The production of high quality protein crystals for
X-ray crystallography is a major obstacle to 3-D structure determination [1,
2], thus new methods to overcome this are continuously being designed. One such
method is the “oil barrier” technique which enables the
slowing down of the crystallisation process when poor crystals are formed due
to crystallisation progressing too rapidly [3]. This
method has been successful in improving crystal quality but has a few problems:
This technique involves manually placing a layer of oil (100-500 microlitre of oil) over the reservoir of vapour diffusion trials thereby consuming microlitre quantities of protein and reagents
An improved oil barrier method that overcomes the limitations of the above method is presented. The principle of the oil barrier remains the same, but the key difference is dispensing the oil in quantities of between 100-250 nanolitres directly on top of the drop in vapour diffusion trials. This creates a localised system of controlled evaporation between each drop and the precipitant reservoir. This improved method is easily customisable allowing for the use of nanolitre volumes of samples and making high throughput trials possible.
When tested on two medically related proteins – an antibody-peptide complex and alpha-crustacyanin; a noticeable difference in crystal number, size and quality was observed. Results show that this “oil-on-drop” method produces fewer crystals and these crystals grow to a larger size. Crystals of the antibody-peptide complex achieved diffraction of 3.6 Å compared to diffraction of 7 Å in previous studies. In addition, successful crystallisation of alpha-crustacyanin, which crystallises with 30% PEG-5000 as the main precipitant has produced fewer and larger crystals with the improved method that was not replicated with the traditional oil barrier method. Work with other proteins and precipitants is on-going.