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Method for improving yield of recombinant human interleukin-2 (rhIL-2)

A technology of human interleukin and yield, applied in chemical instruments and methods, depsipeptides, peptides, etc., can solve the problems of disulfide bond mismatch biological activity, reduction and other problems

Inactive Publication Date: 2019-05-07
江苏金丝利药业股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, a disulfide bond must be formed between Cys-58 and Cys-105 to ensure that the polypeptide molecule has biological activity, but due to the existence of Cys-125, it is easy to cause disulfide bond mismatch or dimer to reduce its biological activity.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] A method for improving recombinant human interleukin-2 (rhIL-2) yield, comprising the steps of:

[0022] (a) Take 500 mg of rhIL-2 before purification, and the protein yield after purification by reverse-phase high-performance liquid chromatography (HPLC) is 45.5%;

[0023] (b) removing the acetonitrile by rotary evaporating the eluted dimer and mismatch mixture at 40° C. to obtain the first mixture;

[0024] (c) Dilute the first mixture with PBS buffer and SDS buffer into 60ml of the second mixture containing 50mmol / L PBS and 1% SDS by mass fraction, leave it at room temperature for 2 hours, and detect the protein concentration to be 3.56mg / ml ;

[0025] (d) diluting the second mixture to a protein concentration of 1.5 mg / mL to obtain a third mixture;

[0026] (e) adding dimercaptoethanol to the third mixture so that the concentration of dimercaptoethanol is 5 mmol / L, and heating in a water bath at 60° C. for 10 minutes to obtain a fourth mixture;

[0027] (f) Put t...

Embodiment 2

[0035] A method for improving recombinant human interleukin-2 (rhIL-2) yield, comprising the steps of:

[0036] (a) Take 100 mg of rhIL-2 before purification, and the protein yield after purification by reverse-phase high-performance liquid chromatography (HPLC) is 45%;

[0037] (b) removing the acetonitrile by rotary evaporating the eluted dimer and mismatch mixture at 35° C. to obtain the first mixture;

[0038] (c) Dilute the first mixture with PBS buffer and SDS buffer into a solution containing 40mmol / L PBS and 0.5% SDS by mass fraction. The second mixture is 15ml, left at room temperature for 2 hours, and the detected protein concentration is 3.40mg / ml ;

[0039] (d) diluting the second mixture to a protein concentration of 1.5 mg / mL to obtain a third mixture;

[0040] (e) adding dimercaptoethanol to the third mixture so that the concentration of dimercaptoethanol is 4mmol / L, and heating in a water bath at 50°C for 5 minutes to obtain a fourth mixture;

[0041] (f) Pu...

Embodiment 3

[0049] A method for improving recombinant human interleukin-2 (rhIL-2) yield, comprising the steps of:

[0050] (a) Take 1000 mg of rhIL-2 before purification, and the protein yield after purification by reverse-phase high-performance liquid chromatography (HPLC) is 46%;

[0051] (b) removing the acetonitrile by rotary evaporating the eluted dimer and mismatch mixture at 45° C. to obtain the first mixture;

[0052] (c) Dilute the first mixture with PBS buffer and SDS buffer to contain 60mmol / L PBS and 1.5% SDS solution of the second mixture 60ml, room temperature for 3 hours, the detection protein concentration is 3.63mg / ml ;

[0053] (d) diluting the second mixture to a protein concentration of 2 mg / mL to obtain a third mixture;

[0054] (e) adding dimercaptoethanol to the third mixture so that the concentration of dimercaptoethanol is 6 mmol / L, and heating in a water bath at 70° C. for 15 minutes to obtain a fourth mixture;

[0055] (f) Put the fourth mixture on the G-25 ...

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PUM

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Abstract

The invention discloses a method for improving the yield of recombinant human interleukin-2 (rhIL-2). The method comprises the steps of purification, eluent recovery after the purification, reductionof rhIL-2 dimer and wrong ligand by using 2-mercaptoethanol, reoxidation renaturation, and purification. The method performs reduction of rhIL-2 dimer and wrong ligand by using the 2-mercaptoethanol,opens disulfide bonds, and performs reoxidation renaturation and purification, so that correctly-paired disulfide bonds with biological activity can be formed so as to obtain the rhIL-2 with the biological activity, and the yield is improved by about 20 percent.

Description

technical field [0001] The invention relates to the field of preparation and purification of recombinant human interleukin-2, in particular to a method for increasing the yield of recombinant human interleukin-2 (rhIL-2). Background technique [0002] Human interleukin-2 is a lymphokine that can effectively activate immune effector cells and induce them to produce a variety of effector factors, so it has a wide range of biological activities, and it has anti-tumor, anti-infection and enhanced body immunity clinically. functions etc. Natural human interleukin-2 consists of 133 amino acids, with a cysteine ​​(Cys) at positions 58, 105, and 125. Among them, a disulfide bond must be formed between Cys-58 and Cys-105 to ensure that the polypeptide molecule has biological activity, but due to the existence of Cys-125, it is easy to cause disulfide bond mismatch or dimer to reduce its biological activity. . [0003] Recombinant human interleukin-2 (rhIL-2) is currently available...

Claims

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Application Information

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IPC IPC(8): C07K14/55C07K1/20
Inventor 翁夏蒙
Owner 江苏金丝利药业股份有限公司
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