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Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
Published on: January 16, 2012
Human glutaredoxin 2 affinity tag for recombinant peptide and protein purification
Mathias Lundberg1, Arne Holmgren, Magnus Johansson
1Karolinska Institute, Division of Clinical Virology, Karolinska University Hospital at Huddinge, S-141 86 Stockholm, Sweden.
Protein Expression and Purification
|August 23, 2005
Summary
Human glutaredoxin 2 (Grx2) can purify short peptides via glutathione affinity chromatography. However, Grx2 fusion with larger proteins like GFP or beta-galactosidase failed, limiting its use as a general recombinant protein purification tag.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Purification
Background:
- Recombinant protein expression often utilizes affinity tags for purification.
- Human glutaredoxin 2 (Grx2) is a glutathione-binding protein with potential as a novel affinity tag.
Purpose of the Study:
- To evaluate the efficacy of human glutaredoxin 2 (Grx2) as an affinity tag for recombinant protein purification.
- To determine the limitations of Grx2 as an affinity tag for different protein sizes.
Main Methods:
- Purification of Grx2 from bacterial extracts using glutathione-Sepharose affinity chromatography.
- Fusion of Grx2 with short peptides, green fluorescent protein (GFP), and beta-galactosidase.
- Assessment of fusion protein binding to glutathione-Sepharose.
Main Results:
- Grx2 purified efficiently using a one-step glutathione affinity chromatography.
- Short peptides fused to Grx2 did not impede glutathione binding.
- Fusion of Grx2 with GFP (27 kDa) or beta-galactosidase (116 kDa) abolished glutathione-Sepharose binding.
- Linker sequences did not restore binding for larger fusion proteins.
Conclusions:
- Grx2 is suitable as an affinity tag for purifying short peptides.
- The utility of Grx2 as an affinity tag is limited for larger recombinant proteins, such as GFP and beta-galactosidase, due to loss of binding.
- Further research may explore modifications to enable Grx2 purification of larger proteins.
