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Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
Published on: January 16, 2012
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CP5 system, for simple and highly efficient protein purification with a C-terminal designed mini tag
Hiroyuki Takeda1, Wei Zhou1, Kohki Kido1
1Proteo-Science Center, Ehime University, Matsuyama, Ehime, Japan.
Plos One
|May 26, 2017
Summary
A novel CP5 tag and antibody system enables efficient purification of recombinant proteins. This method offers mild elution conditions, preserving protein activity for downstream applications.
Area of Science:
- Biochemistry
- Structural Biology
- Protein Purification
Background:
- Affinity purification using monoclonal antibodies targeting linear epitopes is crucial in biochemistry and structural biology.
- Existing methods can be limited by tag size and elution conditions, potentially affecting protein integrity.
Purpose of the Study:
- To introduce a new, short CP5 tag and develop a novel affinity protein purification system.
- To demonstrate the system's efficacy in purifying various proteins, including delicate enzymes and membrane proteins.
Main Methods:
- Developed a CP5 tag based on a minimal epitope sequence of the anti-dopamine receptor D1 (DRD1) antibody clone Ra62.
- Utilized the Ra62 antibody for capture and competing peptides with differential affinities for elution.
- Applied the CP5 system to purify deubiquitinase CYLD, E3 ubiquitin ligase MARCH3, and G protein-coupled receptors (GPCRs).
Main Results:
- Successfully purified deubiquitinase CYLD and E3 ubiquitin ligase MARCH3, confirming their catalytic activity.
- Achieved purification of 9 out of 12 cell-free synthesized G protein-coupled receptors (GPCRs), including integral membrane proteins.
- Demonstrated successful purification of CP5-tagged CHRM2 expressed in baculovirus-insect cells.
Conclusions:
- The CP5 system provides a highly specific and efficient method for protein purification with sharp elution under mild conditions.
- The short CP5 tag minimizes impact on protein characteristics, making it suitable for delicate proteins.
- This system offers a promising alternative for protein sample preparation, ensuring high yield, purity, and activity for functional and structural analyses.
