Identification of secretion domain of Neospora caninum profilin
Identification of secretion domain of Neospora caninum profilin
复制标题
犬新孢子虫profilin分泌结构域的鉴定
DOI：
10.1016/j.bbrc.2019.11.056
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发表时间：
2020
影响因子：
3.1
通讯作者：
Park Enoch Y.
中科院分区：
文献类型：
--
作者：
Suhaimi Hamizah;Xu Jian;Kato Tatsuya;Setyo Utumo Doddy Irawan;Sekiguchi Tomofumi;Park Enoch Y.
Profilin（PROF）是存在于顶复门原虫中的一种小肌动蛋白结合蛋白。以往的研究表明，尽管缺乏一个已鉴定的常规分泌信号肽，但新孢子虫前膜蛋白（Neospora caninumprofilin，NcPROF）仍能分泌到家蚕幼虫的血淋巴中。迄今为止，其分泌所需的结构域仍然未知。为此，我们表达了在其N-末端或C-末端与NcPROF融合的荧光蛋白（mCherry）。两种融合蛋白分别在家蚕Bm 5细胞培养上清和家蚕幼虫血淋巴中表达并分泌。为了进一步缩小其分泌所需的C-末端最小结构域，我们构建了三个截短的C-末端结构域构建物，分别为ΔN（aa 41 -163）、ΔN1（aa 50 -163）和ΔN2（aa 144 -163）。在家蚕Bm 5细胞和家蚕血淋巴的培养上清中均检测到3种融合蛋白。令人惊讶的是，20个氨基酸的C-末端α-螺旋结构域促进mCherry的分泌，允许通过亲和层析从家蚕幼虫血淋巴中纯化ΔN2-mCherry。总之，鉴定了NcPROF的分泌结构域，表明其未来可用于重组蛋白的分泌表达。
Profilin (PROF) is a small actin-binding protein presented in apicomplexan protozoa. It was previously reported thatNeospora caninumprofilin (NcPROF) is secreted into the hemolymph of silkworm larvae regardless of the lack of an identified regular secretion signal peptide. To date, which domain is required for its secretion still remains unknown. To this end, we express a fluorescent protein (mCherry) fused with NcPROF at its N-terminus or C-terminus. Both fusion proteins were expressed and secreted into the culture supernatant from Bm5 cells or hemolymph from silkworm larvae, respectively. To further narrow down the C-terminal minimal domain required for its secretion, we constructed three truncated C-terminal domain constructions, ΔN (aa41–163), ΔN1 (aa50–163), and ΔN2 (aa144–163) respectively. All three fusion proteins were detected in the culture supernatant of Bm5 cells and silkworm hemolymph. Surprisingly, a 20-aa C-terminal α-helix domain facilitates the secretion of mCherry, allowing purification of ΔN2-mCherry from silkworm larval hemolymph by affinity chromatography. Taken together, the secretion domain from NcPROF was identified, indicating that can be utilized for the secretory expression of recombinant proteins in the future.