separate purification of the affinity medium and the antibody purification steps.
3Productivity
If protein A is produced intracellularly, then production yield is improved, but purification difficulty and degradation risk increase
Solution Approach 1:
The patent performs preliminary action by engineering the protein A expression system to include a signal peptide that directs secretory pathway targeting. This preliminary routing decision ensures protein A is automatically secreted into the culture medium during expression, eliminating subsequent cell disruption and intracellular purification steps while maintaining high yield.
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 approach results in significantly higher yields of pure protein A with preserved immunoglobulin-binding function, simplifying the purification process and reducing costs by overcoming the limitations of previous methods.
Implementation Method 1
employing a Brevibacillus genus bacterium as a host for the hyperexpression and secretion of protein A, utilizing genetic recombination techniques
Implementation Method 2
The protein A is one kind of cell wall protein... Its structure is composed of seven functional domains... These five immunoglobulin-binding domains (domains E, D, A, B, and C) of the protein A can respectively bind to immunoglobulin through its Fc region
Implementation Method 3
affinity chromatography capable of specifically adsorbing the antibody proteins... Chromatography using a medium comprising an appropriate resin immobilizing thereon proteins such as protein A
Data Source
AI summary
The present invention relates to an efficient and economical process for producing a protein A-like protein. Hosts such as Escherichia coli and Bacillus subtilis have been used in the production of a protein A-like protein using a genetic recombination technique and however, their low productivity has been a big cause of high cost. Thus, it has been desired strongly to immediately establish a technique enabling the inexpensive, large-scale production of a protein A-like protein using recombinant DNA techniques other than Escherichia coli and Bacillus subtilis. The present invention provides a process for producing a protein A-like protein in large amounts, for example, a process comprising allowing a recombinant Brevibacillus genus bacterium to express and secrete the protein in large amounts into a culture solution and separating and collecting the accumulated protein A-like protein from the culture solution.