Targeted Protein Sequence Diversity via RAG-Mediated Recombination
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Solution Overview
Problem
Current methods for generating sequence diversity in proteins, such as those using the V(D)J recombination system, face challenges including large chromosomal deletions, imprecise junctions, and limited utility for introducing diversity in restricted protein sequences, which restricts their application to extended nucleic acid sequences rather than targeted sequences within small loops.
Innovation Solution
Introducing two or more recombination signal sequences (RSSs) into the protein coding sequence and using a recombination-competent host cell capable of expressing RAG-1 and RAG-2 to generate variants, allowing for targeted sequence diversity through controlled recombination and expression, with the use of flanking sequences to minimize deletions and incorporate additional sequences for enhanced functionality.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If V(D)J recombination is used to generate sequence diversity, then a large number of variants can be produced, but large chromosomal deletions and imprecise junctions occur
Solution Approach 1:
The patent introduces a recombination signal sequence (RSS) as an intermediary element flanking the target sequence. This RSS mediates site-specific recombination by the RAG complex, enabling precise integration of diversity at defined locations rather than random V(D)J style recombination, thus maintaining junction precision while still generating diverse variants
Solution Approach 2:
The patent modifies the recombination system by changing the parameters of where recombination occurs - using engineered RSS sequences with specific spacer lengths (12 or 23 bp) at defined positions flanking the target loop, rather than relying on natural V(D)J recombination parameters. This allows control over recombination precision while maintaining productivity
2Adaptability or versatility
If random mutagenesis is used to introduce diversity, then mutations can be introduced throughout the gene, but the mutations are not targeted to specific functional regions
Solution Approach 1:
The patent applies local quality by restricting the recombination activity to a specific local region - a loop structure flanked by RSS sequences - rather than allowing random mutations throughout the entire gene. This ensures diversity is generated precisely where needed (in the loop region) while maintaining the rest of the protein structure intact
Solution Approach 2:
The patent segments the protein into functional regions, identifying the loop as the target for diversity generation. By placing RSS sequences specifically flanking the loop region and using site-specific recombination, the patent achieves targeted mutagenesis of only the relevant segment rather than random mutagenesis of the entire gene
3Productivity
If saturation mutagenesis is used to achieve high mutation rates, then up to 100% mutation rates are possible, but the entire region is mutated rather than specific locations
Solution Approach 1:
The patent achieves high productivity with location specificity by placing RSS sequences to flank only the loop region. The RAG-mediated recombination then acts locally at this defined site, generating high diversity (similar to saturation mutagenesis) but confined precisely to the loop region rather than the entire gene
Solution Approach 2:
The engineered RSS sequences act as intermediaries that direct the RAG recombination machinery to specific locations. This mediator approach enables high mutation rates at targeted locations by providing defined recombination sites, combining the benefits of saturation mutagenesis (high diversity) with site-directed precision
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 generation of a large number of protein variants with desired functionality, allowing for high affinity binding proteins to be identified in a single round, with the ability to introduce mutations at specific locations and control the recombination process to maintain structural integrity and introduce additional functionality.
Implementation Method 1
V(D)J recombination is the process responsible for the assembly of antibody gene segments (V, D and J; or V and J in the case of the light chain) and as part of the assembly process creates the CDR3 of the respective antibody chain
Data Source
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Figure 3A~3B
AI summary
Methods of generating sequence diversity in a protein, such as a ligand-binding protein, are provided. The methods comprise targeted introduction of two or more recombination signal sequences (RSSs) into the protein coding sequence and introduction of the modified protein coding sequence into a recombination-competent host cell, specifically a recombination-competent host cell that is capable of expressing at least RAG-1 and RAG-2, thereby allowing for recombination of the protein coding sequence and expression of variant proteins. Also provided are polynucleotides comprising a nucleic acid sequence encoding a target protein, such as a ligand-binding protein, and comprising two or more RSSs, and compositions and host cells comprising same.