The Study On Vacuum Mambrane Crystallization Of Protein

The important role of X-ray crystallography in molecular biology and hence the need to produce diffraction quality protein crystals have been well documented. However, the properties of biomacromolecule are different from inorganic and organic compound with smaller molecular weights, which include electrostatics property, more complicated shape, the instability of physical and chemical properties and so on. These make crystallization process of biomacromolecule more complex. 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. Firstly, the bench study of lysozyme by vacuum membrane crystallization, using microporous hydrophobic PVDF membrane, was carried out. The influences of vacuum tightness of downstream side, protein concentration, flow velocity of feed solution, additive, pH and ionic strength on transmembrane flux and the shape of lysozyme crystal were examined. The results showed that the vacuume should be controlled under 0.015MPa; the optimum lysozyme concentration and flow velocity were 20mg·mL^-1 and 288μm·s^-1 respectively for vacuum membrane crystallization; the addition of additives such as glycerol and DMSO into the crystallization solutions could increase effectively the transmembrane flux and improve the quality of crystals; Under the same ionic strength, the higher transmembrane flux was obtained under lower pH, different crystal forms could be obtained under different pH.Secondly, mbrane crystallization of several proteins (bovine serum albumin V 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, transmembrane 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: bovine serum albumin V crystals with good quality could be obtained at about 17-25℃, with KH₂PO₄-Na₂HPO₄ solutions at pH6.3 as buffers and NaCl or PEG6000 as precipitant. In addition, the addition of additives such as sodium potassium tartrate could not only increase effectively the transmembrane flux and improve the quality of crystals, which lead to shorter induction time period and larger crystals in size.