Crystal structures of fusion proteins with large‐affinity tags

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.

[1]  Yutaka Kuroda,et al.  Characterization and prediction of linker sequences of multi-domain proteins by a neural network , 2004, Journal of Structural and Functional Genomics.

[2]  K. M. Routzahn,et al.  Differential effects of supplementary affinity tags on the solubility of MBP fusion proteins , 2004, Journal of Structural and Functional Genomics.

[3]  J. Williamson,et al.  Inherent protein structural flexibility at the RNA-binding interface of L30e. , 2003, Journal of molecular biology.

[4]  C. Wolberger,et al.  Insights into binding cooperativity of MATa1/MATα2 from the crystal structure of a MATa1 homeodomain‐maltose binding protein chimera , 2003, Protein science : a publication of the Protein Society.

[5]  David T. Jones,et al.  Rapid protein domain assignment from amino acid sequence using predicted secondary structure , 2002, Protein science : a publication of the Protein Society.

[6]  Seok-Yong Lee,et al.  Detection and characterization of xenon-binding sites in proteins by 129Xe NMR spectroscopy. , 2002, Journal of molecular biology.

[7]  H. Michel,et al.  Crystallisation of membrane proteins mediated by antibody fragments. , 2002, Current opinion in structural biology.

[8]  Yan-Ping Shih,et al.  High‐throughput screening of soluble recombinant proteins , 2002, Protein science : a publication of the Protein Society.

[9]  E. Wright,et al.  Employing Escherichia coli to functionally express, purify, and characterize a human transporter , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[10]  C. Deber,et al.  Expression and purification of two hydrophobic double-spanning membrane proteins derived from the cystic fibrosis transmembrane conductance regulator. , 2002, Protein expression and purification.

[11]  Yanhui Hu,et al.  Proteome-scale purification of human proteins from bacteria , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[12]  D. Waugh,et al.  Electronic Reprint Biological Crystallography Differential Effects of Short Affinity Tags on the Crystallization of Pyrococcus Furiosus Maltodextrin-binding Protein Biological Crystallography Differential Effects of Short Affinity Tags on the Crystallization of Pyrococcus Furiosus Maltodextrin- Bind , 2022 .

[13]  Martin Hammarström,et al.  Rapid screening for improved solubility of small human proteins produced as fusion proteins in Escherichia coli , 2002, Protein science : a publication of the Protein Society.

[14]  J. Kwang,et al.  Vaccination of ducks with recombinant outer membrane protein (OmpA) and a 41 kDa partial protein (P45N') of Riemerella anatipestifer. , 2002, Veterinary microbiology.

[15]  R. Grisshammer,et al.  Purification and characterization of the human adenosine A2a receptor functionally expressed in Escherichia coli , 2002 .

[16]  R. Grisshammer,et al.  Purification and characterization of the human adenosine A(2a) receptor functionally expressed in Escherichia coli. , 2002, European journal of biochemistry.

[17]  Y. Zhan,et al.  Structural analysis of regulatory protein domains using GST-fusion proteins. , 2001, Gene.

[18]  J. Lefèvre,et al.  Formation of soluble inclusion bodies by hpv e6 oncoprotein fused to maltose-binding protein. , 2001, Protein expression and purification.

[19]  Daniel Lévy,et al.  Two‐dimensional crystallization of membrane proteins: the lipid layer strategy , 2001, FEBS letters.

[20]  C. Tate,et al.  Overexpression of mammalian integral membrane proteins for structural studies , 2001, FEBS letters.

[21]  Chris Sander,et al.  Completeness in structural genomics , 2001, Nature Structural Biology.

[22]  Yingfang Liu,et al.  Crystal structure of the SarR protein from Staphylococcus aureus , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[23]  Cheryl H. Arrowsmith,et al.  Protein production: feeding the crystallographers and NMR spectroscopists , 2000, Nature Structural Biology.

[24]  H. Saibil,et al.  Macromolecular structure determination by cryo-electron microscopy. , 2000, Acta crystallographica. Section D, Biological crystallography.

[25]  R C Stevens,et al.  Design of high-throughput methods of protein production for structural biology. , 2000, Structure.

[26]  O. Nureki,et al.  Crystallization and preliminary X-ray diffraction analysis of the extracellular domain of the cell surface antigen CD38 complexed with ganglioside. , 2000, Journal of biochemistry.

[27]  Joseph J. Falke,et al.  [16] Purification of proteins using polyhistidine affinity tags , 2000 .

[28]  J. Mccoy,et al.  [21] Thioredoxin as a fusion partner for production of soluble recombinant proteins in Escherichia coli , 2000 .

[29]  T. Schmidt,et al.  Use of the Strep-Tag and streptavidin for detection and purification of recombinant proteins. , 2000, Methods in enzymology.

[30]  J. Falke,et al.  Purification of proteins using polyhistidine affinity tags. , 2000, Methods in enzymology.

[31]  J. Beckwith.  The all purpose gene fusion. , 2000, Methods in enzymology.

[32]  J. Chirgwin,et al.  Fusions to maltose-binding protein: control of folding and solubility in protein purification. , 2000, Methods in enzymology.

[33]  D. Smith.  Generating fusions to glutathione S-transferase for protein studies. , 2000, Methods in enzymology.

[34]  J. Mccoy,et al.  Thioredoxin as a fusion partner for production of soluble recombinant proteins in Escherichia coli. , 2000, Methods in enzymology.

[35]  E. Wright,et al.  Purification and functional reconstitution of a truncated human Na+/glucose cotransporter (SGLT1) expressed in E. coli , 1999, FEBS letters.

[36]  F. Baneyx.  Recombinant protein expression in Escherichia coli. , 1999, Current opinion in biotechnology.

[37]  G. Mosser,et al.  Two-dimensional crystallization on lipid layer: A successful approach for membrane proteins. , 1999, Journal of structural biology.

[38]  R. Center,et al.  Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[39]  B. Kieffer,et al.  Expression of delta, kappa and mu human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins. , 1999, European journal of biochemistry.

[40]  H. Kamata,et al.  Overexpression of the alanine carrier protein gene from thermophilic bacterium PS3 in Escherichia coli. , 1999, Journal of biochemistry.

[41]  A. Engel,et al.  Two-dimensional crystallization of Escherichia coli lactose permease. , 1999, Journal of structural biology.

[42]  D. Waugh,et al.  Escherichia coli maltose‐binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused , 1999, Protein science : a publication of the Protein Society.

[43]  T. Bergfors.  Protein crystallization : techniques, strategies, and tips : a laboratory manual , 1999 .

[44]  R. Center,et al.  Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose‐binding protein , 1998, Protein science : a publication of the Protein Society.

[45]  S. Islam,et al.  Crystallization of an intact GST-estrogen receptor hormone binding domain fusion protein. , 1998, Acta crystallographica. Section D, Biological crystallography.

[46]  V. Stoll,et al.  A thioredoxin fusion protein of VanH, a D‐lactate dehydrogenase from Enterococcus faecium: Cloning, expression, purification, kinetic analysis, and crystallization , 1998, Protein science : a publication of the Protein Society.

[47]  Winfried Boos,et al.  Maltose/Maltodextrin System of Escherichia coli: Transport, Metabolism, and Regulation , 1998, Microbiology and Molecular Biology Reviews.

[48]  J. Chirgwin,et al.  Solubility of proteins isolated from inclusion bodies is enhanced by fusion to maltose-binding protein or thioredoxin. , 1998, Protein expression and purification.

[49]  Arnaud Ducruix,et al.  Crystallization of Nucleic Acids and Proteins: A practical Approach , 1998 .

[50]  F A Quiocho,et al.  Extensive features of tight oligosaccharide binding revealed in high-resolution structures of the maltodextrin transport/chemosensory receptor. , 1997, Structure.

[51]  T. Hakoshima,et al.  Use of a fusion protein to obtain crystals suitable for X‐ray analysis: Crystallization of a GST‐fused protein containing the DNA‐binding domain of DNA replication‐related element‐binding factor, DREF , 1997, Protein science : a publication of the Protein Society.

[52]  H. Michel,et al.  Crystallization of membrane proteins. , 1983, Current opinion in structural biology.

[53]  L. Raymond,et al.  A recombinant Chlamydia trachomatis major outer membrane protein binds to heparan sulfate receptors on epithelial cells. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[54]  Chen Gq,et al.  Overexpression of bacterio-opsin in Escherichia coli as a water-soluble fusion to maltose binding protein: efficient regeneration of the fusion protein and selective cleavage with trypsin. , 1996 .

[55]  J. Gouaux,et al.  Overexpression of bacterio‐opsin in Escherichia coli as a water‐soluble fusion to maltose binding protein: Efficient regeneration of the fusion protein and selective cleavage with trypsin , 1996, Protein science : a publication of the Protein Society.

[56]  M. Billeter,et al.  MOLMOL: a program for display and analysis of macromolecular structures. , 1996, Journal of molecular graphics.

[57]  G. Privé,et al.  Engineering the lac permease for purification and crystallization , 1996, Journal of bioenergetics and biomembranes.

[58]  C. Tate,et al.  Overexpression of integral membrane proteins for structural studies , 1995, Quarterly Reviews of Biophysics.

[59]  J. Tainer,et al.  Crystal structures of a schistosomal drug and vaccine target: glutathione S-transferase from Schistosoma japonica and its complex with the leading antischistosomal drug praziquantel. , 1995, Journal of molecular biology.

[60]  D. Case,et al.  High-resolution solution structures of oxidized and reduced Escherichia coli thioredoxin. , 1994, Structure.

[61]  G. Gilliland,et al.  Fusion Proteins as Alternate Crystallization Paths to Difficult Structure Problems , 1994 .

[62]  R. Grisshammer,et al.  Expression of rat NK-2 (neurokinin A) receptor in E. coli. , 1994, Receptors & channels.

[63]  R. Duckworth,et al.  Expression of a rat neurotensin receptor in Escherichia coli. , 1993, The Biochemical journal.

[64]  I. B. Ivanov,et al.  Two-dimensional crystallization , 1993, Nature.

[65]  F. Quiocho,et al.  Crystallographic evidence of a large ligand-induced hinge-twist motion between the two domains of the maltodextrin binding protein involved in active transport and chemotaxis. , 1992, Biochemistry.

[66]  F. Quiocho,et al.  The 2.3-A resolution structure of the maltose- or maltodextrin-binding protein, a primary receptor of bacterial active transport and chemotaxis. , 1992, The Journal of biological chemistry.

[67]  H. Eklund,et al.  Crystal structure of thioredoxin from Escherichia coli at 1.68 A resolution. , 1990, Journal of molecular biology.

[68]  M. Chaudhury,et al.  Solubility of proteins , 1986 .

[69]  W. Wooster,et al.  Crystal structure of , 2005 .

[70]  H. Lipson.  Crystal Structures , 1949, Nature.