A method for removing cytochrome c and endotoxin thereof

Abstract

The invention provides cytochrome C and an endotoxin removing method thereof, and relates to the technical field of purification. The cytochrome C is prepared by removing endotoxin by the endotoxin removing method: anion resin is put into a chromatographic column; buffer liquid is used for balancing the chromatographic column, so that the pH value and the electrical conductivity of effluent liquid are consistent with the pH value and the electrical conductivity of the buffer liquid of the chromatographic column; after the pH value of a fine solution of the cytochrome C with unqualified endotoxin is regulated to be consistent with the pH value of the buffer liquid, filtering is performed to obtain clear sample loading liquid; after the sample loading liquid passes the column, flow through liquid is obtained; the collected flow through liquid is sequentially subjected to dialysis and filtering. The qualified rate of the endotoxin of the cytochrome C prepared by the method is high; the preparation process is simple; the separation effect is good; the repeatability is high; the sample recovery rate is high; the separation conditions are mild; higher industrial popularization prospects are realized.

Description

technical field

[0001] The invention relates to the technical field of purification, and in particular to a method for removing cytochrome C and its endotoxin. Background technique

[0002] Cytochrome is a very common protein in various organisms. It is a kind of electron transfer protein containing iron porphyrin group. It only exists in hypoxic cells and widely exists in the mitochondrial inner membrane and endoplasm of eukaryotes. network, in the chloroplasts of plants, and in photosynthetic microorganisms and bacteria. Cytochrome plays the role of transferring electrons on the inner mitochondrial membrane. Cytochrome C is a kind of cytochrome, which is located between cytochrome B and cytochrome oxidase on the mitochondrial respiratory chain, and is an important part of the respiratory chain. Each molecule of cytochrome C contains a heme and a polypeptide chain. Cytochrome C is a stable soluble protein that is easily soluble in water and acidic solutions.

[0003] In ...

Examples

Embodiment 1

[0054] This embodiment provides a kind of cytochrome C, which is prepared after removing endotoxin by the following method:

[0055] Put QXL into the chromatography column;

[0056] Equilibrate the chromatographic column with PBS buffer solution so that the pH value and conductivity of the effluent are consistent with the pH value and conductivity of the buffer solution on the column;

[0057] Use 0.1M hydrochloric acid to adjust the pH of the fine solution of cytochrome C with unqualified endotoxin to be consistent with the pH value of the buffer solution, and then filter through the first filter membrane with a filter pore size of 0.3-0.5 μm to obtain a clarified sample solution;

[0058] Pass the sample solution through the column at a constant speed of 2mL / min to obtain the flow-through solution;

[0059] The collected flow-through solution was dialyzed and filtered sequentially, and the dialysis was performed with a 5kd membrane until the conductivity of the solution was...

Embodiment 2

[0061] This example provides a kind of cytochrome C, which is different from the cytochrome C provided in Example 1 in that the cytochrome C provided in this example is obtained after removing endotoxin by the following method:

[0062] Put DEAE into the chromatography column;

[0063] Equilibrate the chromatography column with TRIS buffer so that the pH value and conductivity of the effluent are consistent with the pH value and conductivity of the buffer on the column;

[0064] Use 0.15M hydrochloric acid to adjust the pH of the fine solution of cytochrome C to be consistent with the pH value of the buffer solution, and then filter through the first filter membrane with a filter pore size of 0.45 μm to obtain a clarified sample solution;

[0065] Pass the sample solution through the column at a constant speed of 3mL / min to obtain the flow-through solution;

[0066] The collected flow-through solution was dialyzed and filtered sequentially, and the dialysis was performed with...

Embodiment 3

[0068] This example provides a kind of cytochrome C, which is different from the cytochrome C provided in Example 1 in that the cytochrome C provided in this example is obtained after removing endotoxin by the following method:

[0069] The m-phenylenediamine-formaldehyde resin is placed in the chromatographic column;

[0070] Equilibrate the chromatography column with TRIS buffer so that the pH value and conductivity of the effluent are consistent with the pH value and conductivity of the buffer on the column;

[0071] Use 0.2M hydrochloric acid to adjust the pH of the fine solution of cytochrome C to be consistent with the pH value of the buffer solution, and then filter through the first filter membrane with a filter pore size of 0.5 μm to obtain a clarified sample solution;

[0072] Pass the sample solution through the column at a constant speed of 4mL / min to obtain the flow-through solution;

[0073] The collected flow-through solution was dialyzed and filtered sequentia...

technical field

[0001] The invention relates to the technical field of purification, and in particular to a method for removing cytochrome C and its endotoxin. Background technique

[0002] Cytochrome is a very common protein in various organisms. It is a kind of electron transfer protein containing iron porphyrin group. It only exists in hypoxic cells and widely exists in the mitochondrial inner membrane and endoplasm of eukaryotes. network, in the chloroplasts of plants, and in photosynthetic microorganisms and bacteria. Cytochrome plays the role of transferring electrons on the inner mitochondrial membrane. Cytochrome C is a kind of cytochrome, which is located between cytochrome B and cytochrome oxidase on the mitochondrial respiratory chain, and is an important part of the respiratory chain. Each molecule of cytochrome C contains a heme and a polypeptide chain. Cytochrome C is a stable soluble protein that is easily soluble in water and acidic solutions.

[0003] In ...

Examples

Embodiment 1

[0054] This embodiment provides a kind of cytochrome C, which is prepared after removing endotoxin by the following method:

[0055] Put QXL into the chromatography column;

[0056] Equilibrate the chromatographic column with PBS buffer solution so that the pH value and conductivity of the effluent are consistent with the pH value and conductivity of the buffer solution on the column;

[0057] Use 0.1M hydrochloric acid to adjust the pH of the fine solution of cytochrome C with unqualified endotoxin to be consistent with the pH value of the buffer solution, and then filter through the first filter membrane with a filter pore size of 0.3-0.5 μm to obtain a clarified sample solution;

[0058] Pass the sample solution through the column at a constant speed of 2mL / min to obtain the flow-through solution;

[0059] The collected flow-through solution was dialyzed and filtered sequentially, and the dialysis was performed with a 5kd membrane until the conductivity of the solution was...

Embodiment 2

[0061] This example provides a kind of cytochrome C, which is different from the cytochrome C provided in Example 1 in that the cytochrome C provided in this example is obtained after removing endotoxin by the following method:

[0062] Put DEAE into the chromatography column;

[0063] Equilibrate the chromatography column with TRIS buffer so that the pH value and conductivity of the effluent are consistent with the pH value and conductivity of the buffer on the column;

[0064] Use 0.15M hydrochloric acid to adjust the pH of the fine solution of cytochrome C to be consistent with the pH value of the buffer solution, and then filter through the first filter membrane with a filter pore size of 0.45 μm to obtain a clarified sample solution;

[0065] Pass the sample solution through the column at a constant speed of 3mL / min to obtain the flow-through solution;

[0066] The collected flow-through solution was dialyzed and filtered sequentially, and the dialysis was performed with...

Embodiment 3

[0068] This example provides a kind of cytochrome C, which is different from the cytochrome C provided in Example 1 in that the cytochrome C provided in this example is obtained after removing endotoxin by the following method:

[0069] The m-phenylenediamine-formaldehyde resin is placed in the chromatographic column;

[0070] Equilibrate the chromatography column with TRIS buffer so that the pH value and conductivity of the effluent are consistent with the pH value and conductivity of the buffer on the column;

[0071] Use 0.2M hydrochloric acid to adjust the pH of the fine solution of cytochrome C to be consistent with the pH value of the buffer solution, and then filter through the first filter membrane with a filter pore size of 0.5 μm to obtain a clarified sample solution;

[0072] Pass the sample solution through the column at a constant speed of 4mL / min to obtain the flow-through solution;

[0073] The collected flow-through solution was dialyzed and filtered sequentia...