Preparation method of RNasin
A buffer and protective agent technology, applied in the field of RNasin preparation, can solve the problems of difficulty in obtaining active RNasin, inapplicability to industrial production, and long expression cycle, so as to overcome expression difficulties, increase yield, and short expression cycle Effect
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[0017] The present invention relates to a kind of preparation method of RNasin, comprising:
[0018] 1) Escherichia coli transfected with the RNasin expression gene was induced and expressed in LB medium at 20°C to 26°C, and the induced expression system contained a reducing protective agent;
[0019] 2) The lysate of Escherichia coli is contacted with a refolding buffer; in the refolding buffer, the concentration of the main substance that plays a refolding role is ≤5M.
[0020] Routinely use LB medium at 37°C as the expression condition, but the expression is basically in the form of inclusion bodies. The present invention uses LB medium to express under low temperature conditions, and adds a reducing protective agent to the medium to promote the correct folding of RNasin, and the expressed inclusion bodies are not so compact and can be redissolved by low-concentration renaturation reagents.
[0021] In some embodiments, the temperature for inducing expression in step 1) is...
Embodiment 1
[0059] Optimization of expression conditions in embodiment 1
[0060] (1) Selection of medium and optimization of expression temperature.
[0061] Recombinant RNasin was expressed in Escherichia coli using BL21(DE3) strain. Take 100ul glycerol bacteria and inoculate them into 150ml LB medium. After activation at 37°C and 200rpm for 6h, the OD600 value reaches 0.3, then transfer. Take 10ml of the above seed bacteria liquid and transfer them to 500ml LB medium respectively (on the basis of the original LB medium Supplement 100g / L of glucose), TB medium and self-induction medium for culture, when the OD value reaches 0.6 at 37°C and 200rpm, the induction starts, the induction temperature is set at 23°C, 28°C and 37°C respectively, and the induction The time is 4h, 8h and 12h respectively.
[0062] The results showed that the expression levels were large and inclusion bodies were formed under all expression conditions in TB medium. In the self-inducing medium, the strain grew s...
Embodiment 2
[0065] The optimization of embodiment 2 purification conditions
[0066] (1) Cracking conditions
[0067] Since the intracellular environment of Escherichia coli is a reducing condition, when the cell is broken, RNasin is released, and it is easily oxidized in the external environment, so the lysis condition is particularly important. The most critical point is that a high concentration of DTT is required to maintenance, otherwise inactive RNasin will be formed. Since most of the expressed cells are inclusion bodies, the lysis buffer is: 50mM PB, 8% glycerol, 2mM EDTA, 8mM DTT, 200mM NaCl, pH7.2. Treat with 1mg / ml lysozyme, let stand at room temperature for 30min, and then centrifuge. The broken precipitate was washed twice with washing buffer, which was: 50 mM PB, 8% glycerol, 2 mM EDTA, 8 mM DTT, 200 mM NaCl, 0.5% TritonX-100, pH 7.0. The washed inclusion bodies were directly reconstituted with reconstitution buffer containing urea, the reconstitution buffer was: 50mM PB,...
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