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

A technology for separation and purification of cathepsin, applied in the field of separation and purification of cathepsin L in carp back muscle, achieving high yield, good separation effect and good purity

Inactive Publication Date: 2010-11-17
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 the back white meat of fresh and alive carp, add 500 mL of 20 mM Tris-HCl buffer solution (pH 7.0), homogenize, and centrifuge at 6000×g for 20 min at 3°C ​​to obtain a supernatant. The supernatant was placed in a water bath at 60° C. for 4 minutes, cooled in an ice bath, and centrifuged at 8000×g for 20 minutes at 3° C. to obtain a supernatant. Load the supernatant as a crude enzyme solution onto a Phenyl-Sepharose column (inner diameter 1.5cm, height 20cm) that has been equilibrated with the eluent, and elute 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 against 20 mM Tris-HCl buffer (pH 7.5). The obtained sample was then loaded onto a pre-equilibrated DEAE-Bio-Gel A column (inner diameter 1.5 cm, height 15 cm), eluted with 20 mM Tris-HCl buffer (pH 7.5) overnight, and washed with a gradient of 0-300 mM NaCl Remove (total volume 300ml). Coll...

Embodiment 2

[0026] Take 50 g of the back white meat of fresh and alive carp, add 250 mL of 20 mM Tris-HCl buffer solution (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 water bath at 55° C. for 5 minutes, cooled in an ice bath, and centrifuged at 8300×g for 15 minutes at 4° C. to obtain a supernatant. Load the supernatant as a crude enzyme solution onto a Phenyl-Sepharose column (inner diameter 1.5cm, height 20cm) that has been equilibrated with the eluent, and elute 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 against 20 mM Tris-HCl buffer (pH 7.5). The obtained sample was then loaded onto a pre-equilibrated DEAE-Bio-Gel A column (inner diameter 1.5 cm, height 15 cm), eluted with 20 mM Tris-HCl buffer (pH 7.5) overnight, and washed with a gradient of 0-300 mM NaCl Remove (total volume 300ml). Co...

Embodiment 3

[0028]Take 45g of the back white meat of fresh and alive carp, add 360mL of 20mM Tris-HCl buffer solution (pH7.5), homogenize, and centrifuge at 6500×g for 18min at 5°C to obtain the supernatant. The supernatant was placed in a water bath at 65° C. for 3 minutes, cooled in an ice bath, and centrifuged at 8500×g for 17 minutes at 5° C. to obtain a supernatant. Load the supernatant as a crude enzyme solution onto a Phenyl-Sepharose column (inner diameter 1.5cm, height 20cm) that has been equilibrated with the eluent, and elute with 20mM Tris-HCl buffer (pH7.5) at a flow rate of 0.5ml / min. The eluates with enzyme 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 a pre-equilibrated DEAE-Bio-Gel A column (inner diameter 1.5 cm, height 15 cm), eluted with 20 mM Tris-HCl buffer (pH 7.5) overnight, and washed with a gradient of 0-300 mM NaCl Remove (total volume 300ml). Collect the elua...

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PUM

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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, cathepsin S 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 carp back muscle. Background technique [0002] Cathepsins are a class of proteases found in the cells of various animal tissues, especially in the lysosomal fraction. The name comes from the Greek word for "digestion" and was named by R. Willstāatter (1929). It is an enzyme widely present 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 class of glycoproteins that are unstable in alkaline and neutral solutions (except cathepsin D, E, and S). So far, cathepsins A to Z have been reported, basically named according to the order of discovery. According to the catalytic active center of cathepsin, it can be divided into three types, serine protease (...

Claims

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Application Information

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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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