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Expression, purification and crystallization of TGW6, which limits grain weight in rice
Protein Expression and Purification ( IF 1.6 ) Pub Date : 2021-09-16 , DOI: 10.1016/j.pep.2021.105975
Tatsuki Akabane 1 , Nobuhiro Suzuki 2 , Wataru Tsuchiya 2 , Takuya Yoshizawa 3 , Hiroyoshi Matsumura 3 , Naoki Hirotsu 1 , Etsuko Katoh 2
Affiliation  

Rice is the staple food for over half the world's population. Genes associated with rice yield include THOUSAND GRAIN WEIGHT 6 (TGW6), which negatively regulates the number of endosperm cells as well as grain weight. The 1-bp deletion allele of tgw6 cloned from the Indian landrace rice cultivar Kasalath, which has lost function, enhances both grain size and yield. TGW6 has been utilized as a target for breeding and genome editing to increase the yield of rice. In the present study, we describe an improved heterologous expression system of TGW6 in Escherichia coli to enable purification of the recombinant protein. The best expression was achieved using codon optimized TGW6 with a 30 amino acid truncation at the N-terminus (Δ30TGW6) in the Rosetta-gami 2(DE3) host strain. Furthermore, we found that calcium ions were critical for the purification of stable Δ30TGW6. Crystals of Δ30TGW6 were obtained using the sitting-drop vapor-diffusion method at 283 K, which diffracted X-rays to at least 2.6 Å resolution. Herein, we established an efficient procedure for the production and purification of TGW6 in sufficient quantities for structural and functional studies. Detailed information concerning the molecular mechanism of TGW6 will enable the design of more efficient ways to control the activity of the enzyme.



中文翻译:

限制水稻粒重的 TGW6 的表达、纯化和结晶

大米是世界上一半以上人口的主食。与水稻产量相关的基因包括千粒重6 ( TGW6 ),它负向调节胚乳细胞数量和粒重。从失去功能的印度地方水稻品种 Kasalath 克隆的tgw6的 1 bp 缺失等位基因提高了籽粒大小和产量。TGW6 已被用作育种和基因组编辑的目标,以提高水稻产量。在本研究中,我们描述了一种改进的 TGW6 在大肠杆菌中的异源表达系统,以实现重组蛋白的纯化。使用密码子优化的TGW6实现了最佳表达在 Rosetta-gami 2(DE3) 宿主菌株的 N 端 (Δ30TGW6) 处有 30 个氨基酸截断。此外,我们发现钙离子对于稳定 Δ30TGW6 的纯化至关重要。Δ30TGW6 晶体是在 283 K 下使用坐滴蒸汽扩散法获得的,该法将 X 射线衍射到至少 2.6 Å 的分辨率。在这里,我们建立了一个有效的程序来生产和纯化足够数量的 TGW6 以进行结构和功能研究。有关 TGW6 分子机制的详细信息将有助于设计更有效的方法来控制酶的活性。

更新日期:2021-09-22
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