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Method for purifying proteins and kit

A purification method and protein technology, applied in the fields of biochemistry and fermentation engineering, can solve the problems of unsuitability for industrial production, low renaturation rate, and strict requirements on sample volume

Active Publication Date: 2018-07-10
SICHUAN KELUN BIOTECH BIOPHARMACEUTICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, this method has the disadvantages of low refolding rate (30%-40%) and low yield (only 300 mg of finished product is obtained per liter)
In addition, this process uses molecular sieve chromatography to remove small molecules, but molecular sieve chromatography has strict requirements on column height, column efficiency and sample volume, and is mostly used in the laboratory stage, which is not suitable for industrial production

Method used

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  • Method for purifying proteins and kit
  • Method for purifying proteins and kit
  • Method for purifying proteins and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Embodiment 1: the fermentation amplification of bacterial strain

[0133] The strain used is recombinant Escherichia coli GM221 capable of expressing thrombopoietin-mimetic peptide fusion protein, purchased from ATCC (strain preservation number 98957, plasmid preservation number 98113)), and the strain expresses the protein represented by formula A of the present invention.

[0134] Cultivate under conventional fermentation conditions to express the desired peptide. The medium used is LB medium, which can be prepared according to the "Molecular Cloning Experiment Guide".

[0135] You can also follow the steps below:

[0136] Thaw the preserved strain, inoculate 50 μl into a 250ml Erlenmeyer flask containing 50ml LB medium for activation, and cultivate overnight at 30°C with a rotation speed of 150-300r / min, then transfer the bacterial solution into a triangle flask containing 2L LB medium In the bottle, 37°C, 250-400r / min, replenish the medium in time, cultivate ove...

Embodiment 2

[0145] Example 2: Protein Purification

[0146] (1) Bacterial destruction: mix and stir the bacterium and the bacteriostasis solution 1:5 (w:v), the bacteriostasis solution is a solution with 20mM Tris, 10mM EDTA, and its pH is 8.0; Under the condition of 1000bar and 10°C, the bacteria were broken twice with a low-temperature ultra-high pressure continuous flow cell disruptor.

[0147] (2) Inclusion body collection and washing: centrifuge the sterilized solution in step (1) at 10°C at 9500rpm for 20min, collect the inclusion body precipitate, and then add the precipitate into washing buffer I at a ratio of 1:10 (w:v) and mix Stirring, the washing buffer I is a solution with 20mM Tris, 0.1%-0.2% Triton X-100, 0.1M NaCl, 1mM EDTA, 1M urea, its pH is 8.5, and the precipitate is collected by centrifugation; then the collected precipitate Washing buffer II was added at a ratio of 1:10 (w:v) for mixing and stirring. The washing buffer II was a solution with 20 mM Tris and 1 mM ED...

Embodiment 3

[0158] Example 3: Protein Purification

[0159] Steps (1)-(3) of Embodiment 3 With reference to steps (1)-(3) of Embodiment 2, steps (4)-(11) are as follows:

[0160] (4) Dialysis: dialyze the supernatant according to 1:30 (v:v) with the dialysate, and the dialysate is 30mM HCl solution;

[0161] (5) Oxidative derivatization: Then add an appropriate amount of urea to the dialysis harvest solution (retentate in the bag) so that the final concentration of urea is 10M, stir until the solution is clear, then add cystamine and 1g / L final concentration of 2.0g / L After stirring and dissolving the EDTA, adjust the pH to 8.5 with 2.5M Tirs, and let stand for oxidative derivatization for 4 hours;

[0162] (6) Refolding: Then dilute the protein solution with 10M urea solution to a concentration of 15g / L, then add the protein solution dropwise to the refolding solution at a ratio of 1:10 (v:v) to the refolding solution, so that the protein is finally The concentration is about 1.5g / L,...

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Abstract

The invention belongs to the field of biochemistry and fermentation engineering, and relates to a method for purifying proteins and a kit. Particularly, the method for purifying the proteins comprisesthe step that chromatography is carried out on refolding products of protein inclusion bodies. The method for purifying the proteins has the advantages that the renaturation rate is high, the yield is high, and the application prospect is good.

Description

technical field [0001] The invention belongs to the field of biochemistry and fermentation engineering, and relates to a protein purification method and a kit. Specifically, the protein is a thrombopoietin mimetic peptide or a fusion protein thereof. Background technique [0002] Idiopathic thrombocytopenic purpura (ITP) is a common bleeding disorder in which patients are at significant risk of bleeding. In August 2008, the U.S. FDA approved romigrastim (containing a thrombopoietin-mimicking peptide) to be marketed for the treatment of idiopathic thrombocytopenic purpura. Romigrastim is produced from Escherichia coli. It is an Fc peptide fusion protein that can stimulate platelet production using recombinant DNA technology. It contains 2 identical single-chain subunits, and each single chain contains IgG Fc region and platelet production. Thrombopoietin (TPO) mimics the peptide with a molecular weight of about 59kDa, in which the Fc segment can effectively prolong the half...

Claims

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Application Information

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IPC IPC(8): C07K14/52C07K1/16C07K1/18C07K1/22C07K1/34C07K1/113C07K1/36A61K38/19A61P7/04
CPCA61K38/00C07K14/524C07K2319/00
Inventor 薛彤彤肖亮邓春楼俊文刘立平成裕王利春王晶翼
Owner SICHUAN KELUN BIOTECH BIOPHARMACEUTICAL CO LTD
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