Hoang Mai Ngoc, Peterbauer Clemens
Institute of Food Technology, Department of Food Science and Technology, BOKU University, 1190 Vienna, Austria.
Int J Mol Sci. 2024 Dec 30;26(1):219. doi: 10.3390/ijms26010219.
is a potential bacterial cell factory to develop delivery systems for vaccines and therapeutic proteins. Much progress has been made in applications using engineered against, e.g., inflammatory bowel disease and cervical cancer, but the improvement of secretion and cell anchoring efficacy is still desirable. A double-labeling method based on biarsenical hairpin binding and nickel-polyhistidine affinity was used for visualization of protein trafficking and the quantification of targeted proteins on the cell surface and in the cytoplasm. To investigate the importance of mature domain targeting signals (MTSs), we generated truncated constructs encoding 126, 66, and 26 amino acid residues from the N-terminus of the basic membrane protein A (BmpA) and fused those with the gene for the human papillomavirus serotype 16 (HPV16) E7 oncoprotein. Overexpression of fusion proteins was observed to come at the cost of cell proliferation. cells produced and displayed the shortest fusion protein only with difficulty, suggesting that the entire absence of a homologous sequence containing MTSs significantly impedes the export and surface anchoring of fusion proteins. With 40 amino acids following the signal peptide and containing one MTS, effective translocation was possible. Mutations of MTSs towards increased hydrophobicity resulted in increased secreted and surface-displayed fusion protein, suggesting the potential to design rationally improved constructs.
是一种有潜力的细菌细胞工厂，可用于开发疫苗和治疗性蛋白质的递送系统。在使用工程化的[具体细菌名称未给出]针对例如炎症性肠病和宫颈癌的应用方面已经取得了很大进展，但分泌和细胞锚定效率的提高仍然是可取的。一种基于双砷发夹结合和镍 - 多聚组氨酸亲和力的双标记方法用于可视化蛋白质运输以及定量细胞表面和细胞质中的靶向蛋白质。为了研究成熟结构域靶向信号（MTSs）的重要性，我们构建了截短的构建体，其编码来自碱性膜蛋白A（BmpA）N端的126、66和26个氨基酸残基，并将它们与人乳头瘤病毒16型（HPV16）E7癌蛋白基因融合。观察到融合蛋白的过表达是以细胞增殖为代价的。[具体细菌名称未给出]细胞仅难以产生和展示最短的融合蛋白，这表明完全缺乏包含MTSs的同源序列会显著阻碍融合蛋白的输出和表面锚定。信号肽后有40个氨基酸且包含一个MTS时，有效转运是可能的。MTSs向更高疏水性的突变导致分泌和表面展示的融合蛋白增加，这表明有潜力合理设计改进的构建体。
