Crystal structures of fusion proteins with large‐affinity tags

• Published in: Protein science Vol. 12; no. 7; pp. 1313 - 1322
• Main Authors: Smyth, Douglas R., Mrozkiewicz, Marek K., McGrath, William J., Listwan, Pawel, Kobe, Bostjan
• Format: Journal Article
• Language: English
• Published: Bristol Cold Spring Harbor Laboratory Press 01.07.2003; Wiley-Blackwell
• Subjects: 2D, two‐dimensional; 3D, three‐dimensional; Affinity Labels - chemistry; Carrier Proteins - chemistry; Chimera; Crystallography, X-Ray; Dimerization; fusion protein; Genomics - methods; Glutathione Transferase - chemistry; GST, glutathione‐S‐transferase; His‐tag, hexahistidine‐tag; Maltose-Binding Proteins; MBP, maltose‐binding protein; membrane proteins; Membrane Proteins - chemistry; molecular replacement; MR, molecular replacement; PEG, polyethylene glycol; Protein Conformation; protein crystallization; protein expression; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification; Review; structural genomics; Thioredoxins - chemistry; TRX, thioredoxin; X‐ray crystallography
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.0243403

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.