MOESM1 of An improved secretion signal enhances the secretion of model proteins from Pichia pastoris

Additional file 1: Figure S1. The S. cerevisiae α-factor secretion signal. Depicted is the 5′ portion of the MFα1 gene encoding pre-pro-α-factor. This sequence encodes the Leu42 variant. Cleavage sites for signal peptidase and Kex2 are marked, and Leu42 and Asp83 are highlighted. This image was generated using SnapGene software. Figure S2. Low variation in secreted protein levels between clones. Each of the indicated constructs was transformed into P. pastoris cells, and six independent clones with confirmed single integrations were cultured and then analyzed as in Fig. 2a to measure extracellular protein. For each construct, the average protein level for the six clones was defined as 1.0. Two representative clones of a given construct were chosen for further analysis. The red boxes represent E2-Crimson constructs, and the black boxes represent BTL2 constructs. Figure S3. Immunoblot showing cell-associated and secreted E2-Crimson. Strains that expressed E2-Crimson fused to the indicated secretion signals were grown and induced. Fractions containing cell-associated E2-Crimson (“C”) or E2-Crimson in the extracellular medium (“M”) were subjected to SDS-PAGE and immunoblotting. The molecular weight (“MW”) marker was the Precision Plus Protein Dual Color Standards (Bio-Rad). Molecular weights of the protein standards are indicated. Figure S4. Large image fields of cells expressing the various constructs. From the experiment of Fig. 3, large image fields were captured to illustrate that the fluorescence patterns were consistent among the cells in a population, and that cells expressing constructs with the α-factor signal sequence were often unusually large. Scale bar, 2 μm. Figure S5. Distribution of ER-targeted GFP-HDEL before induced expression of E2-Crimson constructs. The same cultures shown in Fig. 4b were examined by fluorescence microscopy before methanol-induced expression of the E2-Crimson constructs. All of the strains showed a typical ER pattern for GFP-HDEL. Scale bar, 2 μm. Figure S6. Theoretical prediction of aggregation propensities in the two allelic variants of the α-factor pro region. Left, the Leu42 variant of the α-factor pro region was analyzed using the online AGGRESCAN tool ( http://bioinf.uab.es/aap/ ). The position of Leu42 in a predicted aggregation-prone region is marked. Right, the same analysis was performed for the Ser42 variant of the α-factor pro region.

补充文件1：图S1. 酿酒酵母（S. cerevisiae）α因子分泌信号肽。图示为编码前原α因子的MFα1基因的5'端区域，该序列携带Leu42变异体。图中标注了信号肽酶（signal peptidase）与Kex2的切割位点，并高亮显示了Leu42与Asp83。本图像使用SnapGene软件生成。
补充文件1：图S2. 不同克隆间分泌蛋白水平差异极小。将各指定构建体转化至毕赤酵母（P. pastoris）细胞中，选取6株经验证为单整合的独立克隆进行培养，参照图2a的实验流程分析胞外蛋白水平。以每个构建体的6个克隆的平均蛋白水平设为1.0，选取每个构建体的2个代表性克隆用于后续分析。红色框代表E2-Crimson构建体，黑色框代表BTL2构建体。
补充文件1：图S3. 免疫印迹展示细胞结合型与分泌型E2-Crimson。将携带指定分泌信号肽融合的E2-Crimson的菌株进行培养并诱导，收集细胞结合型E2-Crimson组分（"C"）或胞外培养基中的E2-Crimson组分（"M"），进行SDS-PAGE电泳与免疫印迹分析。分子量（"MW"）Marker采用Precision Plus Protein Dual Color Standards（Bio-Rad），并标注了蛋白标准品的分子量。
补充文件1：图S4. 各构建体表达细胞的大视野图像。参照图3的实验，拍摄大视野图像以展示细胞群体内荧光模式的一致性，同时可见携带α因子信号序列的构建体表达的细胞通常体积异常偏大。比例尺：2 μm。
补充文件1：图S5. E2-Crimson构建体诱导表达前内质网靶向GFP-HDEL的分布情况。在甲醇诱导E2-Crimson构建体表达前，对图4b中所用的相同培养物进行荧光显微镜观察。所有菌株均呈现GFP-HDEL的典型内质网定位模式。比例尺：2 μm。
补充文件1：图S6. α因子前区域两种等位变异体的聚集倾向理论预测。左侧：使用在线AGGRESCAN工具（http://bioinf.uab.es/aap/）分析α因子前区域的Leu42变异体，标注了Leu42在预测的易聚集区域中的位置。右侧：对α因子前区域的Ser42变异体进行相同分析。