International
Tables for
Crystallography
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. 3.2, p. 96

Section 3.2.4. Common purification pitfalls and prioritized alternative strategies

J. A. Ernst,a,b D. G. Yansurac and C. M. Kothd*

aDepartment of Protein Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, USA,bDepartment of Protein Engineering, Genentech, 1 DNA Way, South San Francisco, California 94080, USA,cDepartment of Antibody Engineering, Genentech, 1 DNA Way, South San Francisco, California 94080, USA, and dDepartment of Structural Biology, Genentech, 1 DNA Way, South San Francisco, California 94080, USA
Correspondence e-mail:  koth.christopher@gene.com

3.2.4. Common purification pitfalls and prioritized alternative strategies

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3.2.4.1. Poor solubility of the target protein

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If the target protein does not remain soluble during purification, an obvious alternative strategy is to test different detergents or detergent combinations. Given that just a handful of different detergents have been used to solubilize and crystallize most membrane proteins, testing alternatives is usually not a daunting task. The most common method of detergent exchange is SEC (Fig. 3.2.3.1[link]).

The zwitterionic lyso-lipid mimetic detergents such as FOS-CHOLINE 12 (dodecylphosphatidylcholine) or 14 (FC12 and FC14; Anatrace, Maumee, Ohio, USA) will solubilize many membrane proteins (Eshaghi et al., 2005[link]) and there are a few reports of their use in crystallography [MscS mechanosensitive channel (Bass et al., 2002[link]); protein-conducting channel (van den Berg et al., 2004[link])]. Should solubilization screening with the more commonly used detergents prove ineffective, it may be possible to solubilize first with a `stronger' more lipid-like detergent, such as FC12, and then exchange to a non-charged detergent, such as DDM, during purification.

3.2.4.2. The isolated target protein does not crystallize

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Although an in-depth analysis of membrane-protein crystallization methods is beyond the scope of this chapter (see instead Chapter 4.2[link] ), there are many purification parameters that can be varied to improve crystallization outcomes should initial trials fail to yield `hits' or well diffracting crystals. Perhaps the foremost method is to exchange the protein into an alternative detergent or detergent mixture, typically by SEC, and repeat the crystal trials. Since the vast majority of membrane-protein structures have used one of only a handful of detergents (see above), it is likely that sampling even just a small number of other detergents would dramatically increase the likelihood of obtaining or optimizing crystals. Also, an increase in the alkyl-chain length by one methylene group (i.e. nonyl- versus octyl-β-D-glucoside) often leads to an increase in protein stability, and this too can improve crystallization outcomes (Wiener, 2004[link]). If significant covalent disulfide aggregation of the protein is observed during purification, alkylation of free cysteines should be considered during the early stages of target-protein isolation. Other parameters that could be varied include the solution pH, salt concentration and temperature, or supplementing with known ligands, to name just a few.

References

Bass, R. B., Strop, P., Barclay, M. & Rees, D. C. (2002). Crystal structure of Escherichia coli MscS, a voltage-modulated and mechanosensitive channel. Science, 298, 1582–1587.
Berg, B. van den, Clemons, W. M., Collinson, I., Modis, Y., Hartmann, E., Harrison, S. C. & Rapoport, T. A. (2004). X-ray structure of a protein-conducting channel. Nature (London), 427, 36–44.
Eshaghi, S., Hedrén, M., Nasser, M. I., Hammarberg, T., Thornell, A. & Nordlund, P. (2005). An efficient strategy for high-throughput expression screening of recombinant integral membrane proteins. Protein Sci. 14, 676–683.
Wiener, M. C. (2004). A pedestrian guide to membrane protein cryst­allization. Methods, 34, 364–372.








































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