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Gene-expressed human tissue kallikrein protein separating and purifying method

A kallikrein, separation and purification technology, which is applied in the field of separation and purification of gene expression human tissue kallikrein protein, can solve the problems of large loss, complicated, unsuitable for large-scale production in multi-step operations, and reduce operating costs. , the effect is good, the effect is conducive to preservation

Inactive Publication Date: 2004-04-14
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Its disadvantage is that salting out and chromatographic separation are completed under multi-step operation, which is very complicated, and the multi-step operation has a large loss, so it is not suitable for large-scale production

Method used

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  • Gene-expressed human tissue kallikrein protein separating and purifying method
  • Gene-expressed human tissue kallikrein protein separating and purifying method
  • Gene-expressed human tissue kallikrein protein separating and purifying method

Examples

Experimental program
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Effect test

Embodiment 1

[0044] The separation and purification method of the present invention comprises steps such as centrifugation, ion exchange chromatography, affinity chromatography; Specific process comprises:

[0045] 1. Separation of cell culture supernatant:

[0046] Place the cell culture solution in a polycarbonate centrifuge tube, balance with a Pasteur pipette; centrifuge in a high-speed centrifuge at 4°C, 2000-000r / min for 20-30min.

[0047] Pour the supernatant into a polypropylene funnel with a filter cloth, filter it into a polypropylene container, and store it in cold storage for further ion exchange chromatography.

[0048] 2. Ion exchange chromatography separation:

[0049] DEAE-Sepharose ion exchange chromatography method: chromatographic column: 16mm × 20cm glass packed column; filler: DEAE-Sepharose ion exchange filler; peristaltic pump: miniplus3, Gilson company; detection wavelength: 280nm; flow rate: 5ml / min

[0050] ①Equilibrate the column with 20mM Tris-HCl pH7.6 buffer...

Embodiment 2

[0083] 1. Separation of cell culture supernatant: same as Example 1;

[0084] 2. Ion exchange chromatography separation:

[0085] DEAE-Sepharose ion exchange chromatography method: chromatographic column: 16mm × 20cm glass packed column; filler: DEAE-Sepharose ion exchange filler; peristaltic pump: miniplus3, Gilson company; detection wavelength: 280nm; flow rate: 5ml / min

[0086] ①Equilibrate the column with 20mM Tris-HCl pH6 buffer solution, then dilute the supernatant with water and pump it into the column. Elute with 20mM Tris-HCl pH6 buffer containing 0.1M NaCl, collect fraction a; change to 20mM Tris-HCl pH6 buffer containing 0.3M NaCl for elution after the detected value is lower than 0.05, and collect fraction b. After detection by SDS-PAGE, it was found that human tissue kallikrein was contained in passing through peak, fraction a and fraction b. The result is as Figure 10 As shown, the sixth lane from the left in the figure is fraction b.

[0087] Following steps ...

Embodiment 3

[0090] 1. Separation of cell culture supernatant: same as Example 1;

[0091] 2. Ion exchange chromatography separation:

[0092] DEAE-Sepharose ion exchange chromatography method: chromatographic column: 16mm × 20cm glass packed column; filler: DEAE-Sepharose ion exchange filler; peristaltic pump: miniplus3, Gilson company; detection wavelength: 280nm; flow rate: 5ml / min

[0093] ①Equilibrate the column with 20mM Tris-HCl pH8.5 buffer, then dilute the supernatant with water and pump it into the column. Elute with 20mM Tris-HCl pH8.5 buffer containing 0.1M NaCl, collect fraction a; change to 20mM Tris-HCl pH8.5 buffer containing 0.3M NaCl after the detection value is lower than 0.05, Fraction b is collected.

[0094] Following steps are with embodiment 1.

[0095] Yield 82%. However, the content of impurity proteins in the first step component b detected by SDS-PAGE increased significantly, which brought a lot of trouble to the follow-up work.

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Abstract

The present invention relates to extraction of human tissue kallikrein, which is a separation and purification method of gene expressed human tissue kallikrein protein, and its operation is as follows: (1). supernatant separation; (2) chromatography; 1). step gradient elution; firstly, using buffer solution with 20-30mM and pH 7-8 to balance column, then pumping the supernatant into the column; using buffer solution containing 0.1-0.2 M of NaCl to make elution, then using the buffer solution containing 0.3-0.5 M of NaCl, collecting fraction for stand-by; firstly, using NaAc with 10-20 mM and pH 5.0-6.0 to balance column, then making step gradient elution and collecting fraction; and making linear gradient elution, in eluent salt component range is 0.1M NaCl to 0.5M NaCl, collecting fraction and storing; (3). utilizing affinity chromatography to make separation and purification.

Description

technical field [0001] The invention relates to the extraction of human tissue kallikrein, in particular to a method for separating and purifying gene-expressed human tissue kallikrein protein. Background technique [0002] In 1930, someone first discovered a protein with special functions in urine and pancreas, and called it kallikrein. Later, the kallikrein system was also found in plasma and many tissues. In addition to participating in inflammatory response and pain-inducing effects, this system also has an important endogenous antihypertensive effect, and is also related to the pathogenesis of hypertension, kidney disease and other diseases. In the 1980s, foreign countries had noticed the various physiological functions of kallikrein and conducted systematic research on it. There have been many reports on the study of its gene locus, tissue expression and physiological function. [0003] Kallikrein is a serine protease that belongs to a subclass of the serine pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/18C07K1/22C07K1/36C12N9/64
Inventor 张玉奎张维冰杨青徐茂兰
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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