High Throughput Approaches for Crystallisation of Biological Macromolecules

 

Christine L. Gee,a Richard D. Kidd,a Anna Aagaard,a Fiona M. McMillan,a Niranjali U. Gamage,a Paul R. Young,b David. A. Humeb and Jennifer L. Martina

 

aInstitute for Molecular Bioscience; bSchool of Molecular and Microbial Sciences,

The University of Queensland, St Lucia, 4072, Australia (c.gee@imb.uq.edu.au).

 

 

Structural genomics programs generate hundreds of proteins for structural analysis.  To keep pace with this, rapid and economical methods for high throughput screening of crystallisation conditions are required.  Here we present a protocol for semi-automated 96 well format screening as a high throughput approach for crystallisation.

We use a Packard multiPROBE II liquid handling robot to dispense solutions into a master 96 deep-well tray.  Once solutions are dispensed into 96 well format, multi-channel pipettes are used to dispense aliquots into microtitre plates and to set up hanging drops on plastic sealing film.  The plastic film is then sealed over the rims of the individual wells.  This method removes the need for grease sealing and individual manipulation of cover slips used in the standard 24 well crystallisation tray, thus saving time.  The smaller footprint trays are space saving, occupying almost half the space of the standard 24 well trays.  They are inexpensive and also save reagents, as they use one-tenth the reservoir volume of the old 24 well format, thus lowering costs.

The robot is also used to mix solutions for grid optimisation screens.  These are generally dispensed in 24 well format into a tissue culture plate and the crystallisation hanging drops are again dispensed with a multi-channel pipette onto sealing film. 

Several proteins have been crystallised in our laboratory using these techniques and these will be described in more detail.