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1 Department of Biochemistry and Molecular Biology, Institute for Molecular Bioscience, and Special Research Centre for Functional and Applied Genomics and
2 Cooperative Research Centre for Chronic Inflammatory Disease, University of Queensland, St Lucia, Queensland 4072, Australia
3 Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA
Reprint requests to: Bostjan Kobe, Department of Biochemistry and Molecular Biology, University of Queensland, Brisbane, Queensland 4072, Australia; e-mail: b.kobe{at}mailbox.uq.edu.au; fax: 61-7-3365-4699.
The fusion of a protein of interest to a large-affinity tag, such as the maltose-binding protein (MBP), thioredoxin (TRX), or glutathione-S-transferase (GST), can be advantageous in terms of increased expression, enhanced solubility, protection from proteolysis, improved folding, and protein purification via affinity chromatography. Unfortunately, crystal growth is hindered by the conformational heterogeneity induced by the fusion tag, requiring that the tag is removed by a potentially problematic cleavage step. The first three crystal structures of fusion proteins with large-affinity tags have been reported recently. All three structures used a novel strategy to rigidly fuse the protein of interest to MBP via a short three- to five-amino acid spacer. This strategy has the potential to aid structure determination of proteins that present particular experimental challenges and are not conducive to more conventional crystallization strategies (e.g., membrane proteins). Structural genomics initiatives may also benefit from this approach as a way to crystallize problematic proteins of significant interest.
Keywords: Chimera; fusion protein; protein crystallization; protein expression; membrane proteins; molecular replacement; structural genomics; X-ray crystallography
Abbreviations: 2D, two-dimensional • 3D, three-dimensional • GST, glutathione-S-transferase • His-tag, hexahistidine-tag • MBP, maltose-binding protein • MR, molecular replacement • PEG, polyethylene glycol • TRX, thioredoxin
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