CN110498847B - Preparation and crystallization method of rice receptor protein RGA5A _ S - Google Patents

Abstract

The invention discloses a preparation and crystallization method of rice receptor protein RGA5A _ S. The preparation method of the protein RGA5A _ S comprises the following steps: expressing a rice receptor protein RGA5A _ S by using a prokaryotic expression system, and sequentially passing the cracked supernatant through an affinity chromatography column and a molecular sieve chromatography column after thallus cracking to finally obtain a purified target protein; aiming at the characteristic that protein RGA5A _ S contains a metal ion structure domain, 100 mu M Zn is added into a culture medium when recombinant bacteria are induced to shake culture²⁺And the stability of the protein is improved. The present invention also provides methods for crystallizing RGA5A _ S. The protein crystallization conditions are screened and optimized by a shotgun method and a Sudoku method, and the result is that the protein RGA5A _ S is in the MNH range of 0.18-0.22₄NO₃And 18-22% of PEG3350, the obtained crystal is large, the quality is good, the resolution ratio is high, and the repeatability of the method is good.

Description

Preparation and crystallization method of rice receptor protein RGA5A _ S

Technical Field

The invention belongs to the field of protein crystallization, and particularly relates to a preparation method and a crystallization method of rice receptor protein RGA5A _ S.

Background

Rice is one of the three major economic crops in the world, and is the staple food for over 50% of the population worldwide (Liu and Wang, 2016). Food safety is the first major thing in global economic production today, and the continuous increase in population is the original power of the development of the world rice industry (zhongying et al, 2010), and the world rice has increased by at least 38% to meet the human needs by 2030 (zhang et al, 2014). Therefore, the method ensures the stable yield and quality of the rice, directly relates to the problems of safe grain supply, people's temperature fullness and the like, and has very important strategic significance. However, the safe production of rice is endangered by diseases and pests throughout the year, wherein the rice blast caused by Magnaporthe oryzae is the most common and extremely harmful fungal disease in rice diseases and pests. The occurrence of rice blast germs has the characteristics of multiple spots, wide range, large disease occurrence area, centralized occurrence area, serious harm degree, large inter-area disease occurrence characteristic difference and the like. Not only can infect rice, but also can infect various gramineous crops and weeds such as barley, wheat, teff and the like, so that the quality of the crops is reduced. On average, rice blast causes a yield loss of 10% to 30% of rice per year, and this is sufficient to solve the 2.12 to 7.42 million population satiety problem (Fisher et al, 2012). Thus, rice blast is rated as the first of ten pathogenic fungi (Dean et al, 2012). At present, disease-resistant varieties and chemical bactericides are mainly adopted in agricultural production to prevent and control rice blast germs. With the improvement of living standard and environmental awareness of people, food safety and quality are more and more emphasized, and the green and environmental life is realized, and the use of chemical pesticides is more and more limited. Therefore, the breeding of disease-resistant varieties becomes crucial in the work of controlling rice blast; the breeding of disease-resistant varieties has the defects of long period, poor resistance stability and the like. In order to meet the increasing grain requirements of people, the synergistic molecular mechanism of the rice receptor and the rice blast germs needs to be deeply understood, and a scientific and reasonable theoretical basis is provided for disease-resistant variety breeding and variety layout.

Proteins are basic organic substances constituting cells, and are also main contributors and executives of life activities, and analysis of their molecular structures is of great importance for understanding their functions. The structural biology mainly uses biological functions as a main line, analyzes three-dimensional structures of biological macromolecules and compounds thereof by means of molecular biophysics and biochemistry, and explains molecular mechanisms of proteins, nucleic acids and the like which play biological functions from the atomic perspective. Therefore, the deep understanding of the three-dimensional structure of the protein is not only beneficial to people to deep understanding of the biological function of the protein, but also provides a theoretical basis for the application fields of genetic improvement and reasonable layout of rice disease-resistant varieties, drug design, vaccine research and development and the like. X-ray crystallography is the most reliable method for analyzing the three-dimensional structure of the protein. On one hand, the technology has strict requirements on protein purity (95-99%), stability, biological activity and the like; another bottleneck is the crystallization process. Therefore, obtaining high-quality protein samples and high-quality crystals with good stability and repeatability has important significance for promoting the structure biological analysis of biomacromolecule interaction mechanisms such as protein-protein, protein-nucleic acid and the like.

Rice and Pyricularia oryzae are one of the main research model systems of plant and pathogen interaction. At present, 25 rice R genes and 11 rice blast germ avirulence genes are cloned, and the interaction of the two genes conforms to a typical gene-pair gene hypothesis (Flor, 1971). Research has shown that a single R gene in rice is not enough to resist the invasion of pathogenic bacteria, and needs to rely on paired R genes to participate in defense reaction together. The rice disease-resistant gene Pia consists of two R genes, namely RGA4 and RGA5, and is translated into CC-NBS-LRR protein; homologous and heterologous complexes can be formed in the host (Cesari et al, 2014; Cesari et al, 2013; de Guillen et al, 2015). When RGA4 alone is present, it spontaneously induces a cellular allergic necrosis in the host, and this activation activity is suppressed by RGA 5. Further analysis shows that a non-conserved non-LRR domain (RGA5A _ S domain, including metal ion binding domain RATX1) exists at the C-terminal of RGA5 protein and is directly involved in recognizing two inconsistent effector factors AVR1-CO39 and AVR-Pia. RGA5RATX1 is a core binding domain involved in pathogen recognition, and through direct binding with AVR1-CO39 and AVR-Pia, the inhibition of RGA4 is released, thereby triggering effector-induced disease resistance (Cesari et al, 2014; Cesari et al, 2013). RGA5 also has additional domains that are directly involved in the recognition of the effector AVR-Pia, regulating the host immune response (Ortiz et al, 2017). AVR1-CO39 stimulates the active oxygen burst innate immune response produced by Pia rice plants, not only controlling the invasion of Magnaporthe grisea (M.oryzae), but also inhibiting bacterial blight of rice and bacterial leaf streak of rice (Hutin et al, 2016) caused by the gram-positive bacteria Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzae (Xoc). At present, the interaction mechanism of RGA5A _ S for recognizing rice blast fungus effector AVR1-CO39 is not clear and needs to be further researched. Therefore, there is a need to provide a crystal preparation and growth technology for rice receptor protein Pia RGA5A _ S, which provides a structural basis for the in-depth understanding of the recognition of fungal effectors by plant receptor R proteins.

Early researches found that the rice receptor protein RGA5A _ S has the problems of sensitivity to temperature, easy precipitation and the like. The problems of pipeline blockage of a crystal growth manipulator, serious protein precipitation, low crystal quality and the like are easily caused by adopting the conventional protein crystallization method.

Disclosure of Invention

The invention provides a preparation method and a crystallization method of rice receptor protein RGA5A _ S, aiming at the problems that RGA5A _ S protein is sensitive to temperature, is easy to precipitate during concentration, has poor crystallization efficiency, low crystal diffraction quality and the like.

In order to realize the purpose of the invention, the invention provides a preparation method of a rice receptor protein RGA5A _ S, which comprises the steps of expressing the rice receptor protein RGA5A _ S by a prokaryotic expression system, cracking thalli, and sequentially passing the cracked supernatant through an affinity chromatography column and a molecular sieve chromatography column to finally obtain the purified target protein. Wherein, 100 MuM Zn is added into the culture medium when the recombinant bacteria are induced and shaken²⁺(ZnCl₂)。

In the method, the recombinant strain is constructed by transforming an expression vector carrying a rice receptor protein RGA5A _ S coding gene into escherichia coli.

The amino acid sequence of the protein RGA5A _ S of the invention is shown in SEQ ID NO 2. Preferably, the sequence of the rice receptor protein RGA5A _ S coding gene is shown as SEQ ID NO. 1.

Preferably, the expression vector is pETM 13. Coli BL21(DE 3).

Specifically, the preparation method of the rice receptor protein RGA5A _ S comprises the following steps:

(1) inducing and expressing the recombinant bacteria: the recombinant strain is shaken at 37 ℃ and 220rpm until the OD of the strain liquid₆₀₀The value is 0.6-0.8, and the final concentration of ZnCl is 100 MuM₂And 0.1mM IPTG, inducing at 18 ℃ and 180rpm for 16-18 h;

(2) and (3) cracking thalli: collecting IPTG induced bacteria liquid, centrifuging at room temperature of 6000rpm for 8min, discarding supernatant, and collecting thallus; resuspend the cells in lysis buffer (20mM PBS, pH 7.0, 500mM NaCl); ultrasonic cracking, turning on for 2s, turning off for 4s, and turning on at power of 400-; 2 cycles; centrifuging the lysate for 25-30min at 12000-13000 rpm and 4 ℃ to obtain cell lysis supernatant;

(3) affinity chromatography: 100mL of cell lysis supernatant was divided into 3 portions and added to 3 portions of 200mM ZnCl solution₂Pretreated affinity chromatography columns (chelating)

fast flow agar column); hanging the column once again by using the penetrating liquid; then gradient elution is carried out by using elution buffer solutions containing imidazole with different concentrations of 0, 20, 60 and 500 mM;

(4) molecular sieve chromatography: collecting 60 mM and 500mM imidazole eluates respectively, adding DTT (dithiothreitol) with the final concentration of 4mM, and concentrating to 1mL respectively; then is added to

7510/300 GL gel column, eluting with chromatography buffer solution, mixing the collected eluates, and collecting the eluates containing purified target protein.

Preferably, the affinity chromatography column used in the present invention is a chromatography column

fast flow agarose column。

The base material of the elution buffer used in the step (3) is as follows: 20mM PBS, pH 7.0, 500mM NaCl.

Wherein, the elution volumes of the elution buffers with imidazole concentrations of 0, 20, 60 and 500mM are 40, 10 and 10 mL.

The formula of the chromatography buffer solution used in the step (4) is as follows: 20mM Tris-HCl, pH 8.0,150mM NaCl,5mM DTT and 4mM EDTA.

The steps (2) to (4) should be carried out on ice or at 4 ℃ as much as possible.

The invention also provides a crystallization method of the rice receptor protein RGA5A _ S, which comprises preparing the target protein prepared by the method into a protein solution with the concentration of 6.4mg/mL by using a buffer solution, and then preparing the protein solution at 0.2M NH₄NO₃And 20% PEG3350(v/v), and standing and culturing in a 16 deg.C incubator for 3-5 days to obtain micro crystal; then using it as seed crystal, under the condition of 0.18-0.22M NH₄NO₃And in the presence of 18-22% of PEG3350(v/v), standing and culturing for 3-5 days in a constant temperature incubator at 16 ℃ to obtain the rice receptor protein RGA5A _ S single crystal.

Preferably, the seed crystal is at 0.18M NH₄NO₃And 20% PEG3350, and obtaining the single crystal.

Wherein the formula of the buffer solution is as follows: 20mM PBS, pH 7.0, 150mM NaCl,5mM DTT and 4mM EDTA.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

(I) aiming at the characteristic that the target protein RGA5A _ S contains a metal ion structure domain (with MxCXXS motif, x represents any amino acid), Zn is added into a culture medium during the shaking culture of recombinant bacteria²⁺Has important function for stabilizing the state of the protein.

(II) the protein crystallization conditions are screened and optimized by adopting a shotgun method and a Sudoku method, and the result shows that the protein RGA5A _ S is at 0.18M NH₄NO₃And 20% PEG3350, large amount of single crystals can be obtained, the obtained crystals are large, the quality is good, the resolution is high, and the method has good repeatability.

(III) due to RGA5A _ S (not containing 6 XHis tag), by virtue of the non-conserved metal ion binding motif MxCXS with Zn²⁺The binding force of the column binding is worse than that of 6 XHis-tag, therefore, the invention selects the elution buffer solution containing imidazole with different concentrations to carry out gradient elution on the target protein of the hanging column so as to ensure that the target protein is removed under low concentrationAnd (3) foreign protein, wherein the high-purity target protein is completely eluted at high concentration.

Drawings

FIG. 1 is a polarization microscope photograph of minute crystals of rice receptor protein RGA5A _ S obtained in example 2 of the present invention.

FIG. 2 is a polarization microscope photograph of a single crystal of rice receptor protein RGA5A _ S obtained in example 2 of the present invention.

FIG. 3 is X-ray diffraction data of the crystalline rice receptor protein RGA5A _ S in example 2 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

EXAMPLE 1 preparation of the Rice receptor protein RGA5A _ S

1. Construction of recombinant expression vectors

(1) Primer design

RGA5_ RATX1 (GenBank: BAK39930.1) target sequence is obtained from NCBI, and the gene sequence is synthesized after codon optimization. Primers were designed by the Sticky-end method, restriction enzymes (NcoI/XhoI) were selected according to restriction sites of the selected vector and restriction enzyme analysis of the target sequence, and two sets of primers were designed by DNAMAN 8.0.

F1-NcoI：catg CTGTCTAACA TGGAATCTGT

R1-XhoI：g CATAGTGCTG CACGGGTT

F2-NcoI：CTGTCTAACA TGGAATCTGT AGTAG

R2-XhoI：tcgag CATAGTGCTG CACGGGT

(2) PCR amplification of fragments of interest and product recovery

Using the method of Sticky-end (Pham et al, 1998), the PCR amplification product was annealed to generate cohesive ends complementary to the ends of the vector.

(3) Annealing to yield sticky ends

The concentration of the PCR purified product is detected by a NaNo Drop K550 micro-spectrophotometer and mixed according to the molar ratio of 1: 1. The double-stranded DNA with the protruding ends is obtained by performing renaturation by a PCR instrument.

And (3) denaturation and renaturation conditions: denaturation at 94 deg.C for 5min, denaturation at 65 deg.C for 15min, and cooling at 16 deg.C.

(4) Enzyme digestion expression vector pETM13

Performing enzyme digestion on the target fragment, performing double enzyme digestion on the vector, incubating for 4-5h at 37 ℃,

(5) ligation of the vector to the fragment of interest

The PCR product which generates cohesive ends after renaturation is ligated to the vector with the same ends after cleavage by T4DNA ligase and incubated overnight at 16 ℃.

(6) Heat shock transformation and resistance selection

Coli JM109/DH5 α cells were removed from the freezer at-80 ℃ and lysed on ice;

adding 50 μ L of competent cells to the ligation product, and standing on ice for 30 min;

heating in 42 deg.C water bath for 90s, and immediately standing on ice for 2 min;

adding 500 μ L SOC culture medium into the super clean bench, and performing shake culture at 37 deg.C and 220rpm for 40 min;

uniformly coating the culture medium on an LB plate with the same resistance as the carrier, and air-drying (operation in a super clean bench);

the cells were grown overnight by inverting in a 37 ℃ incubator.

(7) Recombinant validation

Randomly picking a single clone and placing the single clone in 1mL of LB liquid culture medium with corresponding resistance;

carrying out shaking culture at 37 ℃ and 220rpm for 5-10 h;

PCR (polymerase chain reaction) of bacterial liquid is carried out to verify whether the insert fragment is correct;

after the amplification product is detected by agarose gel, bacterial liquid with the band consistent with the size of the target fragment (SEQ ID NO:1) is selected and sent to the company for sequencing. And comparing the sequencing result with the target sequence by using DNAMAN8.0 or NCBI, and confirming that the enzyme cutting sites carried by the target sequence in the recombinant plasmid are consistent, and whether code shifting and point mutation exist among bases is ensured, so that the success construction of the recombinant expression vector is indicated, and the recombinant expression vector can be used for subsequent experiments.

2. Recombinant bacterium induced expression and separation and purification of target protein

(1) The recombinant expression vector was transformed into E.coli BL21(DE3), and cultured at 37 ℃ and 220rpm until the OD of the bacterial liquid₆₀₀Value 0.8, final concentration of 100. mu.M ZnCl addition₂And 0.1mM IPTG, 18 ℃, 180rpm induction for 18 h.

(2) And (3) cracking thalli: collecting IPTG induced bacteria liquid, centrifuging at room temperature of 6000rpm for 8min, discarding supernatant, and collecting thallus; resuspend the cells in lysis buffer (20mM PBS, pH 7.0, 500mM NaCl); ultrasonic cracking, turning on for 2s, turning off for 4s, and turning on at power of 400-; 2 cycles; the lysate was centrifuged at 12000-13000 rpm at 4 ℃ for 25-30min to obtain cell lysis supernatant.

(3) Affinity chromatography: 100mL of cell lysis supernatant was divided into 3 portions and added to 3 portions of 200mM ZnCl solution₂On the pretreated affinity chromatographic column; hanging the column once again by using the penetrating liquid; then gradient elution is carried out by using elution buffer containing 0, 20, 60 and 500mM of imidazole at different concentrations.

Wherein the affinity chromatography column is a chelating column

fast flow agarose column。

The base materials of the elution buffer used were: 20mM PBS, pH 7.0, 500mM NaCl.

Elution volumes were 40, 10mL for elution buffers with imidazole concentrations of 0, 20, 60, 500 mM.

(4) Molecular sieve chromatography: collecting 60 mM and 500mM imidazole eluates respectively, adding DTT with the final concentration of 4mM, and concentrating to 1mL respectively; then is added to

7510/300 GL gel column, eluting with chromatography buffer solution, and the eluate contains purified target protein.

Wherein the formula of the used chromatography buffer solution is as follows: 20mM Tris-HCl, pH 8.0,150mM NaCl,5mM DTT and 4mM EDTA.

Example 2 method for crystallizing the Rice receptor protein RGA5A _ S

1. Shotgun method for screening protein crystallization condition

The protein RGA5A _ S prepared in example 1 was dissolved in a buffer (20mM PBS, pH 7.0, 150mM NaCl,5mM DTT, 4mM EDTA) to give a protein solution at a concentration of 6.4 mg/mL. And then carrying out shotgun method crystal kit screening. Standing and culturing at 16 deg.C for 5 days in a constant temperature incubator, and adding NH into protein solution under the condition of JCSG + kit (Hampton research) C4₄NO₃And PEG3350 at final concentrations of 0.2M and 20 (v/v)%]Tiny crystals can be observed (fig. 1). Whereas under other crystal growth reagent conditions, crystals are hardly obtainable.

2. Sudoku and inoculation strategy to optimize crystal growth conditions

To obtain a high quality diffractive crystal, NH is optimized using a Sudoku method₄NO₃And PEG3350 concentration gradient, and crystal growth condition optimization is carried out by taking the microcrystal obtained in the step 1 as a crystal seed. As a result, RGA5A _ S was found to be at 0.18M NH₄NO₃And 20% PEG3350(v/v) a large number of single crystals were obtained (FIG. 2). Diffraction under X-ray can obtain diffraction data (generally, diffraction data with higher resolution)

Left and right resolution) (fig. 3). Finally, the collected diffraction data is subjected to indexing (Index), intensity Integration (Integration) and normalization (Scaling) by using related analysis software, and the crystal structure of the protein can be obtained.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Reference to the literature

Cesari,S.,et al.(2014).″A novel conserved mechanism for plant NLR protein pairs:the“integrated decoy”hypothesis.″Frontiers in plant science 5.

Cesari,S.,et al.(2013).″The rice resistance protein pair RGA4/RGA5recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39by direct binding.″The Plant Cell 25(4):1463-1481.

de Guillen,K.,et al.(2015).″Structure analysis uncovers a highly diverse but structurally conserved effector family in phytopathogenic fungi.″PLoS pathogens 11(10):e1005228.

Dean,R.,et al.(2012).″The Top 10fungal pathogens in molecular plant pathology.″Molecular plant pathology 13(4):414-430.

Fisher,M.C.,et al.(2012).″Emerging fungal threats to animal,plant and ecosystem health.″Nature 484(7393):186-194.

Flor,H.H.(1971).″Current status of the gene-for-gene concept.″Annual review of phytopathology 9(1):275-296.

Hutin,M.,et al.(2016).″Ectopic activation of the rice NLR heteropair RGA4/RGA5confers resistance to bacterial blight and bacterial leaf streak diseases.″The Plant Journal 88(1):43-55.

Liu,W.and G.-L.Wang(2016).″Plant innate immunity in rice:a defense against pathogen infection.″National Science Review 3(3):295-308.

Ortiz,D.,et al.(2017).″Recognition of the Magnaporthe oryzae effector AVR-Pia by the decoy domain of the rice NLR immune receptor RGA5.″The Plant Cell Online 29(1):156-168.

ZJING, et al (2014). "2030 world grain development and its impact on the choice of national strategy in both countries of the United statesWorld agriculture(3):I0001-I0001.

Zhongxiong, et al (2010)' the current development situation, trend and revelation of China for the world rice industry presence of Research on generations31(5):525-528.

Sequence listing

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Claims (7)

1. The preparation method of the rice receptor protein RGA5A _ S is characterized in that a prokaryotic expression system is utilized to express the rice receptor protein RGA5A _ S, and after thallus cracking, the cracked supernatant sequentially passes through an affinity chromatography column and a molecular sieve chromatography column to finally obtain the purified target protein;

wherein, when the recombinant bacteria are induced to shake culture, 100 MuM Zn is added into the culture medium²⁺；

The recombinant strain is constructed by transforming an expression vector carrying a rice receptor protein RGA5A _ S coding gene into escherichia coli;

the sequence of the rice receptor protein RGA5A _ S coding gene is shown in SEQ ID NO:1, the expression vector is pETM13, and the Escherichia coli is Escherichia coli BL21(DE 3);

specifically, the method comprises the following steps:

(1) inducing and expressing the recombinant bacteria: the recombinant strain is shaken at 37 ℃ and 220rpm until the OD of the strain liquid₆₀₀The value is 0.6-0.8, and the final concentration of ZnCl is 100 MuM₂And 0.1mMIPTG, and inducing at 18 ℃ and 180rpm for 16-18 h;

(2) and (3) cracking thalli: collecting IPTG induced bacteria liquid, centrifuging at room temperature of 6000rpm for 8min, discarding supernatant, and collecting thallus; resuspending the cells with lysis buffer; ultrasonic cracking, turning on for 2s, turning off for 4s, and turning on at power of 400-; 2 cycles; centrifuging the lysate for 25-30min at 12000-13000 rpm and 4 ℃ to obtain cell lysis supernatant;

(3) affinity chromatography: 100mL of cell lysis supernatant was divided into 3 portions and added to 3 portions of 200mM ZnCl solution₂On the pretreated affinity chromatographic column; hanging the column once again by using the penetrating liquid; then gradient elution is carried out by using elution buffer solutions containing imidazole with different concentrations of 0, 20, 60 and 500 mM;

(4) molecular sieve chromatography: collecting 60 mM and 500mM imidazole eluates respectively, adding DTT with the final concentration of 4mM, and concentrating to 1mL respectively; then is added to

7510/300 GL gel column, eluting with chromatography buffer solution, mixing collected eluate containing purified target protein;

wherein, the lysis buffer used in the step (2) is: 20mM PBS, pH 7.0, 500mM NaCl.

2. The method according to claim 1, wherein the base material of the elution buffer of step (3) is: 20mM PBS, pH 7.0, 500mM NaCl; elution volumes were 40, 10mL for elution buffers with imidazole concentrations of 0, 20, 60, 500 mM.

3. The method of claim 1, wherein the formula of the chromatography buffer used in step (4) is: 20mM Tris-HCl, pH 8.0,150mM NaCl,5mM DTT and 4mM EDTA.

4. The method according to any one of claims 1 to 3, wherein steps (2) to (4) are carried out on ice or at 4 ℃.

5. A method for crystallizing the rice receptor protein RGA5A _ S, characterized in that the target protein prepared by the method of any one of claims 1 to 4 is prepared in a buffer solution at a concentration of 6.4mg/mL and then subjected to NH treatment at 0.2M₄NO₃And 20% PEG3350, and standing in a constant temperature incubator at 16 deg.C for 3-5 days to obtain micro crystal; then using it as seed crystal, under the condition of 0.18-0.22M NH₄NO₃And 18 to 22 percent of PEG3350, and standing and culturing for 3 to 5 days in a constant temperature incubator at the temperature of 16 ℃ to obtain the rice receptor protein RGA5A _ S single crystal.

6. The method of claim 5, wherein the seed crystal is seeded at 0.18M NH₄NO₃And 20% PEG3350, and obtaining the single crystal.

7. The method according to claim 5 or 6, wherein the formulation of the buffer is: 20mM PBS, pH 7.0, 150mM NaCl,5mM DTT and 4mM EDTA.

Images