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Method for preparing polypeptide by utilizing thermally-stabilised fusion protein

A technology for thermostable proteins and fusion proteins, which is applied in the field of using thermostable fusion proteins to prepare polypeptides, can solve the problems of high energy consumption of ultrasonic method breaking cells, unsuitable for industrial scale-up production, inability to achieve the effect of scale, and long time required to achieve suitable The effect of mass production, low cost and low equipment requirements

Active Publication Date: 2019-12-13
CHENGDU ENZPRO BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many methods for breaking cells, such as ultrasonic method, high-pressure homogenization, organic solvent method, enzymatic method, freeze-thaw method, etc. Each of these methods has certain limitations. The ultrasonic method consumes a lot of energy and is not suitable for industrial scale-up. In production, the freezing and thawing method takes a long time to break the cells, and the heat generated by the high-pressure homogeneous breaking is easy to cause damage to the enzyme. The disadvantages of the organic solvent method are also very obvious. The added organic solvent will bring additional environmental protection pressure. At the same time Organic solvents may also cause damage to the target polypeptide, and the enzymes used for cleavage may degrade the proteins released after cell cleavage
The existing cell-breaking methods cannot achieve the best results in terms of cost or scale, so it is urgent to find a low-cost and easy-to-industrial production of cell-breaking extraction methods

Method used

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  • Method for preparing polypeptide by utilizing thermally-stabilised fusion protein
  • Method for preparing polypeptide by utilizing thermally-stabilised fusion protein
  • Method for preparing polypeptide by utilizing thermally-stabilised fusion protein

Examples

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preparation example Construction

[0067] The specific polypeptide preparation method of the present invention includes:

[0068] First-level seed culture conditions: inoculation amount: 0.1%; culture temperature 37℃, shaker rotation speed 220rpm, culture 7-9h, to OD 600 It is 3.0-7.0.

[0069] The composition of the primary seed culture medium:

[0070] raw material Composition ratio Tryptone1％ Yeast extract0.5％ Sodium chloride1％

[0071] Secondary seed culture conditions: inoculum amount: 0.1%; culture temperature 34℃, shaker speed 220rpm, culture 10-12h, to OD 600 For 2.0-4.0.

[0072] The composition of the secondary seed culture medium:

[0073] raw material Composition ratio Diammonium Phosphate0.200％ Potassium Dihydrogen Phosphate0.675％ Citric acid monohydrate0.093％ Magnesium Sulfate Heptahydrate0.070％ Trace solution0.25％ Dextrose monohydrate2％ pH6.8

[0074] The composition of the tank fermentation medium:

[0075]

[0076]

[0077] The composition of the trace element solution (Trace solution) i...

Embodiment 1

[0123] Example 1. Fermentation and expression of the fusion protein mTrA-Arg by recombinant E. coli 34 -GLP-1(7-37) (SEQ IDNO.4)

[0124] The prepared recombinant E. coli (E.coli BL21(DE3) / pET28a-mTrA-Arg 34 -GLP-1(7-37)) 2000ml secondary seed culture solution was planted into a 100L fermenter (50L), and 26ml kanamycin solution was added at the same time, and then the first stage fermentation was carried out according to the following parameters: temperature: 37 ℃; pH: 6.8; speed: initial 250rpm; when the dissolved oxygen is lower than 30%, the speed is gradually increased to 750rpm; ventilation: initial 1:0.5vvm (air volume / culture volume / min, ventilation ratio); dissolved oxygen: greater than Or equal to 20%;

[0125] When the dissolved oxygen soars to 80%, it enters the second stage of fermentation. The second stage of fermentation is adjusted according to the following parameters: temperature: 35℃; pH: 6.8; speed: 750rpm; ventilation: initial 1: 1.5vvm; dissolved oxygen: greate...

Embodiment 2

[0127] Example 2. Recombinant fusion protein mTrA-Arg 34 -Extraction of GLP-1 (7-37) (SEQ ID NO. 4)

[0128] Prepare 156kg of 40mM phosphate buffer solution, adjust the pH to 9.5 with sodium hydroxide solution to obtain cell breaking buffer, add it to the fermenter, heat it with jacket steam, and stir at 100rpm at the same time to raise the temperature of the solution to 80℃ Above, 78.3 kg of the fermentation broth obtained in Example 1 was added to the cell destruction buffer, and 500 g of Triton X-100 was added thereto; the stirring was maintained until the cell destruction was completed, and then the centrifugation was started. The cleaned disc centrifuge is turned on, and the wet weight of the supernatant after centrifugation is controlled within 2% by adjusting the feeding speed, slagging cycle and slagging time, and the temperature of the centrifuged supernatant is reduced to 25°C , Adjust the pH of the supernatant to 7.5. The supernatant is filtered through a hollow fiber...

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Abstract

The invention relates to a preparation method of fusion protein expressed by gene recombinant escherichia coli, which belongs to the technical field of bioengineering. Aiming at the defects in the preparation process of the fusion protein expressed by various escherichia coli engineering bacteria at present, particularly the difficulty of thallus cell breaking, a solution suitable for industrial mass production is developed. The method is mainly characterized in that after fermentation is finished, on the premise that thalli do not need to be collected in a centrifugal mode, the efficient thalli cell breaking effect is achieved in the mode that fermentation liquor is directly subjected to high-temperature thermal cell breaking, and then a supernate containing the recombinant fusion proteinis obtained through centrifugation of a disc centrifuge, a concentrated recombinant fusion protein solution is obtained by utilizing conventional processes of membrane filtration and membrane concentration, and then enzyme digestion is carried out to obtain the target polypeptide. The method is low in equipment requirement, simple in process flow, and low in cost, and very suitable for industriallarge-scale production.

Description

Technical field [0001] The invention belongs to the field of fermentation engineering, and in particular relates to a method for preparing polypeptides by using thermostable fusion proteins. Background technique [0002] When using the Escherichia coli system to produce protein or polypeptide, it is often necessary to break the bacterial cells during the extraction process to release the target polypeptide, and then further extract it. There are many methods for cell destruction, such as ultrasonic method, high pressure homogenization, organic solvent method, enzymatic method, freeze-thaw method, etc. Each of these methods has certain limitations. Ultrasonic method has high energy consumption and is not suitable for industrial amplification. In production, freeze-thaw cell destruction requires a long time. The high heat generated by high-pressure homogeneous cell destruction is likely to cause damage to the enzyme. The disadvantages of the organic solvent method are also very obv...

Claims

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Application Information

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IPC IPC(8): C12P21/06C12N15/62C12N1/06C07K14/605C12R1/19
CPCC12P21/06C12N15/62C12N1/06C07K14/605
Inventor 刘懿
Owner CHENGDU ENZPRO BIOTECHNOLOGY CO LTD