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Method for preparing pancreatic kallikrein using expanded bed adsorption technology and affinity chromatography

A technology of pancreatic kallikrein and adsorption chromatography, which is applied in the field of preparation of pancreatic kallikrein, can solve the problems of affecting the separation effect of chromatography and the reduction of centrifugation efficiency, so as to improve production efficiency, simplify production steps and improve product quality Effect

Inactive Publication Date: 2009-01-07
NANCHANG WANHUA BIOCHEM PHARMA
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Problems solved by technology

However, when the tissue extract contains a large number of high-viscosity solid particles, the centrifugation efficiency will be greatly reduced, which will affect the subsequent chromatographic separation effect

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  • Method for preparing pancreatic kallikrein using expanded bed adsorption technology and affinity chromatography

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Experimental program
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[0017] 1. Extraction: Take 5kg of fresh or frozen porcine pancreas, remove fat tissue and connective tissue and other debris, cut into pieces, then mince, add 3 (L / kg) times the amount of sodium acetate-acetic acid at pH 6.0 Buffer solution (Ca2+ concentration is 5%), at the same time, add appropriate amount of EDTA to the extract to chelate heavy metal ions, stir slowly (70-100rpm) at 4°C for 3 hours, let stand, filter, and obtain 18L of filtrate, which is pig Pancreatic kallikrein crude extract, sampling and determination of protein concentration is 48.80mg / ml, pancreatic kallikrein potency is 24.89KU / ml;

[0018] 2. Expanded bed adsorption and purification: choose an adsorption column (10em×50cm), install 1000ml of Streamline-DEAE, equilibrate the column with 0.01M phosphate buffer with pH 6.0, and load 18L of the crude extract of porcine pancreatic kallikrein adsorption. After the adsorption is completed, use 20% (V / V) glycerol solution to effectively wash out the unadsor...

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Abstract

The invention relates to a method for using extension bed adsorption technology and affinity chromatography to prepare for pancreas kallikrein which is characterized in that it first uses extension bed adsorption technology to directly initial purify the pig's pancreas residuary product and then uses affinity chromatography to purify it to obtain the pancreas kallikrein with high purity. The obtained pancreas kallikrein unit proficiency>800KU / mg; the specific activity over 1400KU / mg.p; the activity recovery ratio>90%.

Description

technical field [0001] The invention relates to a method for preparing pancreatic kallikrein by expanding bed adsorption and affinity chromatography. Background technique [0002] Pancreatic Kallikrein, also known as Pancreatic Kininogenase or Kallidinogenase, is a subclass of the serine protease family. Pancreatic kallikrein can hydrolyze kininogen to produce active substance kinin, which can mediate a wide range of physiological and biochemical reactions, including expanding capillaries, increasing vascular permeability, improving microcirculation, and adjusting blood pressure. Pancreatic kallikrein is not only used in the treatment of hypertension, cardiovascular and cerebrovascular diseases, diabetes, and male infertility diseases, but also used in the treatment of colic, vasospasm, acral paresthesia, frostbite, burns and other diseases, and It is also receiving more and more attention in the emerging health care products and anti-aging cosmetics industries. [0003] P...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/50C12N9/76C07K1/22
Inventor 张万山杨华英姜春华张志武曾英刘兰花朱月华李菊根杨益辉付三枚
Owner NANCHANG WANHUA BIOCHEM PHARMA