Tables for
Volume F
Crystallography of biological macromolecules
Edited by E. Arnold, D. M. Himmel and M. G. Rossmann

International Tables for Crystallography (2012). Vol. F, ch. 4.3, pp. 131-132   | 1 | 2 |

Section 4.3.5. The use of fusion proteins for crystallization

Z. S. Derewendaa*

aDepartment of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908–0736, USA
Correspondence e-mail:

4.3.5. The use of fusion proteins for crystallization

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Tags are routinely used in heterologous protein expression in order to enhance folding and solubility and to facilitate purification (Uhlen et al., 1992[link]; Malhotra, 2009[link]). They are either short oligopeptides, such as a hexahistidine, with unique affinity properties or well expressed and highly soluble proteins, such as GST (glutathione S-transferase), MBP (maltose-binding protein) or thioredoxin. The tags are inserted into the expression vectors downstream or upstream of the target protein and are often separated from it by a protease-sensitive linker sequence. They are cleaved proteolytically following expression and partial purification of the fusion protein and removed, leaving the isolated target ready for crystallization. However, in some cases the target protein may not be adequately soluble after cleavage or may resist crystallization. One of the possible solutions is to use the intact fusion protein in the crystallization screens in the hope that the carrier protein will both confer solubility on the construct and mediate crystal contacts. Not surprisingly, the canonical carrier proteins, all of which crystallize fairly easily on their own, constitute the obvious first choice. Using this strategy, the DNA-binding domain of DNA replication-related element-binding factor (DREF) was crystallized in fusion with Escherichia coli GST (Kuge et al., 1997[link]) and the U2AF homology motif (UHM) domain of splicing factor Puf60 was crystallized as a fusion with thioredoxin (Corsini et al., 2008[link]). A key problem limiting the utility of this technique is the inherent flexibility of a two-domain fusion protein, which is detrimental to its crystallizability. A possible solution to this problem is shortening the linker between the two proteins until a relatively rigid construct is identified (Smyth et al., 2003[link]). This approach was successfully pioneered for maltose-binding protein (MBP), which was used as a fusion chaperone to crystallize the human T-cell leukemia virus type 1 gp21 ectodomain fragment (Center et al., 1998[link]). The same strategy was employed in the crystallization of the ZP-N domain of ZP3 (Monne et al., 2008[link]), the islet amyloid polypeptide (IAPP; Wiltzius et al., 2009[link]) and the MATα1 homeodomain (Ke & Wolberger, 2003[link]). Recently, a genetically modified version of MBP (see below) was used as an N-terminal fusion chaperone to crystallize the signal transduction regulator RACK1 from Arabidopsis thaliana (Ullah et al., 2008[link]; Fig.[link]). Thus, MBP remains the most successful fusion chaperone for protein crystallization, even though the absolute number of proteins crystallized in this way is still limited.


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An example of the use of a fused carrier protein in crystallization: the crystal structure of the RACK1 protein (green) crystallized in fusion with an engineered variant of the maltose-binding protein (MBP; red); the major crystal contacts are mediated by MBP (PDB code 3dm0 ; Ullah et al., 2008[link]). The yellow spheres show alanines introduced by site-directed mutagenesis (see text for further details). Figs. and were generated using PyMOL ( ).

In addition to the canonical fusion chaperones, which were originally designed as affinity tags, other carrier proteins can be used to assist crystallization. For example, a module made up of two sterile α motif (SAM) domains has been engineered to polymerize in response to a pH drop and was shown to drive the crystallization of 11 target proteins in a pilot study (Nauli et al., 2007[link]). In another example, barnase, a secreted ribonuclease from Bacillus amyloliquefaciens, was recently used as a carrier protein for crystallization of the disulfide-rich protein McoEeTI (Niemann et al., 2006[link]).

An alternative to N- or C-terminal fusions is an insertion fusion, in which a carrier protein is inserted into a loop in the sequence of a poorly soluble target. To date, this approach has exclusively been used in membrane-protein crystallization and was initially pioneered for the E. coli lactose permease, in which cytochrome b562, flavodoxin and T4 lysozyme were tested as carrier proteins inserted into one of the loops (Privé et al., 1994[link]; Engel et al., 2002[link]). In this specific case none of these variants actually yielded useful crystals and the structure of lactose permease was eventually solved using crystals obtained using a variant containing the C154G mutation which stabilized a single conformation in complex with a lactose analogue (Abramson et al., 2003[link]). In contrast, a similar insertion fusion with T4 lysozyme replacing the third intracellular loop of the β2-adrenergic receptor was highly successful and yielded good-quality crystals that allowed structure determination at 2.4 Å resolution (Cherezov et al., 2007[link]; Rosenbaum et al., 2007[link]). This spectacular result attests to the potential of insertion-fusion proteins, but the method is not trivial as the constructs must be carefully evaluated for both structural and functional consequences of the insertion and a number of variants may have to be screened before a suitable one is identified.


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