The Crystallization Of Protein In The System Of Ionic Liquid-water

Protein crystallography is an interdisciplinary subject in the research of the structure and function of the protein. With the increasing demands of precise biomolecular 3-D structures, especially protein structures by X-ray diffraction, people pay more and more attention to the study of protein crystal growth. However, the crystallization of protein is very difficult and become a bottleneck in structural genomics, to some extent.Ionic liquids (ILs) with unique structure and outstanding physical and chemical properties have become the focus in the chemistry field. And it is also used in the field of life science, especially in the biocatalyst reaction as solvent or cosolvent. Recently, ionic liquids are also applied in protein crystallization.In this work, a new system of ionic liquid-water was tested for the protein crystallization. The procedure of crystallization could be controllable conducted in this system. The mechanisms for formations of controlled growth and the relationship among polymorph, morphology, quality and ionic liquid content were investigated. Moreover, the limitation effect of ionic liquid on the rate of crystal growth was disscussed from the view of kinetics.Ionic liquids of [BMIM]Cl, [BMIM]BF₄ and [BMIM]PF₆ were prepared through Direct Method and Two-step Method, respectively. And the chemical structures of the final products were characterized by Infrared spectra. For the further studies on the effects of the ILs on the crystal growth, the solubility of protein was mensurated with ultraviolet-visible light spectrophotometer by the way of BCA method. It was found that ionic liquid had similar effect as precipitants or additions, which was favourable for the single crystals. On the other hand, in order to understand their dissolved relationship among each components in the solution, the equilibrium solubility of a ternary system of [BMIM]BF₄, NaCl and buffer as well as the data of liquid-liquid equilibrium of that system are determined at 25℃and atmospheric pressure. It was used for the protein crystallization, and played a role in the conrol of local supersaturation.Two crystallization methods (hanging drop and sitting drop crystallization) were used for the lysozyme crystallization in the ionic liquids with different buffer/IL proportions. Lysozyme crystal belonged to the tetragonal space group P4₃2₁2 and its 3-D model was analyzed. The influence of growth conditions such as the kind of precipitants, solution supersaturation, ionic liquids content and temperature on polymorph, morphology, quality of crystals was discussed. It showed that [BMIM]BF₄ was more suitable for lysozyme crystallization than other ILs. [BMIM]BF₄ and NaCl were used as union-precipitants in the system of [BMIM]BF₄-water. More lager single crystals with controllable morphologies could be obtained due to the manageable crystal growth velocity. In addition, the morphology of lysozyme crystals were changed from bolck to rod and the L/D ratio increased with the proportion of IL.Futhermore, thaumatin crystal, which was belonged to the tetragonal space group P4₁2₁2, could be obtained with hanging drop crystallization. [BMIM]BF₄ was used as a precipitant, and the effect of ionic liquids content, temperature on polymorph and quality of crystals was investigated.On a whole, ionic liquid was a solvent with special structure and function. More importantly, aqueous two-phase system was appeared with diffusion of water molecules. As a new system, it might play an important role in promoting the growth of single crystals as opposed to polycrystals, in limiting nucleation, and in controlling the supersaturation of solution. The size, quality and growth rate of crystals might be controlled by adjusting content of ionic liquid.