A washing method for genetically engineered inclusion bodies

A washing method and genetic engineering technology, applied in the field of biomedicine, can solve problems such as high cost, cumbersome operation, and easy pollution, and achieve the effects of improving production efficiency, simple process, and convenient operation

Active Publication Date: 2016-08-17
鲁南新时代生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The traditional washing method needs to use high-speed centrifuge or continuous flow centrifuge, which brings a series of problems: such as high cost, cumbersome operation, easy pollution, high maintenance cost, and difficult process scale-up
Although the purity of the inclusion bodies obtained is high, the yield of the inclusion bodies is low
However, there are no literature reports on the use of hollow fiber membranes for inclusion body washing

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Washing of Inclusion Body (0.2 kg) of Escherichia coli Expressing Recombinant Human Proinsulin

[0019] The hollow fiber membrane (made of polyethersulfone, the inner diameter of the fiber tube is 1 mm, and the pore size of the filter membrane is 0.1 μm) is first rinsed with purified water to balance, and the water flow rate is adjusted so that the membrane pressure is 15 bar and the permeation flux is 30 LMH . Add 3 L of washing buffer Ⅰ (20 mM Tris-HCl, 1% Triton-X-100, pH 9.0) to 0.2 kg of Escherichia coli expressing recombinant human proinsulin inclusion body, stir to make a suspension, and remove the inclusion body The suspension was pumped into the hollow fiber membrane, so that the pressure across the membrane was 5 bar, and the permeation flux was 10 LMH. The inclusion body suspension was concentrated by 3 times and then stopped feeding.

[0020] Add 5 L of washing buffer II (100 mM Tris-HCl, 2 M urea, pH 9.0) into the above-mentioned inclusion body c...

Embodiment 2

[0022] Example 2 Washing of Escherichia coli expressing recombinant human proinsulin inclusion body (1 kg)

[0023] The hollow fiber membrane (made of polyethersulfone, the inner diameter of the fiber tube is 1.75 mm, and the pore size of the filter membrane is 0.2 μm) is first rinsed with purified water to balance, and the water flow rate is adjusted so that the membrane pressure is 5 bar and the permeation flux is 10 LMH . Add 15 L of washing buffer Ⅰ (20 mM Tris-HCl, 5% Tween-20, pH 9.0) into inclusion bodies expressing recombinant human proinsulin in Escherichia coli, stir to make a suspension, and pump the inclusion body suspension into the hollow For fibrous membranes, the membrane pressure is 5 bar, the permeation flux is 10 LMH, and the inclusion body suspension is concentrated 5 times before liquid feeding is stopped.

[0024] Add 45 L of washing buffer II (100 mM Tris-HCl, 3 M guanidine hydrochloride, pH 9.0) to the above inclusion body concentrate, stir to form a s...

Embodiment 3

[0026] Example 3 Washing of Escherichia coli expressing recombinant human proinsulin inclusion body (5 kg)

[0027] The hollow fiber membrane (made of polyethersulfone, the inner diameter of the fiber tube is 1 mm, and the pore size of the filter membrane is 0.2 μm) is first rinsed with purified water to balance, and the water flow rate is adjusted so that the membrane pressure is 10 bar and the permeation flux is 20 LMH . Add 75 L of washing buffer Ⅰ (100 mM Tris-HCl, 5% Triton-X-100, pH 9.0) into inclusion bodies expressing recombinant human proinsulin in Escherichia coli, stir to make a suspension, and dissolve the inclusion body suspension Pump into the hollow fiber membrane so that the pressure across the membrane is 15 bar, the permeation flux is 30 LMH, and the inclusion body suspension is concentrated 5 times before stopping the liquid feeding.

[0028] Add 45 L of washing buffer II (100 mM Tris-HCl, 4 M urea, pH 9.0) to the above-mentioned inclusion body concentrate, s...

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Abstract

The invention discloses a washing method of a genetic engineering inclusion body. According to the washing method, the genetic engineering inclusion body is washed by adopting a hollow fiber membrane, and therefore the continuous automatic operation can be realized. The washing method has the advantages of being simple in process and convenient for operation, and improving the production efficiency; and the genetic engineering inclusion body has the advantages of high yield and high purity, and is especially suitable for application in the large-scale industrial production.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a method for washing genetic engineering inclusion bodies, in particular to a method for washing genetic engineering inclusion bodies by using a hollow fiber filter membrane. Background technique [0002] Genetic recombination technology can be used to mass-produce extremely small amounts of functional proteins or polypeptides in nature. Prokaryotic organisms such as Escherichia coli are usually used as the preferred host bacteria for gene recombination technology because of their fast growth and low nutritional requirements. However, recombinant proteins highly expressed in E. coli often form insoluble and inactive inclusion bodies. Inclusion body is a dense structure formed by the misfolding of the secondary structure of the protein during the expression of the recombinant gene. This structure is beneficial to the recombinant protein from being hydrolyzed by intracellular...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/34
Inventor 赵志全熊继元柳常青高倩倩
Owner 鲁南新时代生物技术有限公司
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