BRCA2-Mediated Purification of Recombinase Proteins
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Solution Overview
Problem
Current methods for purifying recombinant Rad51 and Dmc1 proteins are inefficient, often requiring frequent re-preparation and leaving additional residues or necessitating sequence-specific proteases, which are costly and burdensome.
Innovation Solution
Utilizing natural protein-protein interactions to isolate and enrich recombinant RAD51 or DMC1 proteins from soluble lysates, followed by chromatographic separation to obtain full-length, un-tagged proteins without non-native amino acids, through co-overexpression with affinity tags and specific motifs like the BRC4 repeat of BRCA2.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standard purification protocols are used for recombinant Rad51 and Dmc1 proteins, then purification can be achieved, but the process is inefficient and requires frequent re-preparation
Solution Approach 1:
The patent introduces BRCA2 BRC repeat domains as intermediary binding partners that specifically interact with Rad51 and Dmc1 proteins. These intermediaries are fused to affinity tags (GST or MBP) enabling indirect purification of the recombinases through their natural protein-protein interactions, thereby achieving efficient and stable purification results
Solution Approach 2:
The patent creates fusion proteins that copy natural interaction interfaces - the BRC repeat domains are engineered to mimic the natural binding interface between BRCA2 and Rad51/Dmc1. This copying of the natural binding interface allows specific recognition and purification without requiring the actual full-length BRCA2 protein
2Ease of manufacture
If affinity tags are used for purification, then protein isolation is simplified, but additional non-native residues remain on the purified protein
Solution Approach 1:
The patent segments the purification system into three distinct functional modules: (1) the affinity tag (GST or MBP) for capture, (2) the BRC repeat domains for specific protein recognition, and (3) the target recombinase protein. This segmentation allows the affinity tag to be used only during purification while the final product contains only the native protein sequence
Solution Approach 2:
The patent extracts the affinity tag and BRC repeat domains from the final purified product through proteolytic cleavage. The protease recognition sequences are strategically placed between the affinity tag/BRC domains and the recombinase protein, allowing clean separation and removal of the purification helpers while leaving the native protein intact
3Manufacturing precision
If proteases are used to remove tags, then full-length native proteins can be obtained, but the process becomes costly and burdensome
Solution Approach 1:
The patent merges the affinity tag function with the BRC repeat domains into a single fusion construct. This combination allows both the affinity capture and the specific protein recognition functions to be performed by one integrated module, simplifying the overall purification process while maintaining the ability to generate native protein sequences
Solution Approach 2:
The BRC repeat domains serve multiple functions: they provide specific binding to Rad51 and Dmc1 proteins, they facilitate affinity purification when fused to GST or MBP tags, and they can be easily removed by proteases to leave the native protein intact. This multi-functionality reduces the need for separate purification and processing steps
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 method achieves efficient purification of full-length, un-tagged RAD51 or DMC1 proteins, reducing the need for additional residues and proteolytic steps, thereby improving yield and reducing costs while maintaining native protein integrity.
Implementation Method 1
BRCA2 interacts with RAD51 through 2 separate and distinct modes. One mode is characterized by the interaction of RAD51 with the BRC repeat region of BRCA2
Implementation Method 2
The larger hydrophobic residue, F, plugs into a deep hydrophobic pocket located in the globular ATPase domain of a neighboring molecule, while the A residue sits in an adjacent shallower pocket
Implementation Method 3
followed by chromatographic separation to obtain full-length, un-tagged proteins
Data Source
AI summary
The invention is the products and methods associated with purifying overexpressed recombinant recombinases from a host cell line resulting in an un-tagged protein of interest without any additional, non-native amino acids. The invention employs at least one DNA vector that co-expresses a tagged fusion protein and the recombinase protein with the recombinase protein having an affinity for binding to the tagged fusion protein. Isolation methods of the recombinase protein include the targeting of the tagged fusion protein.