Protein Crystallisation in Action
Purified NK1 protein was supplied by Dr J. Hepple (Medical Research Centre, Cambridge), and the HR1 domain was purified as described in Chapter 2. The HR1b and NK1 proteins were concentrated to about 20 and 18 mg/ml respectively, aliquoted in 60 - 100 ml fractions and stored at -20 . Prior to a set of crystallisation experiments, one or more protein aliquots were thawed on ice, and then centrifuged at 11,000 rpm for 5 min at 4 to sediment possible aggregates formed during storage. Repeated thawing and freezing of the protein solution was avoided.
A sparse matrix approach (Crystal Screen I and II by Hampton Research) was used with both NK1 and HR1b proteins in the initial crystallisation trials. The list of reagent formulations for both screens is provided in Appendix B. The precipitant solutions for any other crystallisation trials were prepared using chemical compounds of high-purity grade (Sigma, Hampton research, Fluka) and water produced by Milli-Q system (Millipore). Solutions of PEG, buffers and some of the salts were made with 0.05 % NaN (Sigma) to prevent bacteria growth during storage and sterile filtered (0.22 mm filters, BioRad).
Micro-batch crystallisation trials were performed in Terazaki-type microtiters (Nunc Intermed), schematically shown in figure 2a. The wells of a plate were covered with paraffin oil (Fisher), then 1 ml of protein and 1 ml of precipitant solution were pipetted into each well. Manual mixing was avoided to prevent mechanical stress to the protein. The plates with crystallisation trials were left for equilibration either in the temperature controlled room at 22 or in the incubators at 5 (Sanyo) depending on the experiment set-up. The micro-batch trials were usually discarded after two to three weeks. The observations were carried out using a stereomicroscope (Leica) with fiber-optic light source to prevent overheating of the crystallisation trials.
24-well tissue culture plates (Linbro plates, ICN Biomedicals) were used for vapour-diffusion crystallisation trials. Glass cover slips (BDH Co.) were siliconised to minimise the contact area of the drop with the glass surface of cover slip. Siliconisation was performed by immersing the cover slips into 2 % (v/v) dimethyldichlorosilane (solvent 1,1,1-trichloroethane) for 5 minutes, then in deionised water and finally in ethanol, before drying them overnight. Typically, 1 ml of crystallisation solution containing mixture of precipitant agents and buffer was pipetted into each well of a Linbro plate. The rim of the well was covered with a layer of vacuum grease, and then sealed by placing the cover slip on it. A drop of protein solution with volume ranging from 2 to 4 ml was pipetted onto the prepared cover slip, and an equal volume of precipitant solution was then added. Mechanical mixing of the drop was not applied. The cover slip was then carefully inverted and placed on the top of the well and gently pressed around the rim to ensure good sealing.
The crystals were analysed by SDS PAGE. Several large crystals were harvested from the crystallisation drops where they were originally formed and washed in a 6 - 10 ml drop of stabilising solution. The stabilising solution typically contained 5 % more precipitant agent (e.g. PEG 6000 and PEG 4000, in the case of HR1b and NK1 proteins respectively) than the reservoir solution of the corresponding crystallisation trial. After 2 - 4 minutes, the crystals were transferred again into a fresh drop of stabilising solution. The procedure was repeated 4 - 5 times to ensure that no layer of the original protein solution was still present on the surface of the crystal. The crystals were then dissolved in 5 ml of SDS PAGE protein loading buffer and loaded onto the gel.