Fusion Proteins as Research Tools


Fusion proteins encompass a protein or small peptide moiety which is used as a tag, fused to another protein of interest. Fusion proteins are used in biochemical and in genetic (high‐throughput) applications and are especially suited to study protein–protein interactions, even on a genome‐wide scale, and for the generation of antibodies.

Keywords: fusion proteins; GST system; single‐step purification; yeast two‐hybrid; protein–protein interaction

Figure 1.

Principle of the glutathione‐S‐transferase (GST) system. After transformation of the expression plasmids to Escherichia coli, propagation of transformed cells and induction of expression by (IPTG) extracts are prepared. (a) Protein extracts containing GST (‐fusion) proteins are subsequently incubated in the presence of gluthathione beads, which are then spun down and washed. (b) Purified fusion proteins attach to the beads and can be eluted from the beads by incubation with a buffer containing glutathione, after which they are ready for further use.

Figure 2.

GST pull‐down. GST (‐fusion) proteins are incubated with in vitro translated proteins labeled with, for example, [35S]methionine. After isolation of the fusion proteins plus associating proteins by centrifugation, the isolated proteins are subsequently detached from the beads by boiling in a denaturing buffer, followed by SDS‐PAGE and detection. Lane 1, molecular size marker. Lane 2, input of labeled in vitro translated protein. Lane 3, results of a control pull‐down experiment of GST proteins and the in vitro translated proteins. Lane 4, pull‐down of GST‐fusion proteins with the in vitro translated proteins. The translated protein binds to the GST‐fusion protein (lane 4), but not to the GST control (lane 3).

Figure 3.

Far‐Western analysis. Protein extracts or (partially) purified proteins are loaded on an SDS‐PAGE gel. The proteins are subsequently blotted to a membrane. Optionally, the proteins on the filter can be completely denatured, followed by renaturation. The blot is subsequently incubated with a purified and labeled protein probe, followed by washing of the membrane and detection. Lane 1, molecular size marker; lane 2, (WCE) containing protein X; lane 3, protein fraction lacking proteins X; lane 4, purified protein X. The protein probe can bind directly to protein X. In the WCE, one smaller protein is present which also can directly bind to the protein probe.

Figure 4.

The classic yeast two‐hybrid assay exploits the modular structure of transcriptional activators: a protein–protein interaction is identified by reconstitution of the activities of a transcriptional activator. (a) One yeast expression plasmid encodes the DNA binding domain of the transcription factor GAL4 (GAL4‐D) fused to the protein of interest (B, ‘bait’). This fusion protein binds to the promoter region, but, in the absence of a transcriptional activation domain, this protein will not give rise to significant transcriptional activation of the β‐galactosidase reporter (LacZ) gene. (b) The other yeast expression plasmid encodes the transcriptional activation domain of GAL4 fused to the protein of interest or proteins encoded by a cDNA library (P, ‘prey’). In the absence of a DNA‐binding domain or promoter‐targeting domain, this fusion protein will not give rise to transcriptional activation. (c) Two‐hybrid experiment. The bait plasmid and the prey plasmid are cotransformed. The binding of the prey to the bait gives rise to the presence of a strong transcriptional activation domain in the promoter region, resulting in (strongly) increased transcriptional activation. (d, e) Two additional control experiments are necessary to verify that the interaction does not involve binding to GAL4‐A or GAL4‐D. (d) The bait together with the GAL activation domain alone should not give rise to transcriptional activation. (e) The GAL4 DNA‐binding domain alone with the prey should not result in transcriptional activation.



Brachat A, Liebundguth N, Rebischung C, et al. (2000) Analysis of deletion phenotypes and GFP fusions of 21 novel Saccharomyces cerevisiae open reading frames. Yeast 16: 241–253.

Braun H and Suske G (1999) Vectors for inducible expression of dual epitope‐tagged proteins in insect cells. Biotechniques 6: 1038–1042.

Broder YC, Katz S and Aronheim A (1998) The Ras recruitment system: a novel approach to the study of protein–protein interactions. Current Biology 8: 1121–1124.

Dieci G, Bottarelli L, Ballabeni A and Ottonello S (2000) tRNA‐assisted overproduction of eukaryotic ribosomal proteins. Protein Expression and Purification 18: 346–354.

Du W, Vidal M, Xie JE and Dyson N (1996) RBF, a novel RB‐related gene that regulates E2F activity and interacts with cyclin E in Drosophila. Genes and Development 10: 1206–1218.

Fields S and Song O (1989) A novel genetic system to detect protein–protein interactions. Nature 340: 245–246.

Hager DA and Burgess RR (1980) Elution of proteins from sodium dodecyl sulfate–polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Analytical Biochemistry 109: 76–86.

Harlow E and Lane D (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Hoffmann A and Roeder RG (1991) Purification of his‐tagged proteins in non‐denaturing conditions suggests a convenient method for protein interaction studies. Nucleic Acids Research 19: 6337–6338.

Holt LJ, Enever C, De Wildt RM and Tomlinson IM (2000) The use of recombinant antibodies in proteomics. Current Opinion in Biotechnology 11: 445–449.

Keblusek P, Dorsman JC, Teunisse AF, et al. (1999) The adenoviral E1A oncoproteins interfere with the growth‐regulatory inhibiting effect of the cdk‐inhibitor p21 CIP1/WAF1. Journal of General Virology 80: 381–390.

Maina CV, Riggs PD, Grandea AG, et al. (1988) An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose‐binding protein. Gene 74: 365–373.

Martzen MR, et al. (1999) A biochemical genomics approach for identifying genes by the activity of their products. Science 286: 1153–1155.

Rigaut G, Shevchenko A, Rutz B, et al. (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nature Biotechnology 17: 1030–1032.

Shvarts A, Steegenga WT, Riteco N, et al. (1996) MDMX: a novel p53‐binding protein with some functional properties of MDM2. The EMBO Journal 15: 5349–5357.

Smith DB and Johnson KS (1988) Single‐step purification of polypeptides expressed in Escherichia coli as fusions with glutathione‐S‐transferase. Gene 67: 31–40.

Walhout AJ, Sordells R, Lu X, et al. (2000) Protein interaction mapping in C. elegans using proteins involved in vulval development. Science 287: 116–122.

Further Reading

Becker‐Hapak M, McAllister SS and Dowdy SF (2001) Tat‐mediated protein transduction into mammalian cells. Methods 24: 247–256.

Carlson M (2000) The awesome power of yeast biochemical genomics. Trends in Genetics 16: 49–51.

Clackson T, Yang W, Rozamus LW, et al. (1998) Redesigning an FKBP‐ligand interface to generate chemical dimerizers with novel specificity. Proceedings of the National Academy of Sciences of the United States of America 95: 10437–10442.

Golemis EA (ed.) (2002) Protein–Protein Interactions: A Molecular Cloning Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Gonzalez C and Bejarano LA (2000) Protein traps: using intracellular localization for cloning. Trends in Cell Biology 10: 162–165.

Kemmeren P, van Berkum NL, Vilo J, et al. (2002) Protein interaction verification and functional annotation by integrated analysis of genome‐scale data. Molecular Cell 9: 1133–1143.

MacDonald P (ed.) (2001) Two‐hybrid Systems: Methods and Protocols. Totowa, NJ: Humana Press.

Phizicky EM, Martzen MR, McCraith SM, et al. (2002) Biochemical genomics approach to map activities to genes. Methods in Enzymology 350: 546–559.

Rooney I, Butrovich K and Ware CF (2000) Expression of lymphotoxins and their receptor‐Fc fusion proteins by baculovirus. Methods in Enzymology 322: 345–363.

Smith DB (2000) Generating fusions to glutathione‐S‐transferase for protein studies. Methods in Enzymology 326: 254–270.

Vidal M and Legrain P (1999) Yeast forward and reverse ‘n’‐hybrid systems. Nucleic Acids Research 27: 919–929.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Dorsman, Josephine C(Jan 2006) Fusion Proteins as Research Tools. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0005685]