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
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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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