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Method for separating and purifying cathepsin L in dorsal muscle of carp

A technique for separation and purification of cathepsin, which is applied in the field of separation and purification of cathepsin L in carp back muscle, to achieve good separation effect, good purity and high yield

Inactive Publication Date: 2012-05-02
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the separation and purification of CL from carp dorsal muscle

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Take 50 g of white meat from the back of live carp, add 500 mL of 20 mM Tris-HCl buffer (pH 7.0), homogenize, and centrifuge at 6000×g for 20 min at 3° C. to obtain the supernatant. The supernatant was placed in a 60°C water bath for 4 minutes, then cooled in an ice bath, and centrifuged at 8000×g for 20 minutes at 3°C ​​to obtain the supernatant. The supernatant was used as a crude enzyme solution and loaded onto a Phenyl-Sepharose column (inner diameter 1.5cm, height 20cm) that has been balanced with the eluate, and eluted with 20mM Tris-HCl buffer (pH7.5) at a flow rate of 0.5ml / min. The eluates with enzymatic activity were collected, combined, and further purified by dialysis with 20 mM Tris-HCl buffer (pH 7.5). The obtained sample was then loaded onto the pre-equilibrated DEAE-Bio-Gel A column (inner diameter 1.5cm, height 15cm), eluted with 20mM Tris-HCl buffer (pH7.5) overnight, and washed with a gradient of 0~300mM NaCl Take off (total volume 300ml). Collect ...

Embodiment 2

[0026] Take 50 g of white meat from the back of live carp, add 250 mL of 20 mM Tris-HCl buffer (pH 6.8), homogenize and centrifuge at 6200×g for 15 min at 4°C to obtain the supernatant. The supernatant was placed in a 55°C water bath for 5 minutes, then cooled in an ice bath, and centrifuged at 8300×g for 15 minutes at 4°C to obtain the supernatant. The supernatant was used as a crude enzyme solution and loaded onto a Phenyl-Sepharose column (inner diameter 1.5cm, height 20cm) that has been balanced with the eluate, and eluted with 20mM Tris-HCl buffer (pH7.5) at a flow rate of 0.5ml / min. The eluates with enzymatic activity were collected, combined, and further purified by dialysis with 20 mM Tris-HCl buffer (pH 7.5). The obtained sample was then loaded onto the pre-equilibrated DEAE-Bio-Gel A column (inner diameter 1.5cm, height 15cm), eluted with 20mM Tris-HCl buffer (pH7.5) overnight, and washed with a gradient of 0-300mM NaCl Take off (total volume 300ml). Collect the e...

Embodiment 3

[0028] Take 45 g of white meat from the back of live carp, add 360 mL of 20 mM Tris-HCl buffer (pH 7.5), homogenize and centrifuge at 6500×g for 18 min at 5°C to obtain the supernatant. The supernatant was placed in a 65°C water bath for 3 minutes, then cooled in an ice bath, and centrifuged at 8500×g for 17 minutes at 5°C to obtain the supernatant. The supernatant was used as a crude enzyme solution and loaded onto a Phenyl-Sepharose column (inner diameter 1.5cm, height 20cm) that had been balanced with the eluate, and eluted with 20mM Tris-HCl buffer (pH7.5) at a flow rate of 0.5ml / min. The eluates with enzymatic activity were collected, combined, and further purified by dialysis with 20 mM Tris-HCl buffer (pH 7.5). The obtained sample was then loaded onto the pre-equilibrated DEAE-Bio-Gel A column (inner diameter 1.5cm, height 15cm), eluted with 20mM Tris-HCl buffer (pH7.5) overnight, and washed with a gradient of 0-300mM NaCl Take off (total volume 300ml). Collect the e...

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Abstract

The invention relates to a method for separating and purifying cathepsin L. The method comprises the following steps: dissociating out the cathepsin from crude enzyme liquid of the cathepsin L by thermal treatment, eliminating influence of other enzymes with thermal instability, and then centrifuging to obtain a crude enzyme; eliminating other cathepsin such as cathepsin B, cathepsin H, cathepsinS and the like, the property of which is similar with the cathepsin L by hydrophobic gelchromatography and ion-exchange column chromatography, collecting components with enzymatic activity and then precipitating by 70% of ammonium sulfate; re-dissolving the obtained precipitate by a buffer solution and dissociating the cathepsin L from a complex formed by the cathepsin L and a natural inhibitor thereof at the same time, removing a low molecular weight enzyme inhibitor through ultrafiltration and purification and then concentrating target enzyme; and finally carrying out size-exclusion chromatography and efficient liquid-phase separation on the obtained concentrated solution to obtain the cathepsin L. The cathepsin L (CL) obtained by the method has high purity and better activity maintenance.

Description

Technical field [0001] The invention belongs to the technical field of enzyme separation and purification, and relates to a method for separation and purification of cathepsin L in the dorsal muscle of carp. Background technique [0002] Cathepsin is a type of protease found in the cells of various animal tissues (especially the lysosome). This name is derived from the Greek word for "digestion", named for R. Willstāatter (1929). It is an enzyme widely found in the lysosomes of viruses, bacteria, fungi, protozoa and protozoa, plants, mammals and humans, and belongs to intracellular proteases. It is easily activated in a weakly acidic environment and is a type of glycoprotein that is unstable in alkaline and neutral solutions (except cathepsin D, E, and S). So far, cathepsins A to Z have been reported, basically named in the order of discovery. According to the catalytic activity center of cathepsin, it can be divided into three types, serine proteases (such as A, G, R), aspart...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/64
Inventor 胡亚芹叶兴乾刘东红陈健初吴丹
Owner ZHEJIANG UNIV
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