Development of Signal Peptide Optimization Tool

重组微生物中外源蛋白的分泌水平强烈依赖于信号肽(SPS)与目的蛋白的结合，因此对每个目的蛋白的SP序列进行优化是提高分泌蛋白在原核和真核生物中异源表达效率的关键步骤。在本研究中，我们开发了一种新的方法，利用宿主细胞的内源信号肽文库来调节每个目的蛋白的SP，称为信号肽优化工具(SPOT)，该工具可以方便地将SP与目的蛋白融合在一起，而不需要任何界面序列。以米曲霉的β-半乳糖苷酶为模型系统，对分泌型酵母这一典型的真核表达宿主的分泌物进行了靶向蛋白的筛选，通过对相对较少的独立克隆的筛选，发现了几个能从重组酵母中产生较多分泌性蛋白的SPS。此外，Signal P软件分析每个信号肽的分泌效率与D分数之间存在相关性，提示该软件的使用将有助于提高文库的质量。所开发的方法可用于各种宿主细胞中的各种蛋白质。

The secretion level of foreign protein in recombinant microbe is strongly dependent onthe combination of signal peptides (SPs) and the target protein; therefore optimization of SP sequence to each target protein is a crucial step to maximize the productivity of the secreted protein in heterogenous expression both in prokaryote and eukaryote.In this research, we have developed a novel method to adjust SP for each target protein utilizing the library of endogenous signal peptides of host cells, named signal peptide optimization tool (SPOT), which can easily make a library of SP fused to a target protein without any interfacial sequence. As a model system, beta-galactosidase from Aspergillus oryzae was used as a target protein for the secretion from Saccharomyces secrevisiae, a typical eukaryotic expression host, and demonstrated that several SPs giving a larger amount of secreted protein from the recombinant yeast were found by the screening of relatively a small number of independent clones. In addition, a correlation between the secretion efficiency and the D-score of each signal peptide analyzed by Signal P software was found, suggesting the use of the software would be helpful to increase the quality of the library. The developed method can be used for variety of proteins in a variety of host cells.