CN105969788B - A kind of preparation method of soybean antifreeze protein, application - Google Patents

Abstract

The present invention is applicable to the field of biotechnology, and provides a method for preparing soybean antifreeze protein, comprising the following steps: transforming a plasmid containing the expression sequence of the antifreeze protein into Escherichia coli for protein expression; using an affinity layer The protein is purified by analysis method; after purification, the His tag is cut off with thrombin to obtain antifreeze protein; the antifreeze protein is PM1 protein and PM1‑N short peptide. The present invention also provides the application of PM1 protein or PM1-N short peptide in preparing transgenic frost-resistant or cold-resistant plants; the application of PM1 protein or PM1-N short peptide in preparing cryoprotectant for biological products. The preparation method of the PM1 protein and the PM1-N short peptide provided by the invention has a simple preparation process and is convenient for mass production. The invention also provides the application of PM1 protein or PM1-N short peptide, which not only expands new space for the application of PM1 protein or PM1-N short peptide, but also accelerates the progress of related research on PM1 protein or PM1-N short peptide .

Description

Preparation method and application of soybean antifreeze protein

technical field

The invention belongs to the field of biotechnology, and in particular relates to a preparation method and application of soybean antifreeze protein.

Background technique

When organisms are subjected to low temperature stress, the intracellular environment is dehydrated and crystals are formed, which in turn causes cell membrane structure rupture or protein inactivation, and cells or organelles are damaged, resulting in damage to the organism. Therefore, the most important and basic elements for organisms to resist low-temperature freezing injury and cold injury are: 1. Avoid intracellular freezing; 2. Improve the low-temperature stability of cell membrane (structure) and biological macromolecules. Under low temperature conditions, some organisms will produce proteins that can improve biological antifreeze ability. For example, in the 1960s, a specific protein that can prevent the growth of ice crystals - antifreeze protein (AFP) was found in the serum of marine fish in the polar region. ). Most of the antifreeze proteins discovered so far are ordered proteins, which have certain selective characteristics for the protected objects. The discovery of antifreeze protein has laid an important foundation for a wide range of potential applications in agricultural low-temperature breeding, food industry, and pharmaceutical industry.

Soybean is the main economic crop in my country. Scientists have discovered a new type of antifreeze protein—PM1 protein from soybean seeds. PM1 protein is a kind of LEA protein (late embryogenesis abundant protein, LEA), which belongs to inherently disordered protein. , presents an unstructured state in natural solution. Compared with ordered proteins, disordered proteins are thermally stable, highly soluble, and non-specifically bound to protected objects. The existing PM1 protein extraction methods are relatively cumbersome, and the extracted PM1 protein is of low purity; for PM1 protein, it is generally accepted that it has a wide range of application prospects, but its specific use has not yet been determined.

Contents of the invention

The invention adopts a preparation method and application of soybean antifreeze protein, aiming at expanding the specific application of PM1 protein and PM1-N short peptide (N-terminal short peptide of PM1) as antifreeze protein.

The present invention is achieved in that a kind of preparation method of soybean antifreeze protein comprises the following steps:

Transforming the plasmid containing the antifreeze protein expression sequence into Escherichia coli for protein expression;

Protein purification by affinity chromatography;

After purification, the His tag is cut off with thrombin to obtain the antifreeze protein; the antifreeze protein is PM1 protein or PM1-N short peptide.

Further, the parameters of the chromatography column used in the affinity chromatography are as follows: filler: Chelating Sepharose Fast Flow 4BTM, the flow rate is 3 mL/min.

The invention also provides the application of PM1 protein or PM1-N short peptide in preparing transgenic frost-resistant or cold-resistant plants.

The present invention also provides the application of PM1 protein or PM1-N short peptide for preparing cryoprotectant of biological products.

Further, the biological products include protein and enzyme preparations.

Further, the biological products include cells, embryos, tissues or organs.

Further, the biological product is rabbit red blood cells.

Further, the cryoprotectant also contains glycerin.

Further, the cryoprotectant also contains trehalose.

Further, the PM1 protein or PM1-N short peptide is a recombinant protein.

The beneficial effect of the present invention lies in that the preparation method of the PM1 protein or PM1-N short peptide provided by the present invention has a simple preparation process, the obtained protein has high purity, and is convenient for large-scale fermentation production. The present invention provides the functions of PM1 protein and PM1-N short peptide through research, using the PM1 protein or PM1-N short peptide to prepare transgenic frost-resistant or cold-resistant plants; using the PM1 protein to prepare biological products cryoprotectant, said biological products include cells, embryos, tissues or organs, as well as protein and enzyme preparations. The application of PM1 protein or PM1-N short peptide provided by the present invention not only expands new space for the application of PM1 protein, points out the direction for the application of PM1 protein, but also accelerates the progress of related research on PM1 protein.

Description of drawings

Fig. 1 is the sequence and electrophoresis diagram of PM1 protein and its short peptide PM1-N provided by the embodiment of the present invention;

2 is a schematic diagram of the turbidity detection results of liposomes after freeze-thaw treatment provided in Example 1 of the present invention;

3 is a schematic diagram of the particle size measurement results of liposomes after freeze-thaw treatment provided in Example 1 of the present invention;

Figure 4 is a microscopic view of samples before and after freeze-thaw treatment provided in Example 1 of the present invention, wherein Figure 4a is the group without PM1 and PM1-N short peptides, Figure 4b is the group with PM1 protein, and Figure 4c is the group with PM1- N short peptide group;

Fig. 5 is a schematic diagram of the measurement results of the protective effect of PM1 protein on rabbit erythrocytes provided by Example 2 of the present invention.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

PM1蛋白在NCBI(美国国立生物技术信息中心)上的氨基酸序列为：MQGGKKAGESIKETATNIGASAKAGMEKTKATVQEKAERMTARDPVQKELATQKKEAKMNQAELDKQAARQHNTAAKQSATTAGHMGHGHHTTGTGTGTATYSTTGEYGQPMGAHQTSAMPGHGTGQPTGHVTEGVVGSHPIGTNRGPGGTATAHNTRAGGKPNDYGYGTGGT(SEQ ID NO:1)，

(Available from http://www.ncbi.nlm.nih.gov/protein/NP_001238562.1).

The PM1-N short peptide is the N-terminal short peptide of PM1, and its amino acid sequence is: MQGGKKAGESIKETATNIGASAKAGMEKTKATVQEKAERMTARDPVQKELATQKKEAKMNQAELDKQAARQHNTAAKQSATTAG (SEQ ID NO: 2).

Prepare PM1 protein or PM1-N short peptide according to the technical scheme of the present invention, the process is as follows:

Transform the pET28a/PM1 plasmid and pET28a/PM1-N plasmid into E. coli BL21Star for protein expression;

Protein purification by affinity chromatography;

After purification, the His tag was excised with thrombin to obtain PM1 protein and PM1-N short peptide, respectively.

Specifically, the chromatography column parameters used in the affinity chromatography are as follows: column length: 20cm; diameter: 16mm, packing: Chelating Sepharose Fast Flow 4BTM, packing volume: 10mL; manufacturer: Amersham Biosciences, flow rate is 3mL/min .

The electrophoretic images of the prepared PM1 protein and PM1-N short peptide are shown in Figure 1, from which it can be seen that the molecular weight of PM1 protein is about 20-22kDa, and the molecular weight of PM1-N short peptide is about 11kDa.

PM1 protein and PM1-N short peptide are inherently disordered proteins. They are in a state of disordered structure in natural solution and have no obvious selectivity for the protected objects. The functions of the prepared PM1 protein and PM1-N short peptide are tested through specific examples in order to explore their applications.

Example 1 The preparation of artificial liposomes and the stabilizing effect of PM1 protein and PM1-N short peptide on liposomes after freeze-thaw treatment

The chemical composition of the cell membrane is mainly composed of phospholipids, a small amount of proteins and polysaccharides. We choose POPC (oleoylphosphatidylcholine) to prepare artificial liposomes. Weigh 40 mg of POPC, dissolve POPC completely with 500 μl of chloroform, slowly remove chloroform with nitrogen, place POPC in a vacuum environment for 1 h, then re-dissolve POPC with 1 ml of phosphate buffer (pH 7.4), and squeeze out the dissolved POPC The press is repeatedly extruded to form liposomes with a diameter of about 100 nm through a 100 nm polycarbonate membrane. The prepared liposomes were quantified with a phospholipid quantification kit and diluted to 20 mg/ml. Two groups of 100 μl liposomes were added to equal volumes of phosphate buffer (control group without PM1 protein), 0.8 mg/ml liposomes, respectively. PM1 protein, 0.8 mg/ml PM1-N short peptide (plus PM1 protein, PM1-N short peptide group). After incubating at 28°C for 30 minutes, freeze at -80°C for 30 minutes, dissolve at 28°C for 30 minutes, and repeat 3 times.

(1) Turbidity detection of liposomes after freezing and thawing:

Turbidity is an optical effect that reflects the interaction of incoming light with suspended particles in a solution and characterizes the degree to which light is hindered from passing through a layer of water. The measurement of turbidity reflects the amount of transmitted light or scattered light of the sample, that is, the smaller the intensity of transmitted light or the greater the intensity of scattered light of the solution, the greater the turbidity of the aqueous solution. Take 20 μl of each sample before and after freeze-thaw treatment and dilute to 200 μl with buffer solution, and measure its OD ₄₀₀ value with an ultraviolet spectrophotometer, as shown in Figure 2. The OD ₄₀₀ value of liposomes without PM1 protein and PM1-N short peptide group was lower; after freeze-thaw treatment, the turbidity of the solution increased significantly, indicating that the liposomes after freeze-thaw treatment had changed, meaning The liposomes were destroyed. However, the turbidity of the liposome samples added with PM1 protein and PM1-N short peptide did not change or increased slightly. Very good stabilizing effect.

(2) The particle diameter measurement of liposome after freezing and thawing:

When light passes through an inhomogeneous medium, light scattering occurs, and the scattered light contains information such as particle size, shape, structure, and composition, composition, and concentration. Particle size analyzer is to use light scattering technology to measure the size distribution of particles in solution and determine the diameter of particles in solution. Get 80 μl of freshly prepared liposomes and dilute to 4ml with phosphate buffer, measure its particle size with a particle size analyzer, as shown in Figure 3. The prepared liposome particle size is 100-200nm; After repeated freezing and thawing, 73% of the liposome particle size does not change, and 27% of the larger particle size liposomes (about 1000 nm) -3000nm), showing that the particle size of some liposomes after the freeze-thaw treatment is significantly increased, that is, the freeze-thaw treatment will cause the liposomes to rupture and then fuse to form liposomes with larger particle sizes. Add PM1 protein sample in liposome solution, after freezing and thawing, the particle size of 100% liposome does not change obviously; The plastid particle size range is 100-200nm. The above results show that PM1 protein and PM1-N short peptide can maintain or better maintain the stability of liposomes after freeze-thaw treatment.

(3) Microscopic observation of liposomes after freezing and thawing:

Microscopy can directly reflect the morphological changes of liposomes after freezing and thawing. Take 5 μl of the prepared liposome sample and observe it under a phase-contrast microscope. The observed field of view is homogeneous (left in Figure 4a); after freeze-thaw treatment, there are obvious aggregations of liposomes in the field of view. objects (Fig. 4a right). In the samples of PM1 protein and PM1-N short peptide added to the liposome solution, only a small amount of liposome aggregates appeared in the sample after freeze-thaw treatment (Fig. 4b, Fig. 4c).

Based on the above research results, it can be seen that freezing and thawing can change the volume of liposomes, that is, freezing and thawing can destroy liposomes, and the damaged liposomes can also undergo refusion. The main reason for the increase in particle size. Additionally, there is another possibility that freeze-thawing can cause liposomes to increase in viscosity, allowing them to aggregate to form liposome aggregates. Adding PM1 protein and PM1-N short peptide into the liposome solution can prevent liposome damage and refusion caused by freezing and thawing, and can also prevent liposomes from aggregating with each other.

Example 2 Determination of the protective effect of PM1 protein on rabbit erythrocytes

Prepare 50 μl of a mixture of 0.2% fresh rabbit red blood cells and an equal volume of citrate buffer. Then PM1 (final concentration: 5 mg/ml), glycerol (final concentration: 5%), and PM1+glycerol were respectively added to the solution containing rabbit red blood cells. After the solution was incubated at 4°C for 30 min, the number of rabbit blood cells was counted with a cell counter. Then the above solution was frozen at -20°C for 30 minutes, and thawed at room temperature for 30 minutes. After repeated three treatments, the cells were counted.

The results are shown in Figure 5, the number of rabbit erythrocytes after freeze-thaw treatment was significantly reduced, and the remaining cells were only 54%; after adding PM1 protein to rabbit erythrocytes, the number of remaining cells was 72%, which was significantly higher. The remaining amount of rabbit erythrocytes after freezing and thawing without adding PM1 protein. If 5% glycerol is added to the rabbit red blood cell solution, the remaining amount of rabbit red blood cells after freezing and thawing is about 58%. PM1 protein and 5% glycerol, the remaining cell mass increased to 87%, indicating that they have a more obvious protective effect on rabbit erythrocytes treated by freezing and thawing. It can be seen that PM1 protein and glycerol have a significant synergistic protective effect on rabbit erythrocytes.

From the above results, it can be known that adding PM1 protein and glycerol to rabbit erythrocytes can significantly improve the stability of cells after freeze-thaw treatment. This result is consistent with the protective effect of PM1 on liposomes under freeze-thaw.

Example 3 Protective effect of PM1 protein + trehalose on rabbit erythrocytes

0.2% rabbit red blood cells were mixed with PM1 (concentration 5 mg/mL), trehalose (final concentration 0.75 mg/mL) and PM1+trehalose respectively. Incubate at 4°C for 30 min. Count the number of rabbit blood cells with a cell counter. After the above mixture was added to the PM1 protein, the samples were frozen at -20°C for 30 minutes, and then thawed at room temperature for 30 minutes. After repeated treatment three times, the cells were counted. The ratio of the number of remaining cells after freezing and thawing to the number of unfrozen-thawed red blood cells was calculated and compared.

The results are shown in Figure 5, adding 0.75 mg/ml trehalose to the rabbit red blood cell solution, the remaining rabbit red blood cell volume after freeze-thawing was 61%, that is, the addition of trehalose did not show any effect on the freeze-thawed rabbit red blood cells. protective and stabilizing effect; if PM1 and 0.75mg/mL trehalose are added to the rabbit red blood cell solution, the remaining amount of frozen-thawed rabbit red blood cells is 89%. It can be seen that PM1 and 0.75mg/mL have a significant effect on rabbit red blood cells , Synergistic protective effect. It can be known that adding PM1 protein and trehalose to rabbit erythrocytes can significantly improve the stability of the cells after freeze-thaw treatment. This result is consistent with the protective effect of PM1 on liposomes under freeze-thaw.

Based on the above examples, it can be seen that soybean PM1 protein and PM1-N short peptide can be used as plant antifreeze protein for the production of transgenic antifreeze plants; PM1 protein can also be used as cryopreservative for biological products, such as protein (enzyme preparations), cells (such as egg cells, red blood cells), embryos, tissues, organs (such as donated organs); it can also be used for freezing, storage, transportation, and thawing of food. In addition, the combination of soybean PM1 protein and PM1-N short peptide with glycerol or trehalose can enhance the protective effect.

Since the PM1 protein and PM1-N short peptide are disordered proteins, they are in a state of disordered structure and have no obvious selectivity for the protected objects. Therefore, it has a broader application prospect.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (8)

1.PM1 albumen or PM1-N small peptide are used to prepare the application of the freezing protective agent of biological based article；

The PM1 protein amino acid sequence are as follows:

MQGGKKAGESIKETATNIGASAKAGMEKTKATVQEKAERMTARDPVQKELATQKKEAKMNQAELDKQAARQH NTAAKQSATTAGHMGHGHHTTGTGTGTATYSTTGEYGQPMGAHQTSAMPGHGTGQPTGHVTEGVVGSHPIGTNRGP GGTATAHNTRAGGKPNDYGYGTGGT；

The PM1-N small peptide is the N-terminal small peptide of PM1, amino acid sequence are as follows:

MQGGKKAGESIKETATNIGASAKAGMEKTKATVQEKAERMTARDPVQKEL ATQKKEAKMNQAELDKQAARQ HNTAAKQSATTAG。

2. application as described in claim 1, which is characterized in that the biology based article includes protein formulation.

3. application as claimed in claim 2, which is characterized in that the protein formulation is protease preparation.

4. application as described in claim 1, which is characterized in that the biology based article includes cell, tissue or organ.

5. application as described in claim 1, which is characterized in that the biology based article is rabbit erythrocyte.

6. application as described in claim 1, which is characterized in that also contain glycerol in the freezing protective agent.

7. application as described in claim 1, which is characterized in that also contain trehalose in the freezing protective agent.

8. application as described in claim 1, which is characterized in that the PM1 albumen or PM1-N small peptide are recombinant protein.