The wasabi receptor, also known as the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel, is a potential target for development of repellents for insects, like the pine weevil (Hylobius abietis) feeding on conifer seedlings and causing damage in forestry. Heterologous expression of TRPA1 from pine weevil in the yeast Pichia pastoris can potentially provide protein for structural and functional studies. Here we take advantage of the Green Fluorescent Protein (GFP) tag to examine the various steps of heterologous expression, to get more insight in clone selection, expression and isolation of the intact purified protein. The sequence of HaTRPA1 is reported and GFP-tagged constructs were made of the full-length protein and a truncated version (Δ1-708 HaTRPA1), lacking the N-terminal ankyrin repeat domain. Clones were screened on GFP expression plates, induced in small liquid cultures and in fed-batch cultures, and evaluated by flow cytometry and fluorescence microscopy. The screening on plates successfully identifies low-expression clones, but fails to predict the ranking of the best performing clones in small-scale liquid cultures. The two constructs differ in their cellular localization. Δ1-708 HaTRPA1 is found in a ring at the perimeter of cell, whereas HaTRPA1 is forming highly fluorescent speckles in interior regions of the cell. The pattern is consistent in different clones of the same construct and persists in fed-batch culture. The expression of Δ1-708 HaTRPA1 decreases the viability more than HaTRPA1, and in fed-batch culture it is clear that intact cells first express Δ1-708 HaTRPA1 and then become damaged. Purifications show that both constructs suffer from degradation of the expressed protein, but especially the HaTRPA1 construct. The GFP tag makes it possible to follow expression by flow cytometry and fluorescence microscopy. Analyses of localization, cell viability and expression show that the former two parameters are specific for each of the two evaluated constructs, whereas the relative expression of the constructs varies with the cultivation method. High expression is not all that matters, so taking damaged cells into account, something that may be linked to protein degradation, is important when picking the most suitable construct, clone, and expression scheme.

中文翻译：

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GFP和流式细胞术评价松象甲TRPA1在毕赤酵母中的异源表达

芥末受体，也称为瞬时受体电位锚蛋白 1 (TRPA1) 离子通道，是开发昆虫驱避剂的潜在目标，例如以针叶树幼苗为食并造成林业损害的松象甲虫 (Hylobius abietis)。松象鼻虫在毕赤酵母中异源表达 TRPA1 可能为结构和功能研究提供蛋白质。在这里，我们利用绿色荧光蛋白 (GFP) 标签来检查异源表达的各个步骤，以更深入地了解完整纯化蛋白的克隆选择、表达和分离。报道了 HaTRPA1 的序列，并且 GFP 标记的构建体由全长蛋白和截短版本 (Δ1-708 HaTRPA1) 组成，缺少 N 端锚蛋白重复结构域。在 GFP 表达板上筛选克隆，在小液体培养物和补料分批培养物中诱导，并通过流式细胞术和荧光显微镜进行评估。平板上的筛选成功地识别了低表达克隆，但未能预测小规模液体培养物中表现最佳的克隆的排名。这两种构建体的细胞定位不同。 Δ1-708 HaTRPA1 存在于细胞周边的环中，而 HaTRPA1 在细胞内部区域形成高荧光斑点。该模式在同一构建体的不同克隆中是一致的，并且在补料分批培养中持续存在。 Δ1-708 HaTRPA1 的表达比 HaTRPA1 更能降低活力，并且在补料分批培养中，很明显完整细胞首先表达 Δ1-708 HaTRPA1，然后受损。纯化表明两种构建体都遭受表达蛋白的降解，尤其是 HaTRPA1 构建体。 GFP 标签使得可以通过流式细胞术和荧光显微镜来跟踪表达。定位、细胞活力和表达的分析表明，前两个参数对于两个评估的构建体中的每一个都是特定的，而构建体的相对表达随培养方法的不同而变化。高表达并不是最重要的，因此在选择最合适的构建体、克隆和表达方案时，考虑到可能与蛋白质降解有关的受损细胞非常重要。