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Industrially extracting, renaturation and purifying method for recombined TRAILinclusion body protein

A purification method and protein technology, applied in the field of biomedicine, can solve the problems of difficulty in enlarging the production scale, low renaturation efficiency, and limitation, and achieve the effect of reducing the generation of aggregates and improving the efficiency of renaturation

Inactive Publication Date: 2004-02-11
EAST CHINA UNIV OF SCI & TECH +1
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, recombinant proteins expressed in the E. coli system are usually produced in the form of inactive inclusion bodies, and it is a worldwide technical problem to refold these inactive inclusion bodies into active forms.
The common production process route using Escherichia coli as the expression system is: Escherichia coli fermentation → inclusion body washing → inclusion body renaturation → target product purification, where inclusion body renaturation is usually used in the laboratory at a low concentration of 0.01-0.1mg / ml It is completed under the condition of protein concentration, the result is that the renaturation efficiency is low, the production cost is high, and it is difficult to scale up to form a production scale
Wang Xinjuan, School of Basic Medicine, Peking University and others (Journal of Beijing Medical University, 2000; 32(5): 387-390) made a preliminary study on the clone expression and biological activity of human TRAIL in Escherichia coli, and carried out the inclusion body Preliminary renaturation, but no structural identification of the renaturation results, no explanation on whether the renaturation is complete and the yield, and it is limited to laboratory methods

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Purity ≥ 80% TRAIL inclusion body extraction:

[0031] Take 2.0 kg of wet cells, wash with PBS (phosphate buffer containing 0.15 mol / L NaCl, usually 50 mmol / L) buffer solution with pH=7.0 to remove unused medium components entrained in the cells Wait for impurities, centrifuge and discard the supernatant, then reset it in PBS buffer solution with pH=7.0-8.0, stir it into a uniform slurry under the condition of solid-liquid ratio of 1:3-5, and pump into high-pressure homogenate The bacterium is crushed in the machine, so that the TRAIL inclusion body is fully released from the bacterium, and then 0.8 kg of wet TRAIL inclusion body crude product is obtained by centrifugation, wherein the water content is 70-80%.

[0032] Wash 0.8 kg of the above crude wet inclusion bodies extensively with PBS buffer solution of pH = 7.0-8.0 for 2-3 times, and then wash with phosphate buffer solution of pH = 7.0-8.0 containing 2 mmol DTT and 1 mol NaCl. The consumption of washing solution ...

Embodiment 2

[0034] Refolding by continuous feeding and dilution:

[0035] Take 0.15 kg of TRAIL inclusion body with a purity of 80%, dissolve it with 3 liters of 50 mmol / L Tris buffer solution (adjusted with hydrochloric acid) containing 6 mol / L guanidine hydrochloride and 30 mmol / L LDTT pH 7.5, and obtain a uniform denaturing solution solution, the total protein concentration is about 12.5g / L, and the TRAIL protein is about 10g / L.

[0036] The above-mentioned denatured solution was refolded by continuous feeding and dilution method. The refolding solution used contained 0.5mol / L L-Arg, 0.5mol / L urea, 2mmol / L DTT, 0.2mmol / L ZnSO 4 And the pH of 0.5mol / L NaCl is 27L of 50mmol / L Tris damping solution 27L of 7.5, is placed in a 50L reaction kettle with stirring, and its flow acceleration rate is 5ml / min (referring to the flow rate of denaturing lysate) Acceleration rate), the feed time is about 10.5 hours, the final concentration is 1mg / ml (or 1g / L, some protein aggregates), and slowly stir...

Embodiment 3

[0040] Refolding by intermittent flow plus dilution refolding method:

[0041] Except that the feeding mode adopts intermittent feeding, the rest of the methods, components and steps are the same as in Example 2. When batch-feeding and diluting refolding, the feeding concentration of the denaturing solution is controlled at 100-200 mg / L each time, which is equivalent to adding 0.5 liters of the denatured solution in the refolding solution of 27 L each time, feeding at intervals of 90 minutes Once, (accumulative intermittent fed-batch operation takes 7.5h), the whole operation is stirred slowly at 4°C for about 10h. Finally, "TRAIL refolding solution" was obtained for later use. By the way, it is pointed out that the flow speed is slower or the amount of flow is less each time, that is, the renaturation time is slightly longer, which is generally conducive to the improvement of renaturation quality. The industrial sector can make a choice according to its own conditions and re...

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PUM

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Abstract

The industrial extracting, renaturation and purifying process of recombined TRAIL inclusion body protein includes crushing, washing and other pre-treatment of the recombinant colibacillus thallus expressing TRAIL to obtain TRAIL inclusion body of 80 % over purity; dissolving the TRAIL inclusion body with denaturant to obtain denaturated solution; renaturating the solution of the denaturated inclusion body solution via diluting course, two-step dialysis to eliminate residual denaturant to obtain renaturating solution containing TRAIL active protein, superfiltering and concentrating the renaturating solution, column chromatography to separate and to collect qualified eluted liquid as the destination product, which is one kind of renaturated recombinant TRAIL protein with similar activity with soluble expression protein, in the yield over 40 %.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to an industrial extraction, renaturation and purification method of recombinant TRAIL inclusion body. technical background [0002] Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the newly discovered TNF family members, also known as apoptotin 2 ligand (Apo-2L). In 1996, Pitti first isolated and identified TRAIL and its induced apoptosis. It was found that the human TRAIL gene was located on chromosome 3q26, with a molecular weight of 32.5kD and an isoelectric point of 7.63. In the strain, it is expressed on the cell surface in the form of type II transmembrane protein, which is called membrane-bound TRAIL. The soluble part is the 114th-281st amino acid, the molecular weight is about 24kD, the dimer is formed at about 48kD, and the trimer is about 66kD. Its functional site is mainly located outside the cell, and its biolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/14
Inventor 沈亚领魏东芝夏小霞
Owner EAST CHINA UNIV OF SCI & TECH
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