Modified Protein A Ligand for Protease-Resistant Chromatography
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Solution Overview
Problem
Recombinant protein A used in affinity chromatography is susceptible to protease cleavage, particularly by serine proteases and thermolysin, leading to decreased immunoglobulin-binding activity and instability as a ligand in affinity chromatography columns.
Innovation Solution
Modifying the amino acid sequence of protein A by deleting or substituting lysine at specific ligation sites and avoiding hydrophobic amino acids in ligation elements to enhance resistance to proteases, thereby maintaining immunoglobulin-binding activity and column durability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If recombinant protein A is used as an affinity ligand in chromatography, then immunoglobulin-binding activity is achieved, but the protein is susceptible to protease cleavage leading to decreased stability
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of protein A at specific positions (particularly in the ligation elements connecting domains). The modification involves substituting or deleting lysine residues and avoiding hydrophobic amino acids at ligation sites, which changes the chemical parameters of the protein structure to resist protease recognition and cleavage, thereby improving stability while maintaining immunoglobulin-binding activity
Solution Approach 2:
The patent extracts and removes the vulnerable ligation elements from the protein A structure that are susceptible to protease cleavage. By identifying and modifying specific amino acid sequences at domain ligation sites (removing lysine residues and hydrophobic amino acids), the patent eliminates the weak points that proteases target, thereby protecting the overall structure from degradation
2Reliability
If protein A is modified to enhance alkali resistance and antibody elution at weak acidic pH, then ligand function is improved, but immunoglobulin-binding activity may be reduced
Solution Approach 1:
The patent applies local quality by making targeted modifications only at specific ligation sites (domain connection regions) rather than throughout the entire protein structure. The modifications are localized to amino acid positions involved in domain ligation, leaving the immunoglobulin-binding domains intact and preserving their binding activity while improving overall stability and elution properties
Solution Approach 2:
The patent changes specific chemical parameters at ligation sites by substituting lysine residues and removing hydrophobic amino acids from domain connection regions. These parameter changes improve alkali resistance and enable weak acidic pH elution without affecting the immunoglobulin-binding activity of the functional domains
3Ease of operation
If lysine and cysteine residues are removed from protein A domains to enable antibody desorption at weak acidic pH, then elution capability is improved, but overall protein structure and binding activity may be affected
Solution Approach 1:
The patent segments the protein A structure into distinct functional regions: ligation elements (domain connection regions) and immunoglobulin-binding domains. Modifications are applied only to the ligation elements to enable weak acidic pH desorption, while the binding domains are preserved to maintain structural stability and binding activity. This segmentation allows independent optimization of different functional regions
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 modified protein A exhibits improved resistance to proteases, ensuring stable immunoglobulin-binding activity and prolonged durability as an affinity ligand, even under conditions that would typically cause cleavage, such as increased pH or high temperatures.
Implementation Method 1
a protein which has two or more domains derived from any of E, D, and A domains of protein A... having affinity to immunoglobulin
Data Source
Figure 1
Figure 2
Figure 3A~3C
AI summary
An object of the present invention is to provide a protein which has resistance to a serine protease or thermolysin, has an immunoglobulin-binding activity, and can be favorably used as an affinity ligand. One embodiment of the present invention is a protein having affinity for an immunoglobulin, which is a protein having two or more domains derived from any of the amino acid sequences of E, D, and A domains of protein A, and in the amino acid sequence of at least one of the domains, one or more lysines are included, and the C-terminal lysine is deleted or substituted, or a protein having affinity for an immunoglobulin, which is a protein having two or more domains derived from any of B, C, and Z domains of protein A, and in the amino acid sequence of at least one of the domains, one or more lysines are included, and lysine at position 4 and the C-terminal lysine are deleted or substituted.