Expression, purification and crystallization of TGW6, which limits grain weight in rice
Expression, purification and crystallization of TGW6, which limits grain weight in rice
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DOI：
10.1016/j.pep.2021.105975
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发表时间：
2021-09-22
影响因子：
1.6
通讯作者：
Katoh, Etsuko
中科院分区：
文献类型：
--
作者：
Akabane, Tatsuki;Suzuki, Nobuhiro;Katoh, Etsuko
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负调控胚乳细胞数量和籽粒重的千粒重6 （TGW6）。从失去功能的印度地方稻品种Kasalath中克隆到tgw6基因1 bp缺失等位基因，可以提高籽粒大小和产量。TGW6已被用作水稻育种和基因组编辑的靶点，以提高水稻产量。在本研究中，我们描述了一种改进的TGW6在大肠杆菌中的异种表达系统，以便纯化重组蛋白。在Rosetta-gami 2 （DE3）宿主菌株中，n端截断30个氨基酸的密码子优化TGW6 （Delta 30TGW6）获得了最佳表达。此外，我们发现钙离子对纯化稳定的Delta 30TGW6至关重要。在283 K的温度下，用坐滴气相扩散法获得了Delta 30TGW6的晶体，其x射线衍射分辨率至少为2.6 A。在此，我们建立了一个高效的程序来生产和纯化TGW6，以进行足够数量的结构和功能研究。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该酶的活性。
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 (Delta 30TGW6) in the Rosetta-gami 2 (DE3) host strain. Furthermore, we found that calcium ions were critical for the purification of stable Delta 30TGW6. Crystals of Delta 30TGW6 were obtained using the sitting-drop vapor-diffusion method at 283 K, which diffracted Xrays to at least 2.6 A 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.