Ulinastatin purification method based on hydrophobic column

Abstract

The invention discloses an ulinastatin purification method based on a hydrophobic column. The ulinastatin purification method comprises the following specific steps: S1: dissolving an ulinastatin crude product and pure water in a beaker together; then adding a proper amount of salt into the beaker; controlling the pH (Potential of Hydrogen) of a solution in the beaker to be 5 to 6; putting the beaker on a magnetic stirrer and uniformly stirring for 60min to 150min; then filtering by utilizing a filtering screen to obtain sediment for later use; S2: washing and drying the hydrophobic column andfixing the hydrophobic column on an operation platform; dissolving the sediment prepared by step S1 into 1 to 2 times of de-ionized water; after uniformly mixing, uniformly adding the mixture at theupper end of the hydrophobic column in sequence and then reacting for 1h to 2h; then adding a 1mol/L to 2mol/L salt type buffering solution for washing. According to the ulinastatin purification method based on the hydrophobic column, a test finds out that the average yield is 82 percent or above; the purity of purified ulinastatin reaches 99.9 percent or above and the specific activity is 5100U/mg protein at least; the shape of the ulinastatin is colorless liquid and standard requirements of China are met; a purification technology is simple and feasible and is worthy of being greatly popularized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for purifying ulinastatin based on a hydrophobic column. Background technique [0002] Ulinastatin is an acidic glycoprotein isolated and purified from the fresh urine of healthy adult males. It is a broad-spectrum protease inhibitor. It is mainly synthesized in the liver, metabolized by the kidneys and excreted in the urine. The low molecular weight components formed also have a strong inhibitory effect on hydrolases. Ulinastatin is composed of 143 amino acids, with a relative molecular mass of about 37,000 to 43,000. It belongs to protease inhibitors and is effective against serine proteases such as trypsin and α-chymotrypsin and granulocyte elastase, hyaluronidase, sulfhydrylase, and fibrinolytic enzymes. Enzymes and other enzymes have inhibitory effects, and also have the functions of stabilizing lysosomal membranes, inhibiting the release of lysosomal enzymes, inhibiti...

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Problems solved by technology

[0005] In summary, the current domestic methods for purifying human urinary trypsin inhibitors are mainly the classic bubble extraction method combined with affinity chromatography, ion exchange method, affinity chromatography, molecular sieve chromatography, metal chelation chromatography and hydrophobic Chromatography, etc., as well as the joint production process of urinary tryptin inhibitor and urinary kallikrein, these methods often need to combine multi-step chromatography due to the low adsorption efficiency of the purification method, resulting in many process steps, low product yield and long production cycle Long, not easy to enlarge and other technical defects, the invention of a hydrophobic column-based ulinastatin purification method to solve the above defects, has great application prospects

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