The first bottleneck in obtaining the crystal structure of the protein is the preparation of a large amount of purified protein (> 10mg), whose concentration is usually above 10mg / ml, and on this basis, the crystallization conditions are screened. Using recombinant gene technology, specific genes are embedded in expression vectors in a clone manner. This vector usually has the characteristics of easy regulation. Afterwards, the carrier with specific genes is sent into a fast-growing bacterial cell, such as Escherichia coli. While the bacterial cell grows rapidly, it also produces a large amount of protein expressed by the gene on the expression vector. Generally speaking, the higher the purity of the protein is more likely to form crystals, so the step of protein purification becomes an important determinant.
After obtaining a high-purity protein solution, the next step is the cultivation of crystals. The formation of protein crystals is similar to the formation of crystals of other compounds. It is slowly produced in a saturated solution. The conditions for each protein to grow crystals are different. There are many variables that affect the formation of crystals, including chemical variables, such as pH, precipitation Agent type, ion concentration, protein concentration, etc .; physical variables, such as the rate and temperature at which the solution reaches a supersaturated state; and biochemical variables, such as the metal ions or inhibitors required by the protein, the polymerization state of the protein, etc. Electrical point, etc., are all test conditions during crystal growth. So far, there is no set of theories that can predict the conditions of crystallization, so it is necessary to continuously test the combination of various crystal growth solutions to obtain a perfect single crystal.
The cultivation of protein crystals is usually achieved by the principle of Vapor Diffusion Method; that is, a solution containing a high concentration of protein (10-50mg / ml) is added to an appropriate solvent to slowly reduce the solubility of the protein, so that When it is close to spontaneous precipitation, protein molecules will form crystals under a neat stack. For example, we dissolve protein in a low-concentration (~ 1.0M) ammonium sulfate solution and place it in a closed container containing a high-concentration (~ 2.0M) ammonium sulfate solution. The gas phase equilibrates and can slowly increase The concentration of ammonium sulfate in the protein solution further achieves the purpose of crystallization.
Protein crystals are not significantly different from other crystals in appearance, but inside the crystals, they are very different. Generally speaking, in addition to protein molecules, protein crystals, the other space is filled with about 40% to 60% of the aqueous solution, its liquid composition not only makes the crystal fragile, but also easy to make protein molecules in the lattice arrangement irregular The situation arises, which causes difficulties in crystal processing and collection of diffraction data. However, due to the characteristics of high water content, the state of protein molecules in the crystal is very similar to that in the aqueous solution. Therefore, the protein structure solved by the crystal can basically be regarded as the structure in the natural state.
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