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A kind of purification method of recombinant human lysozyme

A human lysozyme and purification method technology, applied in the field of bioengineering, can solve the problems of high cost, complex process, large investment, etc., and achieve the effects of saving time, improving purification efficiency and high yield

Active Publication Date: 2020-04-24
SHAANXI HUIKANG BIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In China, lysozyme has not been fully used, mainly due to the small quantity and poor quality of domestically produced lysozyme, and the lysozyme used in medicine, food and scientific research still relies on imports
At present, egg white is mainly used as raw material to extract lysozyme, but the cost is high, the investment is large, and the process is complicated

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1. Ultrafiltration coarse and pure

[0025] Put 34L of recombinant human lysozyme fermentation broth (Pichia pastoris fermentation broth containing recombinant human lysozyme) into a decanter centrifuge, centrifuge at 2000r / min for 60min, and collect 21L of supernatant through a skimmer. Pass 21L of supernatant through a 0.2μm hollow fiber membrane for ultrafiltration sterilization, and add 5L of normal saline before the end of ultrafiltration to ensure protein recovery during ultrafiltration, and collect 24L of filtrate.

[0026] 2. Hydrophobic chromatography

[0027] Add NaCl to the 24L filtrate obtained in step 1, so that the NaCl concentration in the filtrate is 1.2mol / L, then pass through the hydrophobic chromatography column for purification by high performance liquid chromatography, and the packing is that the adsorption capacity of the protein is not less than 30mg / L mL, the linear flow rate is not higher than 8mL / cm, and the particle size is 45 ~ 160μm Phenyl ...

Embodiment 2

[0031] 1. Ultrafiltration coarse and pure

[0032] Put 30L of recombinant human lysozyme fermentation broth (Pichia pastoris fermentation broth containing recombinant human lysozyme) into a decanter centrifuge, centrifuge at 2000r / min for 60min, and collect 18L of supernatant through a skimmer. Pass 18L of supernatant through a 0.2μm hollow fiber membrane for ultrafiltration sterilization, and add 5L of normal saline before the end of ultrafiltration to ensure protein recovery during ultrafiltration, and collect 21L of filtrate.

[0033] 2. Hydrophobic chromatography

[0034] Add NaCl to the 21L filtrate obtained in step 1, so that the NaCl concentration in the filtrate is 1.0mol / L, and then pass through a hydrophobic chromatography column for purification by high performance liquid chromatography. The packing is that the adsorption capacity of the protein is not less than 30mg / L mL, the linear flow rate is not higher than 8mL / cm, and the particle size is 45 ~ 160μm Phenyl hi...

Embodiment 3

[0038] 1. Ultrafiltration coarse and pure

[0039] Put 36L of recombinant human lysozyme fermentation broth (Pichia pastoris fermentation broth containing recombinant human lysozyme) into a decanter centrifuge, centrifuge at 2000r / min for 60min, and collect 20L of supernatant through a skimmer. Pass 20L of supernatant through a 0.2μm hollow fiber membrane for ultrafiltration sterilization, and add 5L of normal saline before the end of ultrafiltration to ensure protein recovery during ultrafiltration, and collect 23L of filtrate.

[0040] 2. Hydrophobic chromatography

[0041] Add NaCl to the 23L filtrate obtained in step 1, so that the NaCl concentration in the filtrate is 1.5mol / L, then pass through the hydrophobic chromatography column for purification by high performance liquid chromatography, and the packing is that the adsorption capacity of the protein is not less than 30mg / L mL, the linear flow rate is not higher than 8mL / cm, and the particle size is 45 ~ 160μm Phenyl hi...

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Abstract

The invention discloses a method for purifying recombinant human lysozyme. The method comprises the following steps: performing coarse purification on the recombinant human lysozyme on a large scale by using a hydrophobic chromatographic column; and directly performing refined purification by using a weak cation exchange chromatographic column. Compared with the existing technology, the method disclosed by the invention has the advantages that the process procedure is simple and easy to operate, the recovery rate and activity are greatly improved on the premise of ensuring the purity, and low-cost high-yield industrialization requirements on the recombinant human lysozyme are met.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a purification method of recombinant human lysozyme. Background technique [0002] The earliest research on lysozyme started from the lysozyme in Bacillus subtilis published by Nicolle in 1907. In 1922, Fleming et al. found that there was an enzyme that could dissolve cell walls and kill bacteria in human saliva and tears, so it was named lysozyme. . In 1937, Abraham and Robinson isolated lysozyme crystals from egg protein, which opened a chapter in the history of lysozyme research. In 1965, Philip et al. from the United Kingdom used X-ray diffraction to study and analyze lysozyme, and they were the first to fully clarify the three-dimensional structure of lysozyme. [0003] Lysozyme has antibacterial, antiviral and anti-inflammatory effects, and combined application with antibiotics can enhance the efficacy of antibiotics. It is also a non-specific immune f...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/36
CPCC12N9/2462C12Y302/01017
Inventor 邓晗史瑾王维郝东赵金礼杨小玲
Owner SHAANXI HUIKANG BIO TECH CO LTD