Denaturation and renaturation method of recombined protein
A protein and denaturant technology, applied in the biological field, can solve the problems of affecting product recovery rate, lower refolding rate of recombinant protein, and high cost
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Embodiment 1
[0066] The extraction and renaturation process of embodiment 1 recombinant green fluorescent protein
[0067] 1. Engineering bacteria culture and expression
[0068] Engineering bacteria expressing recombinant green fluorescent protein were obtained from the Institute of Genetics, Fudan University.
[0069] Inoculate the E. coli strain that can induce the expression of the target protein into 5ml LB medium (containing 50ug / ml Kan) the day before, and culture overnight at 37°C and 200rpm. The next day, transfer the cultured bacterial solution to 500ml of LB (Kan containing 50μg / ml) medium, cultivate to OD at 37°C, 200rpm 600 =0.6~0.8. IPTG was added to the cultured bacterial solution to a final concentration of 1 mM, 37° C., 200 rpm, and induction culture was continued for 4 hours.
[0070] 2. Refolding of inclusion body proteins
[0071] The protein purification process is as follows:
[0072]
[0073] 1. Bacterial fragmentation
[0074] (1) Take 3.0 grams of genetica...
Embodiment 2
[0094] The extraction refolding process of embodiment 2 recombinant Schistosoma japonicum elastase (SjE)
[0095] 1. Engineering bacteria culture and expression
[0096] The engineered bacteria expressing Schistosoma japonicum elastase were obtained from the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention. The strain cultivation and the protein expression process were the same as in Example 1.
[0097] 2. Refolding of inclusion body proteins
[0098] 1. Bacterial fragmentation
[0099] (1) Take 3.0 grams of genetically engineered bacteria and add 30ml 20mmol / L Tris-HCL (trishydroxymethylaminomethane-hydrochloric acid, pH8.0), stir and wash the bacteria twice for 30min, centrifuge at 4°C at 7000rpm×10min, discard the supernatant .
[0100] (2) Add lysate buffer to wet bacteria (2) [50mmol / L Tris-HCL, 1mmol / L EDTA, pH8.0 buffer] 30ml, use ultrasonic to break, ultrasonic time 5s, ultrasonic interval 5s, ultrasonic times 140 Repeat ...
Embodiment 3
[0115] Example 3 The extraction and refolding process of human HSPC067 protein (HSPC067)
[0116] 1. Engineering bacteria culture and expression
[0117] Genetically engineered bacteria and inclusion bodies expressing human HSPC067 protein (HSPC067) were obtained from Shanghai Huaguan Biochip Co., Ltd. The strain cultivation and the protein expression process are basically the same as in Example 1.
[0118] 2. Refolding of inclusion body proteins
[0119] 1. Bacterial fragmentation
[0120] (1) Take 3.0 g of genetically engineered bacteria and add 30 ml of 20 mmol / L Tris-HCL, stir and wash the bacteria twice for 30 min, centrifuge at 4°C at 7000 rpm for 10 min, and discard the supernatant.
[0121] (2) Add lysate buffer to wet bacteria (2) [50mmol / L Tris-HCL, 1mmol / L EDTA, pH8.0 buffer] 30ml, in ultrasonic, ultrasonic time 5s, ultrasonic interval 5s, ultrasonic frequency 140 times , until the liquid becomes thin and frothy. The bacterial fragmentation rate of microscopic ...
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