Purification of E. coli proteins using a self-cleaving chitin-binding affinity tag
Michael J Coolbaugh1, David W Wood
1Department of Chemical and Biomolecular Engineering, The Ohio State University, 140 West 19th Ave, Columbus, OH, 43210, USA.
Methods in Molecular Biology (Clifton, N.J.)
|June 20, 2014
Summary
This study presents a novel self-cleaving purification tag system combining a chitin-binding domain (CBD) and an intein. This method simplifies recombinant protein purification by eliminating the need for costly proteolytic digestion.
Area of Science:
- Molecular Biology
- Protein Biochemistry
- Biotechnology
Background:
- Affinity tags are essential for recombinant protein purification.
- Tag removal post-purification, often via proteolytic digestion, presents challenges including complexity and cost.
- Alternative methods for efficient tag removal are needed.
Purpose of the Study:
- To develop and describe a novel self-cleaving purification tag system.
- To provide an alternative to proteolytic digestion for recombinant protein purification.
- To demonstrate the utility of a chitin-binding domain (CBD) and intein combination.
Main Methods:
- Genetically fusing a protein of interest downstream of a chitin-binding domain (CBD)-intein fusion tag.
- Purifying the resulting fusion protein using chitin resin.
- Inducing intein-mediated self-cleavage via pH and temperature shift.
Main Results:
- The developed system enables rapid and easy purification of recombinant proteins.
- Intein self-cleavage efficiently liberates the target protein from the purification tag.
- The system is applicable to proteins expressed in E. coli.
Conclusions:
- The CBD-intein self-cleaving tag offers a simplified and potentially cost-effective alternative for recombinant protein purification.
- This system has broad applicability for various recombinant proteins and potential for diverse expression hosts.
- Further applications with different tags and hosts are feasible.
