Method for removing endotoxin in protein

An endotoxin and protein technology, which is applied in the preparation methods of peptides, chemical instruments and methods, organic chemistry, etc. Effect

Inactive Publication Date: 2015-10-28
TIANJIN RINGPU BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] The present invention aims at the technical defects of the prior art, and provides a method for removing endotoxin in proteins, so as to solve the technical problem of low endotoxin removal efficiency existing in the prior art

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 11

[0035] Embodiment 1.1 (induced expression of recombinant genetically engineered bacteria)

[0036] Inoculate fresh bacterial liquid into LB liquid medium at a ratio of 1:100, cultivate for 3-4 hours, add IPTG for induction, and continue culturing for 4-5 hours. Centrifuge at 6000rpm for 6min to collect the cells. Wash the cells with 1 / 10 of the volume of the bacterial solution in PBS, add lysozyme at a ratio of 100 μg / mL, bathe in water at 30°C for 30 min, and disrupt the cells by ultrasonic on ice. Centrifuge and collect the supernatant.

Embodiment 12

[0037] Embodiment 1.2 (purification of protein)

[0038] Mix the medium evenly and pack the column; use about 4 column volumes of equilibration solution (50mM NaH 2 PO 4 , 300mM NaCl, pH 8.0) to equilibrate the medium until the A280 value of the effluent is close to zero; take the collected supernatant and load the sample at a flow rate of about 1-2mL / min; wash the medium with the equilibrium solution until the A280 value of the effluent tends to balance; Wash with about 2 column volumes (50mM NaH 2 PO 4 , 300mM NaCl, PH 8.0) to clean Ni 2+Column until the A280 value of the effluent tends to balance; the eluent (50mM NaH 2 PO 4 , 10mM Tris-Cl, 250mM imidazole, 8M urea, pH 8.0) to elute the protein, and collect the eluate, which is the target protein.

Embodiment 13

[0039] Embodiment 1.3 (membrane bag concentrates)

[0040] Concentration by tangential flow filtration was carried out using membrane packs with a molecular weight cut-off of 300KD and 5KD. First flush the circuit with deionized water, control the pressure of the liquid inlet to not exceed 0.1MPa, and the pressure of the liquid outlet to not exceed 0.05MPa. Then circulate the buffer in the system for 3 to 5 minutes to reduce the loss of samples. Afterwards, the collected target protein is concentrated by ultrafiltration until the required concentration multiple.

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PUM

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Abstract

The invention provides a method for removing endotoxin in protein. The method adopts a sequential combination of chromatography, ultrafiltration and common filtration, and accords with a protein purification process flow. With the method, protein purification is realized while endotoxin is effectively removed, and the properties of protein is not influenced. The method is especially suitable to be used in treating various genetically engineered bacteria expressed intracellular and extracellular proteins. With optimized designs of the conditions of filtering mode, filtering pressure, membrane pore size and the like, operation steps and operation time are simplified, such that production cost is reduced, and processing capacity is improved. With the method provided by the invention, the endotoxin content can be reduced to an animal clinical application standard range, and the properties of the product is not influenced while the endotoxin is removed. The method is suitable for large-scale productions and applications.

Description

technical field [0001] The invention relates to the technical field of biological products, in particular to a method for removing endotoxin in proteins. Background technique [0002] Endotoxin is a general term for toxic substances present in Gram-negative bacteria, which are released after the bacteria are lysed, also known as "pyrogen". Its chemical composition is phospholipopolysaccharide-protein complex, and its toxic composition is mainly lipid A. Endotoxins are located in the outermost layer of the cell wall, on mucinous peptides that coat the cell wall. Bacterial endotoxins are released when cells lyse, die, or are artificially disrupted. Endotoxins generally exist in the form of polymers, and the chelation of phosphate groups with divalent metal ions can further stabilize the polymerization between endotoxin molecules and promote larger endotoxin complexes. The molecular weight of such complexes can range from several thousand to several Don't wait. [0003] End...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/36C07K1/34C07K1/16
Inventor 张英吕茂杰杨保收付旭彬梁武
Owner TIANJIN RINGPU BIO TECH
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