High-Quality Protein Crystal Growth of Mouse Lipocalin-Type Prostaglandin D Synthase in Microgravity.
High-Quality Protein Crystal Growth of Mouse Lipocalin-Type Prostaglandin D Synthase in Microgravity.
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DOI：
10.1021/cg101370v
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发表时间：
2011-06-01
影响因子：
3.8
通讯作者：
Urade, Yoshihiro
中科院分区：
文献类型：
--
作者：
Inaka, Koji;Takahashi, Sachiko;Aritake, Kosuke;Tsurumura, Toshiharu;Furubayashi, Naoki;Yan, Bin;Hirota, Erika;Sano, Satoshi;Sato, Masaru;Kobayashi, Tomoyuki;Yoshimura, Yoshinori;Tanaka, Hiroaki;Urade, Yoshihiro
脂质运载蛋白型前列腺素（PG）D合成酶（L-PGDS）催化PGH 2异构化为PGD 2，参与疼痛、非快动眼睡眠的调节以及男性生殖器和脂肪细胞的分化等。L-PGDS分泌到各种体液中，与各种亲脂性化合物高亲和力结合，也是细胞外转运蛋白。预先用柠檬酸盐或丙二酸盐作为沉淀剂对具有C65 A突变的小鼠L-PGDS进行结晶，并在2.0 nm分辨率下测定X射线晶体结构。为了获得高质量的晶体，我们尝试在与先前研究相同的条件下在微重力下结晶C65 A突变体，但没有成功。在进一步纯化蛋白质并将沉淀剂改为聚乙二醇（PEG）8000后，在微重力下生长出高质量的晶体。沉淀剂溶液为40%（w/v）PEG 8000、100 mM氯化钠和100 mM HEPES-NaOH（pH 7.0）。2007年，晶体在国际空间站上生长了11周，产生了野生型L-PGDS和C65 A突变体的单晶，两者的衍射分辨率都在1.0 nm左右。通过在微重力下使用高粘度沉淀剂溶液并结合使用高度纯化的蛋白质，晶体质量得到显著改善。首次获得野生型小鼠L-PGDS晶体。晶体衍射约1.0 nm。用高粘度沉淀剂使均质蛋白质样品结晶是微重力下晶体成功生长的关键。
Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) catalyzes the isomerization of PGH2 to PGD2 and is involved in the regulation of pain and of nonrapid eye movement sleep and the differentiation of male genital organs and adipocytes, etc. L-PGDS is secreted into various body fluids and binds various lipophilic compounds with high affinities, acting also as an extracellular transporter. Mouse L-PGDS with a C65A mutation was previously crystallized with citrate or malonate as a precipitant, and the X-ray crystallographic structure was determined at 2.0 Å resolution. To obtain high-quality crystals, we tried, unsuccessfully, to crystallize the C65A mutant in microgravity under the same conditions used in the previous study. After further purifying the protein and changing the precipitant to polyethylene glycol (PEG) 8000, high-quality crystals were grown in microgravity. The precipitant solution was 40% (w/v) PEG 8000, 100 mM sodium chloride, and 100 mM HEPES-NaOH (pH 7.0). Crystals grew on board the International Space Station for 11 weeks in 2007, yielding single crystals of the wild-type L-PGDS and the C65A mutant, both of which diffracted at around 1.0 Å resolution. The crystal quality was markedly improved through the use of a high-viscosity precipitant solution in microgravity, in combination with the use of a highly purified protein. Crystals of wild-type mouse L-PGDS were obtained for the first time. The crystals diffracted around 1.0 Ǻ. The crystallization of homogeneous protein samples with a high-viscosity precipitant was crucial to this successful crystal growth in microgravity.
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影响因子：
1.8
作者：
Tanaka, Hiroaki;Yoshizaki, Izumi;Tanaka, Tetsuo
通讯作者：
Tanaka, Tetsuo
DOI：
10.1107/s0907444993014350
发表时间：
1994-07-01
影响因子：
2.2
作者：
GARCIARUIZ, JM;MORENO, A
通讯作者：
MORENO, A
DOI：
10.1196/annals.1324.002
发表时间：
2004-01-01
期刊：
TRANSPORT PHENOMENA IN MICROGRAVITY
影响因子：
--
作者：
Tanaka, H;Inaka, K;Yoshitomi, S
通讯作者：
Yoshitomi, S
影响因子：
2.5
作者：
Tanaka, Hiroaki;Tsurumura, Toshiharu;Urade, Yoshihiro
通讯作者：
Urade, Yoshihiro
影响因子：
4.8
作者：
URADE, Y;TANAKA, T;HAYAISHI, O
通讯作者：
HAYAISHI, O