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

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.