Ⅰ. induced expression of recombinant protein

1.The transformed colonies were inoculated in 3 ml selective LB liquid medium at 37°C and 250 rpm / min for shaking overnight.

2.On the next day, 500 μ l of the overnight bacterial solution was inoculated into 10 ml (1:20) selective LB liquid medium. When the medium was shaken at 37°C and 250 rpm/min to the optical density (OD₆₀₀=0.6), 1 ml of the sample was taken as the pre induction sample, and 10000 g of the sample was centrifuged for 1 min to collect the bacterial precipitation, which was frozen at -20°C for preparation.

3.The final concentration of IPTG was 1 mm, 37°C, 250 rpm/min for 4-5 hours. Take 1 ml of sample as the induced sample, collect the bacterial precipitation by the same method, and freeze at -20°C for standby.

4.The bacteria were precipitated with 20 ~ 40 μl PBS (pH= 8) before and after induction. The same volume of 2xSDS sample buffer was added, boiled 5 min, SDS polyacrylamide gel (SDS-PAGE) was electrophoretic separated, and Coomassie brilliant blue staining for 3 hours, the results were decolorization.

5.Select the successful bacterial clones, expand the scale of induction, collect the bacterial precipitation, store at - 20 ° C, and prepare for the next step of analysis and purification.

Ⅱ. purification of recombinant protein

soluble identification of recombinant protein

1.The body weight of the bacteria collected after induction and culture according to the above method was suspended in the lysate 1 (Lysis buffer under native conditions), and then at -80°C refrigerate for 10 min.

2.Thawing in ice.

3.In the ice bath, the bacteria were broken 6 times by ultrasonic crusher, 10 sec each time, 10 sec intermittently, and the voltage was 200-300 v.

4.10000g, 4°C, centrifugation for 20min, take the supernatant (solution a), and store it at -20°C; in addition, dissolve the precipitate with the same cracking solution 1 (solution b), and store it at - 20°C for subsequent analysis.

5.SDS-PAGE and Coomassie brilliant blue staining were used to compare the solubility of the recombinant protein. If the induced protein is in solution a, it is soluble protein; if it is in solution B, it is insoluble protein.

separation and purification of the recombinant protein as non soluble protein (denatured condition)

1.Dissolve the bacterial precipitation in a proper amount of lysate 2 (Lysis buffer under denaturing conditions), stirring and blowing at room temperature to avoid foaming.

2.10000g, 4°C, centrifugation for 30 min, collection of supernatant.

3.The column was filled with Ni ntaagarose and connected to the Pharmacia low-pressure liquid chromatography system. A ₂₈₀ value was adjusted to the zero line by balancing Ni ntaagarose with 5 times column volume of cracking liquid 2.

4.Put a proper amount of supernatant into Ni NTA agarose column, and wash it with lysis buffer until A₂₈₀ value is lower than 0.01.

5.Wash the column with cleaning solution 1 and 2 (wash buffer 1 and 2) of 5-10 times of column volume respectively until A₂₈₀ value is lower than 0.01.

6.The recombinant protein was eluted with the eluent buffer. Under the monitoring of A₂₈₀ value, all eluents containing the recombinant protein after the peak line appeared were collected.

Ⅲ. refolding, lyophilization and quantification of recombinant protein

The purified protein was slowly dialyzed with urea solution with gradient reduction, and finally dialyzed with 0.01 × PBS. The dialyzed protein solution was freeze-dried to powder. Bovine serum albumin (BSA) was used as the standard to determine the content of protein.