过表达PDI对毕赤酵母分泌淀粉样蛋白的影响——以胱抑素为例

Effect of PDI overexpressing on secretion of exogenous amyloid protein in Pichia pastoris—take cystatin as an example

摘要:

[目的] 本研究旨在结合酵母菌蛋白质二硫键异构酶（protein disulfide isomerase，PDI）与其底物蛋白鸡胱抑素C （chicken cystatin C，cC）在酵母中的共表达，理解PDI影响外源蛋白合成与表达的调控规律。运用转录组深度测序技术（RNA-Seq）筛选差异基因，调取并鉴定影响cC表达的关键基因，为解析外源蛋白高效表达机制，改造工程菌株提供理论支撑。[方法] 以巴斯德毕赤酵母GS115、GS115-cC为出发菌株，采用电转的方法将携带PDI编码基因的载体pPIC3.5K转入到GS115/GS115-cC菌株，使其在菌株中过表达，研究过表达PDI对cC表达的影响。采用RNA-Seq深度测序方法，研究重组毕赤酵母基因表达差异情况。并结合KEGG注释结果对数据进行分析，挑选差异显著表达基因进行验证，初步明确其在蛋白表达调控方面的功能。[结果] 本研究通过构建过表达PDI重组毕赤酵母菌株，使得外源蛋白cC的表达量显著增加。利用RNA-seq技术分析过表达PDI菌株与正常菌株的差异，最终筛选了373个差异表达基因，其中有122个差异基因注释到KEGG生物通路，包括12个基因注释到蛋白质转运和分解代谢途径，21个基因注释到蛋白质折叠分选和降解途径，以及24个基因参与蛋白质的翻译途径等。[结论] 在毕赤酵母中过表达PDI能显著增加外源蛋白cC的表达量。通过对过表达与正常表达PDI的毕赤酵母基因的表达谱分析，初步确定了其中一些转录情况变化显著的基因，明确了它们参与的细胞途径和信号通路，为改造具有高效率表达淀粉样蛋白的酵母菌株奠定基础。

Abstract:

[Objective] The aim of this study was to investigate the underlying mechanism for the regulation of protein disulfide isomerase (PDI) in protein folding and expression of exogenous proteins in yeast by co-expression of yeast PDI and its substrate protein chicken cystatin (cC). RNA-seq was carried out to screen for genes that are differentially expressed in Pichia pastoris (P. pastoris) and identify the key genes that could affect the expression of cC. This might provide theoretical support for the mechanistic analysis of high-level expression of exogenous protein and the construction of high-yield heterologous protein-expression strains.[Methods] PDI coding gene was transferred into GS115 and GS115 cC strains for the overexpression of PDI. RNA-seq was carried out to study the transcription of gene differences of two recombinant P. pastoris strains. Combined with the result of KEGG annotation, the data was analyzed to identify the differentially expressed genes between control and PDI/cC co-overexpressed cells, the result were verified by qRT-PCR and the functions in protein expression regulation were also clarified.[Results] The expression of cC was significantly increased in the PDI-overexpressing strain. A total of 373 differentially expressed genes were screened by RNA-seq analysis. Among them, 122 differentially expressed genes were assigned to the KEGG biological pathways, including 12 genes annotated into protein transport and catabolism pathway, 21 genes related to the protein folding sorting and degradation pathway, and 24 genes involved in the protein translation pathway.[Conclusion]] Overexpression of PDI in P. pastoris significantly increased the expression of the heterologous amyloidogenic protein cC. By analyzing the expression profiles of cC-overexpressed normal P. pastoris cells or PDI-overexpressed strain that co-overexpressed cC, some of the genes with significant transcriptional changes were preliminarily identified, laying a foundation for the transformation of yeast strains with high expression of amyloid protein.