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Preparation and crystallization method of rice receptor protein rga5a_s
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A receptor protein and rice technology, applied in the field of protein crystallization, can solve the problems of low crystal diffraction quality, poor crystallization efficiency, temperature sensitivity, etc., and achieve high resolution, good quality, and good repeatability
Inactive Publication Date: 2021-06-22
CHINA AGRI UNIV
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[0006] Aiming at the problems that RGA5A_S protein is sensitive to temperature, easy to precipitate when concentrated, poor crystallization efficiency and low crystal diffraction quality, the invention provides a preparation and crystallization method of rice receptor protein RGA5A_S
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Embodiment 1
[0032] The preparation of embodiment 1 rice receptor protein RGA5A_S
[0033] 1. Construction of recombinant expression vector
[0034] (1) Primer design
[0035] The target sequence of RGA5_RATX1 (GenBank: BAK39930.1) was obtained from NCBI, and the gene sequence was synthesized after codon optimization. Primers were designed using the Sticky-end method, and restriction enzymes (NcoI / XhoI) were selected according to the restriction enzyme sites of the selected vector and the restriction enzyme analysis of the target sequence, and two sets of primers were designed using DNAMAN8.0.
[0036] F1-NcoI: catg CTGTCTAACA TGGAATCTGT
[0037] R1-XhoI:g CATAGTGCTG CACGGGTT
[0038] F2-NcoI: CTGTCTAACA TGGAATCTGT AGTAG
[0039] R2-XhoI: tcgag CATAGTGCTG CACGGGT
[0040] (2) PCR amplification target fragment and product recovery
[0041] Using the Sticky-end method (Pham et al., 1998), the PCR amplification product was annealed to generate a sticky end complementary to the end of th...
Embodiment 2
[0070] The crystallization method of embodiment 2 rice receptor protein RGA5A_S
[0071] 1. Screening conditions for protein crystallization by shotgun method
[0072] The protein RGA5A_S prepared in Example 1 was dissolved in a buffer solution (20 mM PBS, pH 7.0, 150 mM NaCl, 5 mM MDTT, 4 mM EDTA) to obtain a protein solution with a concentration of 6.4 mg / mL. Shotgun crystal kit screening was then performed. Cultivate statically in a constant temperature incubator at 16°C for 5 days, and in the C4 condition of the JCSG+ kit (Hampton research) [the protein solution is added with NH 4 NO 3 and PEG3350, the final concentrations were 0.2M and 20 (v / v)%] tiny crystals ( figure 1 ). While under other crystal growth reagent conditions, almost no crystals can be obtained.
[0073] 2. Jiugong grid and inoculation strategy to optimize crystal growth conditions
[0074] In order to obtain high-quality diffractive crystals, the NH 4 NO 3 and PEG3350 concentration gradient, the c...
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Abstract
The invention discloses a preparation and crystallization method of rice receptor protein RGA5A_S. The preparation method of the protein RGA5A_S is as follows: the rice receptor protein RGA5A_S is expressed using a prokaryotic expression system, and after cell lysis, the lysed supernatant is sequentially passed through an affinity chromatography column and a molecular sieve chromatography column to finally obtain a purified target protein; for the protein RGA5A_S Contains the characteristics of the metal ion domain, when the recombinant bacteria are induced by shake culture, add 100μM Zn to the medium 2+ , improved protein stability. The invention also provides a method for crystallizing the protein RGA5A_S. Shotgun method and Jiugongge method were used to screen and optimize protein crystallization conditions. 4 NO 3 And under the conditions of 18%-22% PEG3350, the obtained crystals are large, of good quality and high in resolution, and the method has good repeatability.
Description
technical field [0001] The invention belongs to the field of protein crystallization, and in particular relates to the preparation and crystallization method of rice receptor protein RGA5A_S. Background technique [0002] Rice is one of the three major economic crops in the world and is the staple food of more than 50% of the world's population (Liu and Wang, 2016). Food security has become the top priority in today’s global economic production, and the continuous growth of population is the original driving force for the development of the world’s rice industry (Zhou Xiyue et al., 2010). By 2030, the world’s rice must grow by at least 38% to meet human needs (Zhang Jing et al., 2014) . Therefore, it is of great strategic significance to ensure the stable yield and quality of rice, which is directly related to issues such as food security supply and people's food and clothing. However, the safe production of rice is endangered by pests and diseases all the year round, and ...
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