Automated Membrane Protein Crystallography

The Flexus Crystal IMP is the latest addition to the Flexus automated liquid handling product range. The Flexus Crystal IMP is specifically designed to automate the method of crystallizing proteins using lipidic mesophases. Without the use of automation, this method is almost impossible to perform manually and requires tremendous manual dexterity, excellent visual acuity and attention to detail in order to work with the nanoliter volumes involved.

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The basic Flexus automated instrument is modified according to the procedure described by Cherezov et al.(2004) and utilizes low volume washable pipetting probes to dispense the precipitant and a micropump for dispensing the pre-prepared protein/lipidic cubic phase. The deck is 1m long and provides ample space for housing precipitant reagents, the active wash station, the pipetting stage and humidified park position for the cubic phase syringe.

The lipidic cubic phase is prepared by mixing protein and lipid in two syringes as described by Cheng et al.(1998). The cubic phase is transferred to one syringe which is then mounted on the arm of the Flexus robot.

Once the prepared glass plate has been placed on the adjustable stage, the pipettes and syringe are primed ready for dispensing of the cubic phase and precipitant drops. Automation of this method ensures that the cubic phase bolus is centred within the precipitant drop. This is essential to prevent interference between the cubic phase/precipitant and the surrounding adhesive spacer.

The Flexus in meso robotic platform successfully automates an otherwise manual method, fraught with difficulties. On the Flexus platform, plates can be prepared in less than 7 minutes with excellent reproducibility, allowing high throughput trials with minimum amounts of protein required. The speed of this instrument now enables membrane proteins to be screened against more conditions and moves the bottleneck towards the inspection of the plates for crystals, rather than the preparation.

 Acknowledgements
Anachem would like to thank Professor Martin Caffrey and Dr Bo Sun, of the University of Limerick, for providing their technical expertise with this application, and for supplying the crystal images shown in this document.

Anachem is able to promote this Flexus application as a result of holding the exclusive license for this technology, from the University of Limerick, for the UK, Ireland, USA and Australia.
  

References
1. Cherezov, V., Peddi, A., Muthusubramaniam, L., Zheng, Y.F. and Caffrey, M. (2004) Acta Cryst. D60: 1795-1807
2. Cheng, A. H., Hummel, B. Qui, H. and Caffrey, M. (1998) Chem. Phys. Lip. 95: 11-21.