毕赤酵母全蛋白质组信号肽及分泌蛋白分析

Analysis of signal peptides and secreted proteins in the whole proteome of Pichia pastoris

摘要:

信号肽是影响毕赤酵母异源蛋白分泌效率的关键因素之一。目前最常使用的信号肽是来自酿酒酵母的α交配因子(α-mating factor, MFα)的信号肽，利用这个外源信号肽已经成功分泌表达了多种异源蛋白。然而，MFα可能不适用于所有外源蛋白的分泌表达。毕赤酵母分泌蛋白中具有许多典型的信号肽序列，这些内源信号肽可以提供除MFα信号肽之外更为广泛的信号肽选择。因此，有必要分析预测所有内源信号肽引导外源蛋白分泌效果。本研究利用SignalP、TMHMM、Phobius、WoLF PSORT和NetGPI等生物信息工具对毕赤酵母GS115 (ATCC 20864)的全蛋白组进行内源蛋白信号肽及分泌特征的预测，得到了信号肽分布、长度、氨基酸比例和保守性等规律，同时筛选得到69个分泌蛋白及其信号肽，并通过分泌蛋白组验证给出了10条可能用于外源蛋白表达的内源性信号肽。本研究获得的内源性信号肽或许为毕赤酵母表达外源蛋白提供了新的有力工具。

Abstract:

The signal peptide is a key factor that affects the efficiency of protein secretion in Pichia pastoris. Currently, the most used signal peptide is the α-mating factor (MFα) pre-pro leader from Saccharomyces cerevisiae. This exogenous signal peptide has been successfully utilized to express and secret many heterologous proteins. However, MFα is not suitable for the secretory expression of all heterologous proteins. Many typical signal peptides are present in the secretory proteins of P. pastoris, which provides more options besides MFα. Therefore, it is necessary to analyze and identify more efficient endogenous signal peptides that can guide the secretion of heterologous proteins in P. pastoris. In this study, we employed bioinformatics tools such as SignalP, TMHMM, Phobius, WoLF PSORT, and NetGPI to predict endogenous signal peptides from the entire proteome of P. pastoris GS115 (ATCC 20864). Moreover, we analyzed the distribution, length, amino acid composition, and conservation of these signal peptides. Additionally, we screened 69 secreted proteins and their signal peptides, and through secretome validation, we identified 10 endogenous signal peptides that have potential to be used for exogenous protein expression. The endogenous signal peptides obtained in this study may serve as new valuable tools for the expression and secretion of heterologous proteins in P. pastoris.