Two-Part RP-Tag Affinity Purification for Recombinant Proteins
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Solution Overview
Problem
Existing methods for identifying, expressing, and isolating recombinant proteins are inefficient, requiring multiple techniques and increasing costs and time, with a need for improved compositions and systems for protein expression, isolation, and identification.
Innovation Solution
A two-part peptide tag system derived from bacterial ribose binding protein, specifically RP-Tag Large and RP-Tag Small, with high affinity and stability, allowing for affinity purification and protein identification, and enhanced solubility and expression of recombinant proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If multiple purification techniques are used to isolate recombinant proteins, then purification reliability is improved, but process complexity and time increase
Solution Approach 1:
The ribose binding protein is divided into two separate functional fragments: RP-Tag Large (amino acids 1-136) and RP-Tag Small (amino acids 137-276). This segmentation allows each fragment to serve a specific purpose - RP-Tag Large provides stable binding to the target protein while RP-Tag Small enables high-affinity purification through immobilization on solid supports, eliminating the need for multiple purification techniques
Solution Approach 2:
The two-part RP-Tag system acts as an intermediary between the target recombinant protein and the purification system. RP-Tag Large fused to the target protein mediates specific binding, while RP-Tag Small immobilized on solid support mediates purification capture, simplifying the overall purification process into a single-step affinity chromatography
2Reliability
If multiple purification techniques are used to isolate recombinant proteins, then purification reliability is improved, but time consumption increases
Solution Approach 1:
The invention merges the functions of target protein recognition and purification capture into a single two-part tag system. RP-Tag Large provides specific recognition of the target protein while RP-Tag Small provides the purification handle, combining multiple functions into one integrated system that operates in a single purification step
Solution Approach 2:
The RP-Tag fragments are pre-engineered and characterized to ensure optimal binding affinity and purification performance. RP-Tag Large is designed with high stability and specific binding capacity, while RP-Tag Small is optimized for immobilization and capture efficiency, allowing the purification process to proceed rapidly without requiring multiple sequential steps
3Ease of manufacture
If existing purification reagents are used, then current methods are maintained, but cost and time efficiency deteriorate
Solution Approach 1:
The two-part RP-Tag system is universally applicable to any recombinant protein of interest. RP-Tag Large can be fused to the N- or C-terminus of any target protein, and RP-Tag Small can be immobilized on various solid supports, creating a universal purification platform that simplifies the purification process across different protein targets
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 RP-Tag system provides robust and stable protein-protein interactions under various conditions, enabling efficient protein isolation and purification, with high affinity and resistance to denaturants, and is suitable for diverse cell systems.
Implementation Method 1
the first protein and second protein have an equilibrium binding constant, Kd, of less than 100 nM
Data Source
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AI summary
Disclosed herein are compositions, methods, and systems for the purification and/or detection of recombinant and other proteins. In some embodiments, compositions may comprise recombinant protein with one or more sequences having substantial homology to RP-Tag Small or RP-Tag Large. In some cases, the disclosed compositions may be useful in binding or recognizing target proteins.