Supplementary Material for: Expression of Cell-Penetrating Peptides Fused to Protein Cargo

Cell-penetrating peptides (CPPs) are short peptides that can cross cell membranes. CPPs enable the delivery of biomolecules into cells and can act as drug-delivery vectors. Because recombinant production of CPPs as fusions to protein “cargo” leads to low yields for some CPP-cargo fusions, approaches to enhance the recombinant expression of peptide-cargo fusions need to be identified. We optimized expression conditions in <i>Escherichia coli</i> for fusions of CPPs (SynB, histatin-5, and MPG) to the cargo proteins biotin carboxyl carrier protein, maltose-binding protein, and green fluorescent protein. We used Western blotting to evaluate induction temperatures of 37, 30, and 20°C, and induction times of 6, 10, and 24 h. Glutathione-S-transferase was incorporated as a fusion partner to improve expression. In general, expression at 37°C for 6 and 10 h led to the highest levels of expression for the different CPP-cargo constructs. The improvements in expression of CPP-cargo fusions will allow higher yields of CPP-cargo fusions for studies of their translocation into cells.细胞穿透肽（cell-penetrating peptides，CPPs）是一类可跨细胞膜的短肽。这类肽可介导生物分子向细胞内递送，亦可作为药物递送载体。由于将细胞穿透肽与蛋白质“货物”融合进行重组表达时，部分CPP-货物融合蛋白的产量较低，因此亟需筛选可提升肽-货物融合蛋白重组表达量的方法。本研究在大肠杆菌（Escherichia coli）中优化了细胞穿透肽与货物蛋白的融合表达条件，所测试的细胞穿透肽包括SynB、富组蛋白5（histatin-5）与MPG，货物蛋白则涵盖生物素羧基载体蛋白、麦芽糖结合蛋白以及绿色荧光蛋白（green fluorescent protein）。本研究采用蛋白质免疫印迹（Western blotting，WB）技术，评估了37℃、30℃与20℃三种诱导温度，以及6 h、10 h与24 h三种诱导时长对融合蛋白表达的影响。本研究引入谷胱甘肽S-转移酶（glutathione-S-transferase，GST）作为融合伴侣以提升蛋白表达量。总体而言，在37℃下诱导6 h或10 h时，各类CPP-货物融合构建体的表达量可达最高。本研究实现的CPP-货物融合蛋白表达量提升，可为后续开展其细胞转位相关研究提供更高产量的融合蛋白样品。