Experiments on lysis of recombinant proteins of Escherichia coli from inclusion bodies
Experiments on lysis of recombinant proteins of Escherichia coli from inclusion bodies
Source: Laboratory Guide to Proteins and Proteomics
Operation method
basic program
Principle
Molecular cloning techniques enable bacteria to express proteins at high levels, making prokaryotic expression a very convenient system for obtaining recombinant proteins. Unfortunately these proteins tend to polymerize and precipitate in bacteria, forming insoluble inclusion bodies and thus difficult to purify. Inclusion bodies for non-bacterial proteins are particularly prevalent. Although there is no universal method that can be used for all proteins, there are many strategies that are applicable to the solubilization of inclusion body proteins. This protocol describes one of these strategies. There are many steps to consider when lysing inclusion body proteins: (1) cell lysis, (2) isolation of inclusion bodies, (3) washing of inclusion bodies, (4) lysis of inclusion bodies, and (5) reconstitution of recombinant proteins (if required).
Materials and Instruments
Bacterial cells expressing the target recombinant protein Move ① Lysed cells as described in Experimental protocol 6. ② Centrifuge the cell lysate at 23,000 g for 30 min at 4°C. ③ Pour out the supernatant and determine the wet weight of the precipitate. ④ Resuspend the precipitate with about 10 times the volume of washing buffer and stir the suspension for 1h at room temperature. ⑤ Centrifuge the mixture at 23000g for 30min at 4°C. ⑥ Remove the supernatant and resuspend the precipitate. ⑦ Repeat steps ④ ~ ⑥ more than 3 times (until the purity of recombinant protein >80%). ⑧ Dissolve the precipitate from step ⑦ by adding about 8 ml of dissolution buffer per gram of wet heavy precipitate (determined by step ③), and stir the mixture gently for 16~20 min at 4°C. Experimental tip: The protein concentration should not exceed 2~3mg/ml of other dissolution buffer: a.8 mol/L guanidine hydrochloride, 10 mmol/L DTT, 50 mmol/L Tris-Cl (pH 8.5) Lysed with 15 ml of solution per 12 to 15 g wet weight of collected cells. incubate the mixture at 37°C for 1 h. Denature the proteins as described in step ⑨a. b.8 mol/L urea, 2 mmol/L reduced glutathione/0.2 mmol/L oxidized glutathione. Use 9x volume of buffer per gram of wet weight inclusion body precipitation. Incubate the mixture at room temperature for 1 h. Do not exceed a protein concentration of 2.5 mg/ml. Complex the proteins as described in step ⑨,b. The optimal conditions required for the refolding of a specific target protein must be selected by preexperimentation.⑨ The replication conditions include: a. 25-fold dilution of the soluble precipitate with 1.5 μmol/L copper sulfate, 50 mmol/L Tris-Cl (pH 8.5) solution to a final concentration of 0.04 mol/L DTT, 0.24 mol/L guanidinium hydrochloride, and about 1.4 μg/ml of target protein. incubate the mixture at 20°C for 2h. The mixture was incubated for 2~4h at 25°C. b. 9-fold volume of 50mmol/L phosphate, 50mmol/L NaCl and 1mmol/L EDTA (containing 2mmol/L reduced glutathione/0.2mmol/L oxidized glutathione) was slowly added to the dissolved precipitate. c. The mixture was incubated for 2~4h at 25°C. d. The mixture was incubated for 2~4h at 25°C. ⑩ Recover the refolded protein with a concentration step suitable for the target protein (e.g. RP-HPLC, lyophilization or ultrafiltration). Caveat Wash buffer:100mmol/L Tris-Cl (pH 8.0)2~4.0 mol/L urea1% (v/v) TritonX-100 For more product details, please visit Aladdin Scientific website.
Compounding Buffer Lysis Buffer Wash Buffer
Centrifuge