Mixed carbon source induced pichia pastoris expression recombinant batroxobin and purification method thereof

A Pichia pastoris and mixed carbon source technology, which is applied in the field of efficient production of medicinal enzyme proteins, can solve problems such as difficult removal, confusion of different types of snake venom, and difficulty in quality control

Pending Publication Date: 2021-12-31
BEIJING GREATSUN BIO PHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Natural snake venom products have several defects: first, snake venom comes from different places and different seasons, quality control is difficult, and there is even a risk of confusing different types of snake venom; second, snake venom itself contains a variety of toxins, which is difficult to obtain in the later purification process Removes clean, trace amounts of impurities and toxins that may cause damage to patients taking medication
[0007] In addition

Method used

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  • Mixed carbon source induced pichia pastoris expression recombinant batroxobin and purification method thereof
  • Mixed carbon source induced pichia pastoris expression recombinant batroxobin and purification method thereof
  • Mixed carbon source induced pichia pastoris expression recombinant batroxobin and purification method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Inoculate 1 ml of frozen working seeds of Pichia batroxobin and Pichia engineered bacteria into a 500 ml Erlenmeyer flask containing 100 ml of YPD medium. Under the conditions of 30° C. and 220 rpm, aeration and shaking were carried out for 20 hr. Measure its OD the next day 600 The value is 12.4. Add 24ml YPD medium to dilute and mix well. Take 10ml of the diluted first-class species and inoculate it into 500ml Erlenmeyer flasks containing 100ml of YPD medium, a total of 10 flasks. 30° C., 220 rpm, shake culture for 5 hours, and then inoculate the fermenter as fermentation seeds.

[0043] Prepare 20 liters of FM22 medium, and adjust the pH of the prepared basal salt medium to 4.5 with KOH. Divide into four 10-liter fermenters in 5-liter portions and autoclave. Sterilization conditions: 121°C, 20min. After sterilization, cool down to 30°C and add 10ml of PTM4 to each can. Ammonia water adjusts the pH value of the fermentation medium to 4.5. Maintain a stirring r...

Embodiment 2

[0051] Inoculate 1 ml of frozen working seeds of Pichia batroxobin and Pichia engineered bacteria into a 500 ml Erlenmeyer flask containing 100 ml of YPD medium. Under the conditions of 30° C. and 220 rpm, aeration and shaking were carried out for 20 hr. Measure its OD the next day 600 The value is 13.1. Add 31ml YPD medium to dilute and mix well. Take 10ml of the diluted first-class species and inoculate it into 500ml Erlenmeyer flasks containing 100ml of YPD medium, a total of 10 flasks. 30° C., 220 rpm, shake culture for 5 hours, and then inoculate the fermenter as fermentation seeds.

[0052] Prepare 20 liters of FM22 medium, and adjust the pH of the prepared basal salt medium to 4.5 with KOH. Divide into four 10-liter fermenters in 5-liter portions and autoclave. Sterilization conditions: 121°C, 20min. After sterilization, cool down to 30°C and add 10ml of PTM4 to each can. Ammonia water adjusts the pH value of the fermentation medium to 4.5. Maintain a stirring r...

Embodiment 3

[0060] Inoculate 1 ml of frozen working seeds of Pichia batroxobin and Pichia engineered bacteria into a 500 ml Erlenmeyer flask containing 100 ml of YPD medium. Under the conditions of 30° C. and 220 rpm, aeration and shaking were carried out for 20 hr. Measure its OD the next day 600 The value is 13.5. Add 35ml YPD medium to dilute and mix well. Take 10ml of the diluted first-class species and inoculate it into 500ml Erlenmeyer flasks containing 100ml of YPD medium, a total of 10 flasks. 30° C., 220 rpm, shake culture for 5 hours, and then inoculate the fermenter as fermentation seeds.

[0061] Prepare 20 liters of FM22 medium, and adjust the pH of the prepared basal salt medium to 4.5 with KOH. Divide into four 10-liter fermenters in 5-liter portions and autoclave. Sterilization conditions: 121°C, 20min. After sterilization, cool down to 30°C and add 10ml of PTM4 to each can. Ammonia water adjusts the pH value of the fermentation medium to 4.5. Maintain a stirring r...

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Abstract

The batroxobin is a hemostatic which is widely used at present. The invention relates to production of recombinant batroxobin by continuous fermentation induction of a mixed carbon source of methanol and glucose. And the recombinant batroxobin in a fermentation supernatant is purified by adopting an ionic membrane filtration and high-recovery-rate chromatography method.

Description

technical field [0001] The invention belongs to the technical field of microbial fermentation engineering, and in particular relates to the efficient production of medicinal enzyme proteins by means of continuous fermentation and mixed induction. Background technique [0002] In the fermentation production of gene recombinant protein drug engineering bacteria, different culture methods are often used to control the growth of microbial cells. It is very important to explore the best fermentation culture formula, feed ingredients and control parameters for the expression of the target protein. [0003] The Pichia pastoris expression system is a mature exogenous protein expression system, which has a high level of exogenous protein expression ability, and has the characteristics of rapid growth of Escherichia coli, convenient large-scale fermentation and post-translational modification of mammalian cells. Pichia pastoris can use methanol as the only carbon source, and methanol ...

Claims

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Application Information

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IPC IPC(8): C12N9/64C12N15/81C12N1/19C12R1/84
CPCC12N9/6418C12N15/815C12N1/16C12N2800/22C12N2800/102
Inventor 姜松赵文辉滕灵艳李红杨子义
Owner BEIJING GREATSUN BIO PHARM TECH CO LTD
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