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A heme fusion tag for protein affinity purification and quantification
1Department of Chemistry, University of Rochester, Rochester, New York 14627-0216, USA.
Protein Science : a Publication of the Protein Society
|July 29, 2010
Summary
Researchers developed a novel heme tag purification method for Escherichia coli proteins. This visible affinity tag simplifies protein tracking and quantification, offering an efficient purification strategy.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Chemistry
Background:
- Protein purification is crucial for biochemical and molecular biology research.
- Existing methods can be complex or lack visual tracking capabilities.
- Developing efficient and visible affinity tags is an ongoing need.
Purpose of the Study:
- To introduce a novel, visually traceable affinity-based protein purification method.
- To demonstrate the efficacy of a heme tag for protein purification from Escherichia coli.
- To establish a method that also allows for protein quantification.
Main Methods:
- Utilized a heme tag that coordinates with L-histidine-immobilized sepharose (HIS) resin for affinity purification.
- Purified azurin and maltose binding protein from cell lysate.
- Employed mild binding and elution conditions using phosphate buffer (pH 7.0) and imidazole or buffer pH adjustment.
Main Results:
- Successfully purified target proteins using the heme tag and HIS resin.
- The heme tag provided a visible marker for protein tracking.
- Demonstrated low nonspecific binding of untagged proteins to the HIS resin.
- Showcased the heme tag's utility for protein quantification via the pyridine hemochrome method.
Conclusions:
- The heme tag-HIS resin system offers an effective and visually guided method for protein purification.
- This approach simplifies protein tracking and enables quantification.
- The method is suitable for proteins expressed in Escherichia coli under mild conditions.
