The Study On Membrane Crystallization Of Protein

With the development of recombinant DNA technology and expression system, quantity of known protein increases. X-ray crystallography is a good method for determining a protein 3D structure at atomic resolution, while application of X-ray crystallography is absolutely dependent on crystals of the macromolecule, which requires crystals have adequate size (larger than 0．1mm) and quality to permit accurate data collection. Unfortunately, many macromolecules are reluctant to crystallize and, usually, their lattice is not regular enough to provide diffraction data. Generally, the crystallization methods of small molecules are not suitable for crystallization of biomacromolecules. Therefore, crystal growth on biomacromolecules is of a concern due to the difficulty of some biomacromolecules crystallization, which makes new crystallization method and technique very necessary. Crystals of biomacromolecules are obtained by membrane crystallization technology, which are suitable for X-ray diffraction.In this thesis, firstly, osmotic membrane crystallization of several proteins (lysozyme, papain, albumin egg, bovine serum albumin and lipase) was carried out. The influence of various membrane crystallization conditions on osmotic membrane crystallization process was studied, and crystallization conditions of several proteins were optimized. In addition, transmembranc flux was evaluated, and the mechanism of precipitant and additive during osmotic membrane crystallization of protein was discussed. At last, crystals obtained were analyzed. Results showed that:For osmotic membrane crystallization of lysozyme, NaCl and NaSCN are found to be good precipitants. And the addition of additives such as glycerol, PEG4000 and PEG6000 into the crystallization solutions can increase effectively the transmembrane flux and improve the quality of crystals, which lead to shorter induction time period and larger crystals in size.For continuous osmotic membrane crystallization of lysozyme, protein concentration was found to have a direct influence on the crystallization process; too high protein concentrations let to the appearance of clear granular precipitation and a decline in the yield of crystals produced; whereas very low protein concentration lead to very small transmembrane flux and long crystallization induction time. Generally, transmembrane flux decreases with increasing lysozyme concentration. Within the range of lysozyme concentrations examined, the range from 20mg/mL to 30mg/mL, especially 20mg/mL, are found to be better than other concentrations for continuous membrane crystallization, in which the quantity of crystal nucleations can be controlled and crystals yielded are larger.Precipitant (NaCl) concentration is found to have an important influence on the continuous osmotic membrane crystallization process of lysozyme. Increasing the NaCl concentration from 4 to 6．5% wt/vol resulted in a reduction in the induction time; increasing further the precipitant concentration from 6．5 to 9% resulted in a substantial increase in the induction time period accompanied by a decline in the solvent transmembrane flux. When precipitant concentration is higher than 9%, there occurs a mass of precipitation in the crystallization solutions. Therefore, precipitant (NaCl) concentrations within the range from 4-6．5% wt/vol. were considered as optimum for better osmotic membrane crystallization of lysozyme solutions. For continuous osmotic membrane crystallization of 20mg/mL lysozyme, with 4%NaCl as precipitant, when concentration and flow velocity of stripping solutions MgCl₂ were 25% and 4319μm/s, the optimal flow velocity of crystallization solutions was 288μm/s.For the effects of MgCl₂ concentrations and flow velocities on the membrane crystallization process, when the flow velocity of MgCl₂ is in the range of 2880～4319μm/s, the transmembrane flux and the induction time period were the best for protein crystallization, and perfect crystals were obtained. Furthermore, for MgCl₂ concentrations, it was found that the transmembrane flux increases with the increase of MgCl₂ concentrations. Higher stripping solution concentration was found to reduce the induction time. However, MgCl₂ concentration has to be controlled as higher concentrations may affect produced crystals quality. Higher MgCl₂ concentration may increase the number of crystals formed but on the other hand the obtained crystals were of smaller sizes. As a conclusion an MgCl₂ stripping solution concentration within the range of 20～25% was found to give the best results.For the effect of pH and ion strength on membrane crystallization, it can be found that under the same pH, the higher transmembrane flux was obtained under higher ion strength. Crystal form and habit were mainly influenced by the pH of the crystallization solutions, while the ion strength has an important influence on the transmembrane flux, and the effect of the ion strength on crystals form and habit was very little. Various crystals forms were obtained at various pH levels. It can be found that two crystal forms (tetragonal and orthorhombic forms) were obtained in various crystallization conditions when sodium acetate at pH4．7 was used as buffers. Crystal form changes from tetragonal to orthorhombic with increasing pH. In addition, crystals transit from several to single crystal form with increasing pH. Crystallization at pH4．7 resulted in the formation of tetragonal and orthorhombic crystals, while only orthorhombic crystals were grown at pH6．5 and 8．3．It seems that crystals of orthorhombic forms are easier to grow than other crystal forms.At last, the changes of crystallization solutions concentration with time and the induction time were investigated. It can be found that, when concentration of NaCl is 4% and 6．5%, especially 4%, as a whole, lysozyme concentration in the crystallization solutions increases with time, while lysozyme concentration in the crystallization solutions decreases with time under 9% NaCl. In conclusion, the induction time under 6．5% NaCl is the shortest among the NaCl concentrations tested while other conditions were maintained as in previous experiments.For static osmotic membrane crystallization of papain, (NH₄) ₂SO₄ and Na₃PO₄ are found to be effective precipitants. At 17℃, when 0．1M sodium acetate buffer at pH 4．7 was used as buffers, papain crystals with good quality were obtained in the certain range of precipitants concentration(Na₃PO₄: 4～7%; (NH₄) ₂SO₄: 4～10%). The changes of papain concentration with time in crystallization solutions were monitored, and the influences of precipitants and additives on crystallization process were also investigated. It can be found that when using Na₃PO₄ as precipitant, papain crystals with good quality can be obtained under lower papain concentration. In addition, additives such as PEG600 and PEG4000 can enhance greatly transmembrane flux, reduce induction time period and improve the quality of crystals. Crystal size distribution showed that, papain crystals obtained meet for X-ray diffraction analysis.In addition, static osmotic membrane crystallization of several proteins (albumin egg, bovine serum albumin and lipase) was also studied. For static osmotic membrane crystallization of albumin egg, when PBS solutions at pH7．3 was used as buffers, albumin egg was 30mg/mL, with 3～7% PEG6000 as precipitant and 20%MgCl₂ as stripping solutions, albumin egg crystals with large size and good crystal habit were obtained; For static osmotic membrane crystallization of bovine serum albumin, in the range of 16～25℃, when PBS solutions at pH6．3 was used as buffers, NaCl, PEG600 and PEG6000 were effective precipitant. And the addition of additives such as PEG and DMSO into the crystallization solutions can increase effectively the transmembrane flux and improve the quality of crystals; For static osmotic membrane crystallization of lipase, when PBS solutions at pH6．3 was used as buffers, and NaSCN, glycerol, PEG400 and PEG600 were used as precipitant, lipase crystals were yielded, in which crystals yielded using glycerol as precipitant have best quality.At last, vacuum membrane crystallization of lysozyme was investigated. When lysozyme concentration was 20mg/mL and the flow velocity of crystallization solutions was 288μm/s, 4%NaCl was used as precipitant, under vacuum of 0．015MPa, lysozyme crystals with good quality were yielded. Under higher flow velocity of crystallization solutions, size of crystals yielded was smaller, and crystal aggregated in a degree and was easy to brittle.