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Updated: May 16, 2026
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Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
Published on: January 16, 2012
CHiC, a new tandem affinity tag for the protein purification toolbox
Gro Anita Stamsås1, Leiv Sigve Håvarstein, Daniel Straume
1Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, NO-1432 Aas, Norway.
Journal of Microbiological Methods
|November 17, 2012
Summary
Researchers developed a novel purification tag, CHiC (choline-binding His-tag), for efficiently isolating Streptococcus pneumoniae proteins PcsB and ECL1. This versatile tag enables high-purity recombinant protein production in minimal steps, aiding cell division research.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- PcsB is a peptidoglycan hydrolase involved in bacterial cell division.
- ECL1 is the extracellular domain of FtsX, a membrane protein that interacts with PcsB.
- Understanding the purification of these proteins is crucial for studying bacterial cell division.
Purpose of the Study:
- To construct and validate a new tandem affinity purification tag, CHiC.
- To purify PcsB and ECL1 from Streptococcus pneumoniae using the CHiC tag.
- To assess the efficiency and utility of the CHiC tag for recombinant protein purification.
Main Methods:
- Construction of a novel CHiC tag (6xHis-tag, choline binding domain, TEV protease site).
- Expression of CHiC-tagged PcsB and ECL1 in Escherichia coli.
- Sequential purification using diethylaminoethyl-cellulose and Ni(2+) immobilized metal affinity chromatography.
- TEV protease digestion for tag removal and final purification.
Main Results:
- Successfully purified PcsB and ECL1 from Streptococcus pneumoniae with high purity (≥95%).
- Obtained 4-7 mg of recombinant protein per liter of cell culture.
- Demonstrated the CHiC tag's ability to enhance protein solubility, particularly for PcsB.
- Achieved purification in one to two steps, including tag removal.
Conclusions:
- The CHiC tag is an effective and versatile tool for purifying recombinant proteins.
- This method allows for the efficient isolation of milligram quantities of highly purified proteins.
- The CHiC tag facilitates research into bacterial cell division by providing pure PcsB and ECL1.
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