Engineered p53 Tetramerization Domains for High-Purity Multimer Production
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Solution Overview
Problem
Current methods for producing multimers of effector domains, such as tetramers and octamers, face challenges in achieving high purity and yield, often resulting in mixtures with monomers, dimers, and trimers, which complicates their application in medical and industrial uses.
Innovation Solution
The use of engineered polypeptides comprising self-associating tetramerization domains (TDs) like NHR2, p53, p63, or p73 TDs to form multimers that are specifically designed to self-associate into tetramers or octamers, allowing for higher yields and purity by associating into specific multimeric forms without contamination from lower-order structures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional multimerisation techniques (biotin, dHLX, ZIP, BAD domains) are used to produce multimers of effector domains, then multimer formation is achieved, but the products are mixtures containing monomers, dimers, trimers and multimers resulting in low purity and complicated downstream processing
Solution Approach 1:
The patent changes the structural parameters of the multimerisation domain by using engineered variants of p53 tetramerisation domain with specific amino acid substitutions (e.g., R331A, R331Q, R331K, R331E, R331H, R331R, R331D, R331N, R331S, R331C, R331G, R331P, R331F, R331W, R331Y, R331M, R331V, R331I, R331L, R331A, R331T, R331Q, R331K, R331E, R331H, R331R, R331D, R331N, R331S, R331C, R331G, R331P, R331F, R331W, R331Y, R331M, R331V, R331I, R331L) that enhance tetramer formation stability and specificity, thereby improving manufacturing precision while maintaining high yield
Solution Approach 2:
The patent uses identical copies of the engineered p53 tetramerisation domain in each effector domain fusion protein, ensuring that all monomers have the same self-association capability. This copying approach ensures uniform tetramer formation and eliminates the formation of mixed oligomers, thereby achieving high purity multimer products
2Manufacturing precision
If self-associating tetramerisation domains are used to form multimers, then higher yield and purity are achieved, but the device complexity increases due to engineered domain design
Solution Approach 1:
The engineered p53 tetramerisation domain serves multiple functions: it mediates self-association of effector domains into tetramers, provides stable multimer formation, and enables high-purity product isolation. This multi-functionality reduces the need for additional purification steps and complex processing equipment, thereby offsetting the initial design complexity
Solution Approach 2:
The self-associating tetramerisation domains enable the effector domain fusion proteins to automatically form tetramers through their inherent ability to self-associate. This self-service mechanism eliminates the need for external crosslinking agents, complex assembly protocols, or additional reagents, thereby simplifying the overall manufacturing process despite the engineered domain design
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 enables the production of high-purity multimers that are specifically designed for medical and industrial applications, enhancing antigen binding avidity and providing multivalent binding capabilities, while minimizing contamination from monomers, dimers, or trimers.
Implementation Method 1
Multimerisation domains which cause self-assembly of protein monomers into multimers are known in the art
Implementation Method 2
The tetramerization domain of human p53 extends from residues 325 to 356, and has a 4-helical bundle fold
Data Source
AI summary
The invention relates to multimers such as tetramers of polypeptides and tetramers and octamers of effector domains, such as antigen binding sites (eg, antibody or TCR binding sites that specifically bind to antigen or pMHC, or variable domains thereof) or peptides such as incretin, insulin or hormone peptides.