A kind of mrrp protein, gene, carrier, engineering bacteria and application thereof
A technology of genetically engineering bacteria and proteins, applied in the field of protein biopolymers, to achieve the effects of high yield, uniform length and no chemical pollution
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Embodiment 1
[0023] Step 1: entrust the MRRP expression cassette sequence to Biotechnology Co., Ltd. to carry out the whole gene synthesis (the nucleotide sequence is shown in SEQ ID NO.2, and the amino acid sequence is shown in SEQ ID NO.1), and use restriction enzyme digestion ligation Methods The EcoR Ⅴ site on PUC57 was inserted, and then introduced into Escherichia coli DH5α strain by chemical method or electric shock method, and the MRRP expression strain was obtained by screening on carbenicillin-resistant medium and DNA sequencing.
[0024]Step 2: Inoculate the MRRP expressing strain clone into 1000mL self-induction medium, put it in a Erlenmeyer flask at a loading rate of 200mL / L, ferment and culture it at 16°C at a shaking speed of 220 rpm for 12h, and then press 35g Ratio of / 100mL fermentation broth Add ammonium sulfate gradually to the fermentation broth, precipitate in ice bath for 30min, then centrifuge at 12000×g, 4°C for 10min to collect the precipitate. The self-induction...
Embodiment 2
[0032] Step 1: The preparation of the MRRP expression strain is the same as in Example 1.
[0033] Step 2: Inoculate MRRP-expressing strain clones into 1000mL autoinduction medium, put them in a Erlenmeyer flask at a loading rate of 200mL / L, and culture at 40°C for 24h at a shaking speed of 220 rpm, and then press 35g / L Proportion of 100mL fermentation broth Add ammonium sulfate gradually to the fermentation broth, precipitate in ice bath for 30min, then centrifuge at 12000×g, 4°C for 10min to collect the precipitate. The self-induction medium formula is: yeast extract 6.0g / L, tryptone 10.0g / L, casein peptone 10.0g / L, glucose 2.0g / L, dipotassium hydrogen phosphate 8.0g / L, potassium dihydrogen phosphate 9.0g / L, ammonium phosphate 5.0g / L, magnesium sulfate 1.5g / L, calcium chloride 6.0mg / L, cobalt chloride 4.0mg / L, copper chloride 2.0mg / L, manganese sulfate 8.0mg / L, Sodium molybdate 7.0mg / L, boric acid 0.6mg / L, ferric chloride 7.0mg / L, zinc chloride 2.5mg / L, glycerin 10.0g / L, se...
Embodiment 3
[0040] Step 1: The MRRP expression strain is the same as in Example 1.
[0041] Step 2: Inoculate MRRP-expressing strain clones into 1000mL autoinduction medium, place them in a Erlenmeyer flask at a loading rate of 200mL / L, and culture at 28°C for 18h at a shaking speed of 220 rpm, and then press 35g / L Proportion of 100mL fermentation broth Add ammonium sulfate gradually to the fermentation broth, precipitate in ice bath for 30min, then centrifuge at 12000×g, 4°C for 10min to collect the precipitate. The self-induction medium formula is: yeast extract 4.0g / L, tryptone 7.5g / L, casein peptone 7.5g / L, glucose 1.0g / L, dipotassium hydrogen phosphate 6.0g / L, potassium dihydrogen phosphate 6.0g / L, ammonium phosphate 3.5g / L, magnesium sulfate 0.85g / L, calcium chloride 4.0mg / L, cobalt chloride 2.25mg / L, copper chloride 2.25mg / L, manganese sulfate 4.5mg / L, Sodium molybdate 5.0mg / L, boric acid 0.35mg / L, ferric chloride 4.0mg / L, zinc chloride 1.5mg / L, glycerin 5.5g / L, serine 2.25g / L, gl...
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