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A purification method and membrane protein technology, applied in the field of membrane protein purification, can solve the problems of low extraction rate of target membrane protein, high cost, complicated operation, etc., and achieve the effect of improving purification effect, low cost, simple and fast method
Active Publication Date: 2021-08-20
CUSABIO TECH LLC
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[0004] The present invention aims at the technical problems such as low extraction rate of target membrane protein, complicated operation and high cost in the existing membrane protein purification method, and provides a membrane protein purification method method, by combining low-speed centrifugation and high-speed centrifugation, and screening detergents, the content of the target membrane protein in the total protein is effectively improved, and the operability and high cost of the membrane protein purification process are improved, that is, the present invention provides A membrane protein purification method with simple operation, low cost and high extraction rate
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Embodiment 1
[0043] Membrane protein purification of embodiment 1 Escherichia coli expression system
[0044] In this example, the membrane proteins expressed by the Escherichia coli expression system: endothelin receptor B protein (EDNRB) and Yersinia pestis virulence protein (YopE) were used to optimize the purification steps, as follows:
[0045] 1. Use conventional methods in this field to construct expression vectors according to the following table:
[0046]
[0047] 2. The host cell E.coli BL21(DE3) was cultured in LB medium, cultured at 37°C for 4 hours, and then the expression of the target membrane protein was induced with 0.5mMIPTG. After induction, the temperature was lowered to 18°C, and the bacteria were collected after overnight culture.
[0048] 3. Take the Escherichia coli cells above, after ultrasonically disrupting the cells, centrifuge at 4°C and 490g at low speed for 15 minutes, separate the supernatant and precipitate for SDS-PAGE detection, then take the supernatant...
Embodiment 2
[0067] Membrane protein purification of embodiment 2 mammalian cell expression system
[0068] In this example, on the basis of the optimization steps in Example 1, the purification effect of the method on the membrane protein of the mammalian cell expression system: B lymphocyte antigen (CD20) is verified, as follows:
[0069] 1. Use conventional methods in this field to construct expression vectors according to the following table:
[0070]
[0071] 2. The day before transfection, divide the cells into 1x10 6 cells / mL, the parameters of transfection cells were optimized, and the optimal transfection parameters were: the ratio of plasmid DNA to PEI (MW 25000, Polysciences) was 1:3. Plasmids are per 1x10 6 Add 1 μg of plasmid DNA to cells. On the day of transfection, dilute the plasmid and PEI separately with medium, then pour the PEI into the plasmid. After incubating at room temperature for 10 min, pour the mixed plasmid PEI mixture into the cells. After 2 days of ex...
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Abstract
The invention discloses a method for purifying a membrane protein. The method comprises the following steps: inducing a target membrane protein to be expressed in host cells; crushing the host cells, centrifuging at a low speed of 490-1000g, taking a supernatant, centrifuging the supernatant at at a high speed of 30000g, and taking a precipitate; re-suspending the precipitate by using a buffer, then adding a detergent, shaking and dissolving, centrifuging, and taking a supernatant; and purifying by adopting affinity chromatography, eluting by using an eluent containing a buffer, inorganic salt, dodecyl-beta-D-maltoside, imidazole and glycerol, and carrying out ultrafiltration and centrifugation on the eluent to obtain the target membrane protein. Aiming at the technical problems of membrane protein denaturation, low target membrane protein extraction rate, complex operation, high cost and the like caused by a detergent in the existing membrane protein purification method, low-speed centrifugation and high-speed centrifugation are combined, and the detergent is selected, so that the content of the target membrane protein in total protein is effectively increased, and the membrane protein purification method provided by the invention is simple to operate, low in cost, high in extraction rate and wide in applicability.
Description
technical field [0001] The invention belongs to the technical field of protein purification, and in particular relates to a membrane protein purification method. Background technique [0002] Membrane proteins are the main bearers of biomembrane functions, their expression levels are relatively low, and they are usually purified as protein-lipid-detergent complexes. The solubility of this complex in aqueous environments allows the use of essentially the same separation techniques as for water-soluble proteins, with the exception that membrane protein purification requires the addition of detergents in solution, and that protein-detergent complexes are dynamic, In the absence of free detergent molecules, the detergent molecules dissociate immediately. Water-soluble proteins with 6-histidine tags can be directly purified according to standard procedures, but membrane proteins with 6-histidine tags are weakly bound to immobilized metal ion affinity chromatography (IMAC), makin...
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