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A method for purification and renaturation of fusion protein

A fusion protein and protein technology, applied in chemical instruments and methods, peptide preparation methods, organic chemistry, etc., can solve the problems of no biological activity, spatial conformation error, etc., and achieve the effect of stable antigen activity and high purity

Active Publication Date: 2019-08-23
广州白云山拜迪生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of them (more than 50%) are the expression products of cloned foreign genes, which have the correct amino acid sequence of foreign genes expressed by engineering bacteria, but the spatial conformation is often wrong, so they have no biological activity

Method used

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  • A method for purification and renaturation of fusion protein
  • A method for purification and renaturation of fusion protein
  • A method for purification and renaturation of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Bacteria reconstitution:

[0025] The Mtb72f engineered strain was inoculated into the fermentor to carry out high-density fermentation in a feed-through manner. When the fermentation broth OD650nm=50, IPTG was added for induction at 37°C, and the bacteria were harvested 5 hours after induction. Take a certain amount of bacteria (≥16L of bacteria during fermentation and harvest), add pre-cooled lysis buffer, and adjust the bacterial reconstitution to OD 650 nm≈60. Homogenize the bacterial compound solution with a high-speed homogenizer, and cool to ≤10°C after homogenization.

Embodiment 2

[0027] Broken bacteria:

[0028] The samples after cooling and homogenization are crushed with a high-pressure homogenizer (Ponny of Niro Soavi). The pressure of the high-pressure homogenizer is adjusted to 100-150 bars for the second stage, 850-900 bars for the first stage, and the total crushing pressure is 1000 ±50bars; the inlet sample temperature of the homogenizer should be controlled at ≤10℃, and the outlet sample temperature must be ≤25℃. The same batch of samples were crushed and processed twice, and the cell crushing liquid was obtained after the treatment.

Embodiment 3

[0030] Centrifugation and cleaning of broken bacteria liquid:

[0031] Add the same volume of lysis buffer to the bacterial cell crushing liquid, mix well and collect the inclusion bodies to settle with an industrial-scale centrifuge (such as a tubular high-speed centrifuge). The process temperature is less than 15°C. After centrifugation, the inclusion bodies are collected, and the inclusion body washing buffer is added to the equivalent volume when the cells are reconstituted, and then homogenized with high-speed homogenization (such as Ultra Turrax T50), and then the inclusion body precipitate is collected by a centrifuge. The process temperature is also less than 15°C. Repeat the cleaning and centrifugation steps for inclusion bodies. The inclusion bodies are collected, a small amount of washing buffer liquid is added, and high-speed homogenization is used to prepare an inclusion body suspension for purification.

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Abstract

The invention provides a purification and renaturation method of a fusion protein, which comprises the following steps of: purifying protein; diluting a sample; and preparing, concentrating and replacing membrane packages. The purification and renaturation method provided by the invention can achieve industrialized amplification; and the protein obtained after purification has high purity under the condition of high yield and is stable in antigen activity.

Description

Technical field [0001] The invention relates to a method for preparing a fusion protein, in particular to a method for preparing an Mtb72f fusion protein inclusion body suitable for industrialization and low cost. Background technique [0002] At present, when E. coli engineered bacteria constructed with high-efficiency expression plasmids synthesize a large number of heterologous proteins, they generally exist in the bacteria in the form of inclusion bodies. Inclusion bodies are composed of highly aggregated foreign proteins, nucleic acids, protein synthetase and ribosomes. Most of them (more than 50%) are the expression products of cloned foreign genes. They have the correct amino acid sequence of the foreign genes expressed by the engineered bacteria, but the spatial conformation is often wrong, so they have no biological activity. Since the inclusion bodies are located in bacterial cells and have no biological activity, the active protein can be obtained only after cell lysi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/36C07K1/16C07K1/34
Inventor 丘力功魏荣华韦剑黄明
Owner 广州白云山拜迪生物医药有限公司