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Ribonuclease inhibitor inclusion body renaturation method

A ribonuclease and inhibitor technology, applied in the field of renaturation of ribonuclease inhibitor inclusion bodies, can solve the problems of low renaturation yield, undisclosed degree of solubility, TEV enzyme residual pollution, etc.

Active Publication Date: 2018-08-21
SHANGHAI ZHAOWEI TECH DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Chinese patent application CN102234649A reports that the soluble expression of RI can be performed, but a TrxA thioredoxin fusion tag needs to be added to the 5'-end of the RI sequence, and the tag is removed by TEV enzyme digestion during the purification process, and the TrxA tag exists in this method Incomplete excision, TEV enzyme residual contamination and other problems
[0008] Chinese patent application CN1584031A reports that human-derived ribonuclease inhibitors (RI) can be expressed through prokaryotic systems (E. and the solubility degree of its expression
[0012] From the results, it can be speculated that the dilution effect of renaturation is the best, but the renaturation rate is still low, and most of the inclusion bodies have not been effectively utilized.
Therefore, the low yield of renaturation is the bottleneck problem faced in the large-scale production process of the current ribonuclease inhibitor (RI)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072]Embodiment 1, the purification of RI inclusion body

[0073] (1) Construction of ribonuclease inhibitor RI carrier

[0074] Extract total RNA from discarded placental tissue isolated from human body, then perform RT-PCR to amplify the RI gene sequence (adding 6×His-tag at the N-terminal), recover by enzyme digestion, connect to pET28b vector, and transform into DH5a competent Cells were selected for single clone culture and the extracted plasmid (pET28b-6*His-RI) was transformed into the expression host bacteria.

[0075] (2) Cell preparation

[0076] The constructed BL21DE3 expression strain was inoculated into 100ml LB medium (Kana + ), 37°C, 230rpm, 16hr. Transfer the seed liquid to 2×YT medium according to the inoculation amount of 1%, and carry out fermentation culture. When the OD600 of the bacterial liquid reaches 0.9, add the inducer IPTG to the final concentration of 0.2mM, induce for 4.5 hours, and collect the bacterial cells by centrifugation after the indu...

Embodiment 2

[0084] Embodiment 2, renaturation of RI inclusion body

[0085] (1) Denaturation and dissolution of inclusion bodies

[0086] Take 1 g of the purified inclusion body, add it to 10 ml of resuspension buffer, and stir rapidly for 1 hr, then add the above resuspension into 10 ml of inclusion body denaturation solution stored at 4°C, and stir rapidly for 30 min, at which time the inclusion body is completely dissolved, and it is Transparent.

[0087] Resuspension buffer formula: DTT 1mM, EDTA 1mM, sodium chloride 50mM, glycerol 10% (v / v), Tris-HCl 15mM, pH 7.5.

[0088] Inclusion body denaturing solution formula: DTT 1mM, denaturant urea 6M, Tris-HCl 35mM, EDTA 1mM, sodium chloride 50mM, glycerol 10% (v / v), pH 7.5.

[0089] (2) Refolding of inclusion bodies

[0090] Quickly pour the above-mentioned denaturing solution into 100ml of the refolding solution stored at 4°C, stir rapidly to mix, and then stir at a low speed for 20 hours to obtain the treated refolding solution.

[0...

Embodiment 3

[0093] Example 3, Column Purification of RI Refolding Solution (chromatography at 4°C)

[0094] (1) Sample loading

[0095] Dilute the treated refolding solution obtained above to a protein concentration of 0.5-1 mg / ml, adjust the pH to 8.0, and load the sample into Ni-sepharose FF (10 ml, GE Company) at a flow rate of 2 ml / min.

[0096] (2) Washing to remove RNase A

[0097] After loading the sample, first wash 2 times the column volume with the column equilibration buffer, then wash the column with the washing buffer at a flow rate of 2ml / min, and perform SDS-PAGE electrophoresis at the same time to detect the banding of the washing solution. When the RNase A band appears, stop washing.

[0098] Column equilibration buffer formula: DTT 1mM, EDTA 1mM, sodium chloride 50mM, glycerol 10% (v / v), Tris-HCl 35mM, pH 7.5.

[0099] Washing buffer formulation: DTT 1 mM, EDTA 1 mM, sodium chloride 300 mM, NaAC / HAC 35 mM, pH 5.0, glycerol 5% (v / v).

[0100] (3) Gradient elution of R...

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Abstract

The present invention relates to a ribonuclease inhibitor (RI) inclusion body renaturation method. According to the present invention, RNase A is used during the renaturation so as to substantially improve the renaturation rate of RI, and is easily removed with the inorganic salt with a certain concentration under the slightly acidic condition in the subsequent purification process, and RI can bebound to the purification column through proper purification, such that the purification can be conveniently performed to obtain the high-purity RI.

Description

technical field [0001] The invention belongs to the field of chemical and biological engineering; more specifically, the invention relates to a method for refolding inclusion bodies of ribonuclease inhibitors. Background technique [0002] Ribonuclease inhibitor (Ribonuclease Inhibitor, RI) is an acidic protein in mammalian cytoplasm, with a molecular weight of 50kD. It can bind and inhibit RNase A and angiogenesis factor (Angiogenin, Ang) in the pancreatic ribonuclease superfamily at a ratio of 1:1. The combination of RI and Ang has attracted people's attention because Ang has the activity of promoting the growth of neovascular endothelium, and neovascularization is necessary for the growth of solid tumor tissues. Injecting RI into the transplanted tumor tissue of mice resulted in extensive necrosis of solid tumor tissue and a significant reduction in new blood vessels. According to an important characteristic of the primary structure of RI, all 32 cysteine ​​residues are...

Claims

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Application Information

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IPC IPC(8): C07K14/47C07K1/18
CPCC07K14/4703
Inventor 李新亮
Owner SHANGHAI ZHAOWEI TECH DEV
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