Preparation method and crystallization method of oryza sativa receptor protein RGA5A_S
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A receptor protein, rice technology, applied in the field of protein crystallization, can solve the problems of poor crystallization efficiency, temperature sensitivity, easy precipitation and so on
Inactive Publication Date: 2019-11-26
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 DNAMAN 8.0.
[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 t...
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 method and crystallization method of oryza sativa receptor protein RGA5A_S. The preparation method of the protein RGA5A_S comprises the steps of expressing the oryza sativa receptor protein RGA5A_S through a prokaryotic expression system, performing thalli splitting, and enabling splitting supernatant to sequentially pass through an affinity chromatography column and a molecular sieve chromatography column so as to obtain purified target protein finally. In accordance with the characteristic that the protein RGA5A_S contains a metal ion structural domain,during recombinant strain induced shaking culture, 100 [mu] M of Zn<2+> is added to a culture medium, so that the stability of the protein is improved. The invention also provides the crystallizationmethod of the protein RGA5A_S. The protein crystallization condition is screened and optimized by a shot gun method and a 9-window method, so that crystals obtained from the protein RGA5A_S under thecondition of 0.18-0.22M of NH4NO3 and 18%-22% of PEG3350 are large, good in quality and high in resolution. The method is good in 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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