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Method for separating and purifying curcin from seeds of jatropha curcas

A technology for separation and purification of toxic proteins, applied in the field of separation and purification of plant natural proteins, can solve the problems of long process cycle, increase in column volume cost, unfavorable production cost control and maintenance of end product activity, etc., to achieve the improvement of end product purity , The effect of improving the separation and purification capacity of the process

Inactive Publication Date: 2010-11-24
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Second, due to the slow flow rate of gel filtration chromatography used in this method, the process takes a long time, which is not conducive to the maintenance of Curcin activity
[0009] Third, according to the description of the method, the process takes about 58 hours (dialysis takes 48 hours, and gel filtration takes 10 hours), so the process cycle is longer, which is not conducive to the control of production costs and the maintenance of the activity of the final product
However, gel filtration packing is very expensive, and the increase in column volume will inevitably lead to a substantial increase in cost
At the same time, due to the substantial increase in column volume, the chromatographic column also needs to be replaced with a thicker or longer chromatographic column at the same time, which will inevitably lead to a substantial increase in production costs, so the scalability of this method is not enough

Method used

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  • Method for separating and purifying curcin from seeds of jatropha curcas
  • Method for separating and purifying curcin from seeds of jatropha curcas
  • Method for separating and purifying curcin from seeds of jatropha curcas

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The first step: prepare the crude extract of Curcin. The crude extract of Curcin was prepared according to the method disclosed in prior art 1.

[0041] The second step: desalting and buffer exchange. First use buffer A, that is, Tris-HCl buffer solution with a Tris concentration of 35mM and a pH of 8.2 for column equilibration, equilibrate for 5 column volumes, then load the crude extract of Curcin as a sample, and collect the first sample after loading. 280nm absorption peak components. Among them, the instrument uses AKTA purifier UPC 10 (product of General Electric Medical Group), the filler uses Sephadex G 25 superfine filler, the flow rate is 200cm / h, and the detector wavelength is 280nm.

[0042] The third step: anion exchange chromatography. First use buffer A (the ratio, concentration and pH are the same as the previous step) for column equilibration, equilibrate for 5 column volumes, then load the components collected in the desalting and buffer exchange st...

Embodiment 2

[0046] The first step: prepare the crude extract of Curcin. The crude extract of Curcin was prepared according to the method disclosed in prior art 1.

[0047] The second step: desalting and buffer exchange. First use buffer A, that is, Tris-HCl buffer with a Tris concentration of 20mM and a pH of 8.0 for column equilibration, equilibrate for 5 column volumes, then load the crude extract of Curcin as a sample, and collect the first 280nm absorption peak components. Wherein the flow rate is 150cm / h.

[0048] The third step: anion exchange chromatography. First use buffer A (the ratio, concentration and pH are the same as the previous step) for column equilibration, equilibrate for 5 column volumes, then load the components collected in the desalting and buffer exchange steps as samples, and collect the breakthrough after loading Components (components not bound to fillers). Wherein the flow rate is 150cm / h.

[0049] Step Four: Dilution. The fraction collected in the thir...

Embodiment 3

[0053] The first step: prepare the crude extract of Curcin. The crude extract of Curcin was prepared according to the method disclosed in prior art 1.

[0054] The second step: desalting and buffer exchange. First use buffer A, that is, Tris-HCl buffer with a Tris concentration of 40 mM and a pH of 8.0 for column equilibration, equilibrate for 5 column volumes, then load the crude extract of Curcin as a sample, and collect the first 280nm absorption peak components. Wherein the flow rate is 150cm / h.

[0055] For the chromatographic peak pattern (or chromatogram) corresponding to this step, see figure 1 . See the specific collection location figure 1 In the middle shaded part, the electrophoresis results of the collected fractions are shown in Figure 4 Lane 3.

[0056] The third step: anion exchange chromatography. First use buffer A (the ratio, concentration and pH are the same as the previous step) for column equilibration, equilibrate for 5 column volumes, then lo...

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Abstract

The invention discloses a method for separating and purifying curcin from seeds of jatropha curcas, which comprises the following steps: firstly carrying out desalinization and buffer solution exchange on prepared curcin crude extract solution, collecting a component of a fist 280nm absorption peak after sample loading, then carrying out anion exchange chromatography on the collected component, collecting a penetrating component after the sampling loading, diluting the collected penetrating component by using buffer solution, finally carrying out cation exchange chromatography on the component solution after dilution, and collecting the component of the highest 280nm absorption peak, which is a just final curcin pure product. As the separation and purification capacity of the method is high, the method can not only improve the purity and the activity of the final product and improve the yield of the final product, but also greatly shorten the time consumption of the process cycle, andhave good process scaling-up property and low production cost, thereby providing the powerful support for research and development of various products of the curcin and deep and refined basic research based on the curcin.

Description

technical field [0001] The invention belongs to the technical field of separation and purification of plant natural proteins, and in particular relates to a method for separation and purification of Curcin, a jatropha seed poison protein. Background technique [0002] Jatropha curcas belongs to Euphorbiaceae (Euphorbiaceae) oil plants, widely distributed in Southwest China, is an important resource plant in my country. The oil content of Jatropha curcas seeds is as high as 40%. It is an ideal raw material for biodiesel production and has a good application prospect. At the same time, the protein content in Jatropha curcas seeds is also quite rich. In recent years, it has been reported in the literature that a toxin protein was isolated from Jatropha curcas, named Curcin. It has been proved by experiments that Curcin belongs to type I ribosome inactivating protein (Lin Juan et al., "Separation, purification and mechanism of action of Jatropha curcas ribosome inactivating pr...

Claims

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Application Information

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IPC IPC(8): C07K1/18C07K14/415
Inventor 徐莺蔡峰陈放
Owner SICHUAN UNIV
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