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Separation and purification process of high-activity phytolectin

A plant lectin, separation and purification technology, applied in the fields of plant peptides, organic chemistry, peptide preparation methods, etc., can solve the problems of purity and activity detection, increase of uncertainty rate, unavoidable, etc., and achieve effective removal of impurities and easy production. Scale, the effect of scaling up production

Active Publication Date: 2022-06-10
GUANGZHOU LDEBIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although affinity chromatography can meet the requirements of high purity, high yield, and fewer purification steps, the high cost of packing material and the limitation that it cannot be separated in a long chromatographic column or in batches make the application of affinity chromatography At present, it is only limited to the laboratory stage and cannot be mass-produced industrially
[0005] Although the above methods are all purification and extraction methods for phytohemagglutinin (PHA), the actually obtained PHA is a tetramer mixture including E subunit and L subunit, which cannot separate the E subunit and L subunit of phytohemagglutinin. The L subunit is further isolated and purified, and the purity and activity of PHA-L with mitogen activity are not further tested. When it is used as a non-specific stimulator of immune cells, it will easily lead to uncertainty in the test sample. increase, it is difficult to reflect the true state of immune cells
CN112707958A discloses a method for extracting L-type and E-type lectins, which involves rough extraction of protein through steps such as soaking beans, breaking, centrifuging, and ammonium sulfate fractional precipitation, and then through hydrophobic chromatography and ion exchange chromatography. The L-type and E-type lectins are separated and purified. Although the two subtypes of PHA-L and PHA-E can be separated and extracted separately, the step of ammonium sulfate fractional precipitation is still unavoidable.

Method used

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  • Separation and purification process of high-activity phytolectin
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  • Separation and purification process of high-activity phytolectin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Separation and purification extraction of PHA-L

[0077] Pre-treatment:

[0078] Remove the well-preserved red kidney beans from the refrigerator at 2-8 °C for weighing, weigh about 1000±20g, add pure water to soak at room temperature for 4-6h, peel the peeling machine, add 4 mL 20 mM NaAC-HAc (buffer, pH = 4.5-7.4) per gram of red kidney beans, use the wall breaker to break the wall, and the solution after the wall is placed on a stirrer in the refrigerator at 2-8 °C for 300-400rpm4h or more. After thorough agitation, the slurry was first frozen by at least 12h, and re-thawed, and then the pH of the solution was adjusted using 2M dilute hydrochloric acid to 4.5-5.5, preferably 5.0, and the solution was divided into centrifuges into centrifuges, centrifuged at a centrifugal force of 14000g or more, centrifuged at a centrifugal temperature of 4 ° C for 10-30min, the supernatant was taken.

[0079] Figure 1 Is a different treatment after the supernatant electropho...

Embodiment 2

[0096] Example 2: PBMC cell stimulation activity verification

[0097] The activities of the elution gradients of the pretreatment step, SP ion exchange chromatography (20 mM NaAC-HAc, pH5.0±0.1, 50 mM NaCl, 150 mM NaCl) and the DEAE ion exchange chromatography purification step (20 mM Tris, pH8.0±0.1, 20 mM NaCl, 70 mM NaCl, 1 M NaCl) were compared in activity, as follows:

[0098] The slurry, SP ion exchange chromatography and DEAE ion exchange chromatography purification step of the pretreatment step were taken separately, and the protein concentration (PHA) determined by BCA was the starting concentration, diluted with endotoxin-free PBS to a final concentration of 1ug / 25uL, 0.2ug / 25uL.

[0099] Add 25 ul of the above diluted eluent to a 2 ml centrifuge tube free of endotoxins, respectively.

[0100]Take one tube of frozen PBMC cells for resuscitation, prepare a cell suspension (1 M / mL) and add 600 uL of cell suspension to the centrifuge tubes of the above 2 ml. Gently mix wel...

Embodiment 3

[0111] Example 3: Effect of different preparation processes on PHA-L purity and yield

[0112] The pretreatment process of Example 1 is compared with a conventional PHA purification method (fractional precipitation containing ammonium sulfate).

[0113] The conventional PHA purification method is as follows:

[0114]Step 1: Pre-processing.

[0115] With step one of the present invention, kidney beans do not peel, centrifugation to obtain supernatant.

[0116] Step 2: Ammonium sulfate graded precipitation.

[0117] 1) According to the above supernatant volume, the calculation requires the addition of solid ammonium sulfate to a saturation of 40%. And while adding supernatant while stirring to dissolve, after dissolution placed at room temperature continue to stir for 10min, stand for 1h, the solution is divided into centrifuge tubes for equilibrium, and then use high-speed centrifuge to set the centrifugal force of 10000g, centrifuge at a centrifugal temperature of 4 °C for 30min, ...

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Abstract

The invention discloses a separation and purification process of high-activity phytolectin, which comprises the following steps: fully soaking beans with pure water, peeling, mixing the peeled beans with a buffer solution, and carrying out wall breaking treatment to obtain slurry; fully stirring the slurry to fully dissolve out PHA; preliminarily removing impurities to obtain a PHA crude extract; and purifying by using SP ion exchange chromatography and DEAE ion exchange chromatography in sequence to obtain a PHA-L protein solution. The separation and purification process of some examples is simple and convenient to operate, impurities can be effectively removed, the activity of the phytolectin can be reserved, and the high-purity and high-activity phytolectin can be obtained.

Description

Technical field [0001] The present invention belongs to the field of biotechnology, specifically relates to a highly active plant lectin separation and purification process. Background [0002] Leguminaceous plant lectin (Phytohemagglutinin, PHA) is a glycosylated modified protein extracted from legume seeds, which is a tetrameric glycoprotein composed of two highly homologous and biologically different subunits, which includes erythrocyte lectins (PHA-E) composed of E subunits, and leukocyte lectins (PHA-L) composed of L subunits. PHA-E has a high coagulation activity, which can promote red blood cell agglutination, and PHA-L has a high mitotic activity. Among them, PHA is mostly used as a highly effective immune enhancer in the biomedical field, mainly because PHA-L can bind to the protein glycosyl group on mammalian blood cells (such as lymphocytes), produce non-specific stimulating activity, promote lymphocyte division and value-add, and have a variety of immune effects, such...

Claims

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Application Information

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IPC IPC(8): C07K14/42C07K1/18C07K1/14
CPCC07K14/42
Inventor 杨翔黄彬谢桂华
Owner GUANGZHOU LDEBIO TECH CO LTD
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