Home > Archive>Volume 61, Issue 10, 2021 >3315-3327. DOI:10.13343/j.cnki.wsxb.20210049
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Effect of PDI overexpressing on secretion of exogenous amyloid protein in Pichia pastoris—take cystatin as an example
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    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.

    Reference
    [1] Karbalaei M, Rezaee SA, Farsiani H. Pichia pastoris:a highly successful expression system for optimal synthesis of heterologous proteins. Journal of Cellular Physiology, 2020, 235(9):5867-5881.
    [2] Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM. Heterologous protein production using the Pichia pastoris expression system. Yeast, 2005, 22(4):249-270.
    [3] Ellgaard L, Ruddock LW. The human protein disulphide isomerase family:substrate interactions and functional properties. EMBO Reports, 2005, 6(1):28-32.
    [4] Tian G, Xiang S, Noiva R, Lennarz WJ, Schindelin H. The crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sites. Cell, 2006, 124(1):61-73.
    [5] Bode W, Engh R, Musil D, Thiele U, Huber R, Karshikov A, Brzin J, Kos J, Turk V. The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases. The EMBO Journal, 1988, 7(8):2593-2599.
    [6] Janowski R, Abrahamson M, Grubb A, Jaskolski M. Domain swapping in N-truncated human cystatin C. Journal of Molecular Biology, 2004, 341(1):151-160.
    [7] Janowski R, Kozak M, Abrahamson M, Grubb A, Jaskolski M. 3D domain-swapped human cystatin C with amyloidlike intermolecular beta-sheets. Proteins, 2005, 61(3):570-578.
    [8] Mori K. Tripartite management of unfolded proteins in the endoplasmic Reticulum. Cell, 2000, 101(5):451-454.
    [9] Higgins DR, Cregg JM. Introduction to Pichia pastoris. Methods in Molecular Biology:Clifton, N J, 1998, 103:1-15.
    [10] He JW, Sakamoto T, Song YT, Saito A, Harada A, Azakami H, Kato A. Effect of EPS1 gene deletion in Saccharomyces cerevisiae on the secretion of foreign proteins which have disulfide bridges. FEBS Letters, 2005, 579(11):2277-2283.
    [11] Gasser B, Saloheimo M, Rinas U, Dragosits M, Rodríguez-Carmona E, Baumann K, Giuliani M, Parrilli E, Branduardi P, Lang C, Porro D, Ferrer P, Tutino ML, Mattanovich D, Villaverde A. Protein folding and conformational stress in microbial cells producing recombinant proteins:a host comparative overview. Microbial Cell Factories, 2008, 7:11.
    [12] Zhu TC, Guo MJ, Zhuang YP, Chu J, Zhang SL. Understanding the effect of foreign gene dosage on the physiology of Pichia pastoris by transcriptional analysis of key genes. Applied Microbiology and Biotechnology, 2011, 89(4):1127-1135.
    [13] Gasser B, Maurer M, Gach J, Kunert R, Mattanovich D. Engineering of Pichia pastoris for improved production of antibody fragments. Biotechnology and Bioengineering, 2006, 94(2):353-361.
    [14] Inan M, Aryasomayajula D, Sinha J, Meagher MM. Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase. Biotechnology and Bioengineering, 2006, 93(4):771-778.
    [15] Elowitz MB. Stochastic gene expression in a single cell. Science, 2002, 297(5584):1183-1186.
    [16] Hansen KD, Wu ZJ, Irizarry RA, Leek JT. Sequencing technology does not eliminate biological variability. Nature Biotechnology, 2011, 29(7):572-573.
    [17] Robasky K, Lewis NE, Church GM. The role of replicates for error mitigation in next-generation sequencing. Nature Reviews Genetics, 2014, 15(1):56-62.
    [18] Schulze SK, Kanwar R, Gölzenleuchter M, Therneau TM, Beutler AS. SERE:single-parameter quality control and sample comparison for RNA-Seq. BMC Genomics, 2012, 13:524.
    [19] Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, Katayama T, Kawashima S, Okuda S, Tokimatsu T, Yamanishi Y. KEGG for linking genomes to life and the environment. Nucleic Acids Research, 2008, 36(suppl_1):D480-D484.
    [20] Daly R, Hearn MTW. Expression of heterologous proteins in Pichia pastoris:a useful experimental tool in protein engineering and production. Journal of Molecular Recognition, 2005, 18(2):119-138.
    [21] Montoliu-Gaya L, Esquerda-Canals G, Bronsoms S, Villegas S. Production of an anti-Aβ antibody fragment in Pichia pastoris and in vitro and in vivo validation of its therapeutic effect. PLoS ONE, 2017, 12(8):e0181480.
    [22] Guan B, Chen FX, Su S, Duan ZY, Chen Y, Li HZ, Jin J. Effects of co-overexpression of secretion helper factors on the secretion of a HSA fusion protein (IL2-HSA) in Pichia pastoris. Yeast, 2016, 33(11):587-600.
    [23] Cheng H, Wang L, Wang CC. Domain a' of protein disulfide isomerase plays key role in inhibiting α-synuclein fibril formation. Cell Stress and Chaperones, 2010, 15(4):415-421.
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Tingting Niu, Xuejie Zhou, Xiaobo Yu, Yisheng Cai, Jing Chen, Rui Wang, Shuang Fu, Chuang Yi, Jianwei He. Effect of PDI overexpressing on secretion of exogenous amyloid protein in Pichia pastoris—take cystatin as an example. [J]. Acta Microbiologica Sinica, 2021, 61(10): 3315-3327

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  • Received:January 23,2021
  • Revised:March 30,2021
  • Online: September 29,2021
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