Novel fusion protein NSCR5 and preparation method thereof
A fusion protein and protein technology, applied in the field of biopolymer research, can solve the problems of few applied research reports
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Embodiment 1
[0037] Step 1: Entrust the NSCR5 expression cassette sequence to Biotechnology Co., Ltd. for whole gene synthesis ( figure 2 , SEQ NO:2), and inserted into the EcoRⅤ site on PUC57 by restriction enzyme ligation method, and then introduced into Escherichia coli DH5α strain by chemical method or electric shock method, and was screened by carbenicillin-resistant medium and verified by DNA sequencing to obtain Fusion protein NSCR5 expression strain.
[0038] The restriction enzyme ligation method is as follows: digest the PUC57 plasmid with EcoRⅤ, use agarose gel electrophoresis to separate the linear fragment of PUC57 digested by EcoRⅤ, and then recover the linearized PUC57 plasmid with an agarose gel recovery kit. The artificially synthesized NSCR5 expression frame sequence was amplified with high-fidelity DNA polymerase, the NSCR5 expression frame fragment was also separated by agarose gel electrophoresis, and the NSCR5 expression frame fragment was recovered with an agarose ge...
Embodiment 2
[0047] Step 1: Refer to the method of Step 1 in Example 1 to obtain a strain expressing fusion protein NSCR5.
[0048] Step 2: Inoculate the fusion protein NSCR5 expressing strain clone into 200mL self-induction medium, culture at 40°C for 24h at a shaking speed of 220 rpm, and then centrifuge at 6000×g for 5min at room temperature to collect the bacteria. The self-induction medium formula is: yeast extract 5.0g / L, tryptone 10.0g / L, casein peptone 15.0g / L, glucose 1.5g / L, dipotassium hydrogen phosphate 10.0g / L, potassium dihydrogen phosphate 9.0g / L, ammonium phosphate 6.0g / L, magnesium sulfate 1.6g / L, calcium chloride 5.0mg / L, cobalt chloride 3.5mg / L, copper chloride 1.5mg / L, manganese sulfate 5.0mg / L, Sodium molybdate 8.0mg / L, boric acid 0.5mg / L, ferric chloride 8.0mg / L, zinc chloride 3.0mg / L, glycerin 10.0g / L, serine 2.0g / L, glycine 10.0g / L, tyrosine Acid 1.0g / L, isopropylthio-β-D-galactoside (IPTG) 300mg / L.
[0049] Step 3: The cells collected by centrifugation in step 2 ...
Embodiment 3
[0054] Step 1: Refer to the method of Step 1 in Example 1 to obtain a strain expressing fusion protein NSCR5.
[0055] Step 2: Inoculate the fusion protein NSCR5 expressing strain clone into 200mL self-induction medium, culture at 28°C for 18h at a shaking speed of 220 rpm, and then centrifuge at 6000×g for 5min at room temperature to collect the bacteria. The self-induction medium formula is: yeast extract 3.0g / L, tryptone 7.5g / L, casein peptone 10.0g / L, glucose 0.75g / L, dipotassium hydrogen phosphate 7.0g / L, potassium dihydrogen phosphate 6.0g / L, ammonium phosphate 4.0g / L, magnesium sulfate 1.0g / L, calcium chloride 3.5mg / L, cobalt chloride 2.0mg / L, copper chloride 1.0mg / L, manganese sulfate 3.1mg / L, Sodium molybdate 5.6mg / L, boric acid 0.3mg / L, ferric chloride 4.5mg / L, zinc chloride 1.7mg / L, glycerin 5.0g / L, serine 1.25g / L, glycine 6.0g / L, tyrosine Acid 0.5g / L, isopropylthio-β-D-galactoside (IPTG) 160mg / L.
[0056] Step 3: The cells collected by centrifugation in step 2 we...
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