Expression method of TBX5 protein
An expression method and protein technology, applied in the fields of biotechnology and biopharmaceutical products, can solve problems such as low protein concentration, and achieve the effects of firm binding, increased yield, recovery rate and activity retention
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Embodiment 1
[0030] Embodiment 1 The preparation of the required solution of the present invention
[0031] 1) 10×TBE: Tris base 108g plus ddH 2 O 600ml is fully dissolved, add Boric acid 55g, 0.5M EDTA (pH8.0) 20ml, and dilute to 1L.
[0032] 2) 0.7% agarose gel: 0.7g Agarose, add 1×TBE100ml, boil in microwave.
[0033] 3) 30% acrylamide: Dissolve 29g of acrylamide and 1g of methylenebisacrylamide in 60ml of water, heat at 37°C until completely dissolved, add water to a final volume of 100ml, check that the pH of the solution is less than 7.0, and place it in a brown bottle at room temperature save.
[0034] 4) 10% ammonium persulfate: 1g ammonium persulfate dissolved in 10mlddH 2 O, stored at 4°C.
[0035] 5) 1.5mol / L Tris (pH8.8): 80ml ddH 2 Add 18.15g Tris base to O, add concentrated hydrochloric acid to adjust the pH to 8.8, and dilute to 100ml.
[0036] 6) 1mol / L Tris (pH6.8): Add 12.11g Tris base to 80ml ddH2O, add concentrated hydrochloric acid to adjust the pH to 6.8, and ...
Embodiment 2
[0047] Example 2 Induced expression of recombinant TBX5 protein:
[0048] After the recombinant expression plasmid Pet-30aSH-TBX5 was transformed into BL21, plated, picked and cloned into 1ml LB containing kanamycin and cultured overnight at 37°C with shaking. On the next day, pour all the bacterial solution into 200ml LB containing kanamycin, culture at 37°C and shake at 230rpm until the OD value is 1.5, change the culture medium to M9 minimal medium and continue to shake for 1 hour until the OD value reaches 2- 2.5, then add 0.5mM IPTG, and induce at two different temperatures of 37°C and 18°C respectively. Proteins induced at 37°C were sampled at 3h, 4h, 5h, 6h and 7h for SDS-PAGE analysis of expression. For proteins induced at 18°C, the OD value was detected after 12-16 hours, and samples were taken for SDS-PAGE analysis of expression. The results showed that the expression level of TBX5 protein at 37°C was much higher than that at 18°C, and the protein began to be e...
Embodiment 3
[0049] Example 3 High concentration expression method
[0050] Pick glycerol bacteria into 1ml LB containing kanamycin and culture overnight at 37°C with shaking. The next day, pour all the bacterial solution into 400ml LB containing kanamycin, culture at 37°C, 230rpm shaking, until the OD value is 1.5, convert the culture solution to M9 minimal medium at 2:1, and then add 2 times more Glucose, continue shaking culture for 1 h, until the OD value reaches 3.5-4, then add 0.5 mM IPTG, and induce at 37°C. During the whole induction process, the pH value of the culture medium was always checked and should be maintained above 6.0. After 3 hours, samples were taken for SDS-PAGE to detect protein expression. The results showed that the amount of protein obtained by the high-concentration method was higher than that obtained by ordinary induced expression ( figure 2 ). 1 indicates that no IPTG was added as a negative control, 2 indicates the protein expression induced by adding...
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