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2025年4月3日 20:39 星期四
首页 > 过刊浏览>2024年第40卷第3期 >834-846. DOI:10.13345/j.cjb.230512
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毕赤酵母全蛋白质组信号肽及分泌蛋白分析
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国家重点研发计划(2021YFC2103901)；国家自然科学基金(31970059)；中央高校基本科研业务费专项资金(buctrc202131)


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

    参考文献
    [1] 吴静雯. α-factor信号肽及其N-糖基化修饰对毕赤酵母外源蛋白分泌的影响[D]. 福州:福建师范大学硕士学位论文, 2019. WU JW. Effects of α-factor signal peptides and its N-glycosylation on secretion of heterologous proteins in Pichia pastoris [D]. Fuzhou:Master's Thesis of Fujian Normal University, 2019(in Chinese).
    [2] KARBALAEI M, REZAEE SA, FARSIANI H. Pichia pastoris:a highly successful expression system for optimal synthesis of heterologous proteins[J]. Journal of Cellular Physiology, 2020, 235(9):5867-5881.
    [3] DAMASCENO LM, HUANG CJ, BATT CA. Protein secretion in Pichia pastoris and advances in protein production[J]. Applied Microbiology and Biotechnology, 2012, 93(1):31-39.
    [4] de SCHUTTER K, LIN YC, TIELS P, van HECKE A, GLINKA S, WEBER-LEHMANN J, ROUZÉ P, van de PEER Y, CALLEWAERT N. Genome sequence of the recombinant protein production host Pichia pastoris [J]. Nature Biotechnology, 2009, 27(6):561-566.
    [5] KÜBERL A, SCHNEIDER J, THALLINGER GG, ANDERL I, WIBBERG D, HAJEK T, JAENICKE S, BRINKROLF K, GOESMANN A, SZCZEPANOWSKI R, PÜHLER A, SCHWAB H, GLIEDER A, PICHLER H. High-quality genome sequence of Pichia pastoris CBS7435[J]. Journal of Biotechnology, 2011, 154(4):312-320.
    [6] STURMBERGER L, CHAPPELL T, GEIER M, KRAINER F, DAY KJ, VIDE U, TRSTENJAK S, SCHIEFER A, RICHARDSON T, SORIAGA L, DARNHOFER B, BIRNER-GRUENBERGER R, GLICK BS, TOLSTORUKOV I, CREGG J, MADDEN K, GLIEDER A. Refined Pichia pastoris reference genome sequence[J]. Journal of Biotechnology, 2016, 235:121-131.
    [7] 王庆华, 高丽丽, 梁会超, 巩婷, 杨金玲, 朱平. 影响毕赤酵母高效表达重组蛋白的主要因素及其研究进展[J]. 药学学报, 2014, 49(12):1644-1649. WANG QH, GAO LL, LIANG HC, GONG T, YANG JL, ZHU P. Research advances of the influence factors of high level expression of recombinant protein in Pichia pastoris [J]. Acta Pharmaceutica Sinica, 2014, 49(12):1644-1649(in Chinese).
    [8] RUTKOWSKI DT, OTT CM, POLANSKY JR, LINGAPPA VR. Signal sequences initiate the pathway of maturation in the endoplasmic reticulum lumen[J]. Journal of Biological Chemistry, 2003, 278(32):30365-30372.
    [9] LIACI AM, FÖRSTER F. Take me home, protein roads:structural insights into signal peptide interactions during ER translocation[J]. International Journal of Molecular Sciences, 2021, 22(21):11871.
    [10] LIN-CEREGHINO GP, STARK CM, KIM D, CHANG J, SHAHEEN N, POERWANTO H, AGARI K, MOUA P, LOW LK, TRAN N, HUANG AD, NATTESTAD M, OSHIRO KT, CHANG JW, CHAVAN A, TSAI JW, LIN-CEREGHINO J. The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris [J]. Gene, 2013, 519(2):311-317.
    [11] MURASUGI A, TOHMA-AIBA Y. Comparison of three signals for secretory expression of recombinant human midkine in Pichia pastoris [J]. Bioscience, Biotechnology, and Biochemistry, 2001, 65(10):2291-2293.
    [12] OKA C, TANAKA M, MURAKI M, HARATA K, SUZUKI K, JIGAMI Y. Human lysozyme secretion increased by alpha-factor pro-sequence in Pichia pastoris [J]. Bioscience, Biotechnology, and Biochemistry, 1999, 63(11):1977-1983.
    [13] GHOSALKAR A, SAHAI V, SRIVASTAVA A. Secretory expression of interferon-alpha 2b in recombinant Pichia pastoris using three different secretion signals[J]. Protein Expression and Purification, 2008, 60(2):103-109.
    [14] BARRERO JJ, CASLER JC, VALERO F, FERRER P, GLICK BS. An improved secretion signal enhances the secretion of model proteins from Pichia pastoris [J]. Microbial Cell Factories, 2018, 17(1):161.
    [15] ITO Y, ISHIGAMI M, HASHIBA N, NAKAMURA Y, TERAI G, HASUNUMA T, ISHII J, KONDO A. Avoiding entry into intracellular protein degradation pathways by signal mutations increases protein secretion in Pichia pastoris [J]. Microbial Biotechnology, 2022, 15(9):2364-2378.
    [16] LIANG SL, LI C, YE YR, LIN Y. Endogenous signal peptides efficiently mediate the secretion of recombinant proteins in Pichia pastoris [J]. Biotechnology Letters, 2013, 35(1):97-105.
    [17] KARAOGLAN M, YILDIZ H, INAN M. Screening of signal sequences for extracellular production of Aspergillus niger xylanase in Pichia pastoris [J]. Biochemical Engineering Journal, 2014, 92:16-21.
    [18] SHEN Q, ZHOU XT, GUO Q, XUE YZ, XUE YP, ZHENG YG. Potential of the signal peptide derived from the PAS_chr3_0030 gene product for secretory expression of valuable enzymes in Pichia pastoris [J]. Applied and Environmental Microbiology, 2022, 88(9):e0029622.
    [19] DUAN GD, DING LM, WEI DS, ZHOU HC, CHU J, ZHANG SL, QIAN JC. Screening endogenous signal peptides and protein folding factors to promote the secretory expression of heterologous proteins in Pichia pastoris [J]. Journal of Biotechnology, 2019, 306:193-202.
    [20] CREGG JM, BARRINGER KJ, HESSLER AY, MADDEN KR. Pichia pastoris as a host system for transformations[J]. Molecular and Cellular Biology, 1985, 5(12):3376-3385.
    [21] CHOO KH, RANGANATHAN S. Flanking signal and mature peptide residues influence signal peptide cleavage[J]. BMC Bioinformatics, 2008, 9:1-11.
    [22] 张光荣, 袁再美, 孙丽媛, 邓茗月, 王荣蛟, 和世春, 王寒雪, 毛华明, 杨舒黎. 奤頠牣癡聲繲荩er-dr虩癶繥杮吠剣聲癹畳牴佡恬孬剩杺ation鹛齊汝甮爠光单CrJ, 2016, 3(Pt 1):10-19.
    [38] TIAN J, ZHANG YH, LIU B, ZUO DY, JIANG T, GUO J, ZHANG W, WU NF, FAN YL. Presep:predicting the propensity of a protein being secreted into the supernatant when expressed in Pichia pastoris [J]. PLoS One, 2013, 8(11):e79749.
    [39] WANG P, HUANG L, JIANG H, TIAN J, CHU XY, WU NF. Improving the secretion of a methyl parathion hydrolase in Pichia pastoris by modifying its N-terminal sequence[J]. PLoS One, 2014, 9(5):e96974.ndoplasmic reticulum of Saccharomyces cerevisiae [J]. Journal of Biological Chemistry, 2017, 292(50):20707-20719.
    [24] 王建国, 赵南南, 何志云, 王泽. 白假丝酵母菌(Candida albicans) WO-1分泌蛋白质组的生物信息学分析[J]. 沈阳师范大学学报(自然科学版), 2020, 38(5):463-467. WANG JG, ZHAO NN, HE ZY, WANG Z. Bioinformatics analysis of secretome in Candida albicans WO-1[J]. Journal of Shenyang Normal University (Natural Science Edition), 2020, 38(5):463-467(in Chinese).
    [25] CHU YJ, COREY DR. RNA sequencing:platform selection, experimental design, and data interpretation[J]. Nucleic Acid Therapeutics, 2012, 22(4):271-274.
    [26] YUAN J, TAO WJ, CHENG YY, HUANG BF, WANG DS. Genome-wide identification, phylogeny, and gonadal expression of fox genes in Nile tilapia, Oreochromis niloticus [J]. Fish Physiology and Biochemistry, 2014, 40(4):1239-1252.
    [27] XU N, ZHU JX, ZHU QY, XING YZ, CAI MH, JIANG TY, ZHOU M, ZHANG YX. Identification and characterization of novel promoters for recombinant protein production in yeast Pichia pastoris [J]. Yeast, 2018, 35(5):379-385.
    [28] MASSAHI A, ÇALIK P. Endogenous signal peptides in recombinant protein production by Pichia pastoris:from in-silico analysis to fermentation[J]. Journal of Theoretical Biology, 2016, 408:22-33.
    [29] 杨青, 汪斌, 王亚伟, 张华山, 熊海容, 张莉. 介导两种半纤维素酶分泌表达的信号肽比较[J]. 中国生物工程杂志, 2017, 37(8):15-22. YANG Q, WANG B, WANG YW, ZHANG HS, XIONG HR, ZHANG L. Comparison of signal peptides for two hemicellulase secretory expression[J]. China Biotechnology, 2017, 37(8):15-22(in Chinese).
    [30] 石艺平, 周雪, 胡美荣, 林剑辉, 陶勇, 黄建忠. 不同信号肽对毕赤酵母表达漆酶的影响[J]. 微生物学报, 2014, 54(12):1446-1452. SHI YP, ZHOU X, HU MR, LIN JH, TAO Y, HUANG JZ. Effect of signal peptides on the expression of laccase in Pichia pastoris [J]. Acta Microbiologica Sinica, 2014, 54(12):1446-1452(in Chinese).
    [31] 苗杨利, 李站胜, 韩双艳. 促进米黑根毛霉脂肪酶分泌的毕赤酵母内源信号肽的筛选[J]. 现代食品科技, 2019, 35(10):155-163. MIAO YL, LI ZS, HAN SY. Screening of endogenous signal peptides from Pichia pastoris for promoting lipase secretion by Rhizopus oryzae [J]. Modern Food Science and Technology, 2019, 35(10):155-163(in Chinese).
    [32] 田小梅, 任建洪, 房聪. 不同碳源下毕赤酵母GS115蛋白组学分析[J]. 中国生物工程杂志, 2012, 32(1):21-29. TIAN XM, REN JH, FANG C. Proteomic analysis of Pichia pastoris GS115 cultured in different carbon source mediums[J]. China Biotechnology, 2012, 32(1):21-29(in Chinese).
    [33] GUO H, SUN JH, LI X, XIONG Y, WANG HT, SHU H, ZHU RM, LIU Q, HUANG YM, MADLEY R, WANG YL, CUI JQ, ARVAN P, LIU M. Positive charge in the n-region of the signal peptide contributes to efficient post-translational translocation of small secretory preproteins[J]. Journal of Biological Chemistry, 2018, 293(6):1899-1907.
    [34] OWJI H, NEZAFAT N, NEGAHDARIPOUR M, HAJIEBRAHIMI A, GHASEMI Y. A comprehensive review of signal peptides:structure, roles, and applications[J]. European Journal of Cell Biology, 2018, 97(6):422-441.
    [35] NILSSON I, WHITLEY P, von HEIJNE G. The COOH-terminal ends of internal signal and signal-anchor sequences are positioned differently in the ER translocase[J]. The Journal of Cell Biology, 1994, 126(5):1127-1132.
    [36] XUE SL, LIU XF, PAN YY, XIAO CF, FENG YZ, ZHENG L, ZHAO MM, HUANG MT. Comprehensive analysis of signal peptides in Saccharomyces cerevisiae reveals features for efficient secretion[J]. Advanced Science (Weinheim, Baden-Wurttemberg, Germany), 2023, 10(2):e2203433.
    [37] TING YT, HARRIS PWR, BATOT G, BRIMBLE MA, BAKER EN, YOUNG PG. Peptide binding to a bacterial signal peptidase visualized by peptide tethering an
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朱淩宣,崔鑫,胡善桐,王诗卉,张桂敏. 毕赤酵母全蛋白质组信号肽及分泌蛋白分析[J]. 生物工程学报, 2024, 40(3): 834-846

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  • 收稿日期:2023-07-17
  • 最后修改日期:2023-11-22
  • 在线发布日期: 2024-03-25
  • 出版日期: 2024-03-25
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