疏水蛋白HFBI融合标签能显著提高外源基因在植物中的表达水平

摘要/Abstract

摘要： 目的针对植物生物反应器中外源基因表达水平普遍较低的问题，探索和评价丝状真菌瑞氏木霉（Trichoderma reesei）编码
的小分子疏水蛋白（Hydrophobin, HFBI）作为融合标签在植物生物反应器中提高系统表达量的应用潜力；分析其提高外源基因
表达水平的可能机制。方法以绿色荧光蛋白（GFP）为报道基因，采用基因体外合成技术和亚克隆技术构建GFP和GFP-HFBI
融合植物表达载体。利用农杆菌渗滤技术接种植物本明烟（Nicotiana benthamiana）。通过GFP荧光观察，荧光显微镜镜检，
Western blot，ELISA和RT-PCR等实验手段测定报告基因GFP在植物中的表达情况，探明HFBI融合标签在植物中表达外源基
因的作用效果和特点并分析其可能的作用机制。结果HFBI融合标签对植物细胞无明显的细胞毒性；GFP-HFBI融合蛋白在植
物中的积累水平显著高于对照；GFP-HFBI融合蛋白在细胞内形成致密的蛋白质颗粒。结论HFBI融合标签能够显著提高外源
基因在植物中的积累水平。推测形成的蛋白质颗粒隔绝了细胞内源性蛋白酶对目的表达产物的降解，进而提高了外源基因产
物在细胞中的积累。

Abstract: Objective To explore the mechanisms by which HFBI fusions increase recombinant fusion protein accumulation in
plants. Methods The HFBI sequence from Trichoderma reesei was synthesized and two plant expression vectors for expression
of green fluorescence protein (GFP) and GFP-HFBI were constructed. The vectors were inoculated in Nicotiana benthamiana
plants through agroinfiltration, and the expression levels and mRNA accumulation levels of GFP in Nicotiana leaves were
examined by Western blotting, ELISA and RT-PCR. Results The HFBI fusion tag significantly enhanced the accumulation of
GFP in the leaves of N. benthamiana without causing toxic effects. Endoplasmic reticulum-targeted GFP-HFBI fusion induced
the formation of spherical protein particles in the plant cells. Conclusion HFBI fusions can increase the accumulation of its
fusion partner in plants by forming stable protein particles, which probably shields the target protein from endogenous
protease-induced degadation. HFBI fusion technology provides an alternative to improving recombinant protein expression in
plants from agroinfection-compatible expression vectors.

张西倩，牟红珍，马婷，丁向真，李志英，王盛. 疏水蛋白HFBI融合标签能显著提高外源基因在植物中的表达水平[J]. 南方医科大学学报, 2015, 35(12): 1665-.

ZHANG Xiqian, MU Hongzhen, MA Ting, DING Xiangzhen, LI Zhiying, WANG Sheng. Effect of the hydrophobin HFBI-fusion tag on exogenous protein accumulation in tobacco
plant[J]. Journal of Southern Medical University, 2015, 35(12): 1665-.

参考文献

参考文献：
［1］ Redkiewicz P, Sirko A, Kamel KA, et al. Plant expression systems
for production of hemagglutinin as a vaccine against influenza virus
［J］. Acta Biochim Pol, 2014, 61(3): 551-60.
［2］ Virdi V, Depicker A. Role of plant expression systems in antibody
production for passive immunization［J］. Int J Dev Biol, 2013, 57(6/
8): 587-93.
［3］ Mortimer CL, Dugdale B, Dale JL. Updates in inducible transgene
expression using viral vectors: from transient to stable expression
［J］. Curr Opin Biotechnol, 2015, 32(8): 85-92.
［4］ Bell MR, Engleka MJ, Malik A, et al. To fuse or not to fuse:what is
your purpose［J］. Protein Sci, 2013, 22(11): 1466-77.
［5］Waugh DS. Making the most of affinity tags［J］. Trends Biotechnol,
2005, 23(6): 316-20.
［6］ Mishra S, Yadav DK, Tuli R. Ubiquitin fusion enhances cholera
toxin B subunit expression in transgenic plants and the plantexpressed
protein binds GM1 receptors more efficiently［J］. J
Biotechnol, 2006, 127(1): 95-108.
［7］ Dus Santos MJ, Wigdorovitz A, Trono K, et al. A novel methodology
to develop a foot and mouth disease virus (FMDV) peptide-based
vaccine in transgenic plants［J］. Vaccine, 2002, 20(7/8): 1141-7.
［8］ Kim TG, Ruprecht R, Langridge WH. Synthesis and assembly of a
cholera toxin B subunit SHIV 89.6p Tat fusion protein in transgenic
potato［J］. Protein Expr Purif, 2004, 35(2): 313-9.
［9］ Cañizares MC, Nicholson L, Lomonossoff GP. Use of viral vectors
for vaccine production in plants［J］. Immunol Cell Biol, 2005, 83
(3): 263-70.
［10］Obregon P, Chargelegue D, Drake PM, et al. HIV-1 p24-
immunoglobulin fusion molecule: a new strategy for plant-based
protein production［J］. Plant Biotechnol J, 2006, 4(2): 195-207.
［11］Streatfield SJ. Approaches to achieve high-level heterologous
protein production in plants［J］. Plant Biotechnol J, 2007, 5(1): 2-15.
［12］Rubio V, Shen Y, Saijo Y, et al. An alternative tandem affinity
purification strategy applied to Arabidopsis protein complex
isolation［J］. Plant J, 2005, 41(5): 767-78.
［13］Lichty JJ, Malecki JL, Agnew HD, et al. Comparison of affinity
tags for protein purification［J］. Protein Expr Purif, 2005, 41(1):
98-105.
［14］Linder MB. Hydrophobins: proteins that self assemble at interfaces
［J］. Curr Opin Colloid Interface Sci, 2009, 14(5): 356-63.
［15］Wösten HA, Scholtmeijer K. Applications of hydrophobins: current
state and perspectives［J］. Appl Microbiol Biotechnol, 2015, 99(4):
1587-97.
［16］Linder M, Selber K, Nakari-Setälä T, et al. The hydrophobins HFBI
and HFBII from Trichoderma reesei showing efficient interactions
with nonionic surfactants in aqueous two-phase systems［J］.
Biomacromolecules, 2001, 2(2): 511-7.
［17］Linder MB, Qiao M, Laumen F, et al. Efficient purification of
recombinant proteins using hydrophobins as tags in surfactantbased
two-phase systems［J］. Biochemistry, 2004, 43(37): 11873-82.
［18］Joensuu JJ, Conley AJ, Linder MB, et al. Bioseparation of
recombinant proteins from plant extract with hydrophobin fusion
technology［J］. Methods Mol Biol, 2012, 824(7): 527-34.
［19］Joensuu JJ, Conley AJ, Lienemann M, et al. Hydrophobin fusions
for high-level transient protein expression and purification in
Nicotiana benthamiana［J］. Plant Physiol, 2010, 152(2): 622-33.
［20］Reuter LJ, Bailey MJ, Joensuu JJ, et al. Scale-up of hydrophobinassisted
recombinant protein production in tobacco BY-2
suspension cells［J］. Plant Biotechnol J, 2014, 12(4): 402-10.
［21］Jacquet N, Navarre C, Desmecht D, et al. Hydrophobin fusion of an
influenza virus hemagglutinin allows high transient expression in
Nicotiana benthamiana, easy purification and immune response
with neutralizing activity［J］. PLoS One, 2014, 9(12): e115944.
［22］Phan HT, Hause B, Hause G, et al. Influence of elastin-like
polypeptide and hydrophobin on recombinant hemagglutinin
accumulations in transgenic tobacco plants［J］. PLoS One, 2014, 9
(6): e99347.
［23］Velásquez AC, Chakravarthy S, Martin GB. Virus-induced gene
silencing (VIGS) in Nicotiana benthamiana and tomato［J］. J Vis
Exp, 2009(28): 1-4.
［24］Anandalakshmi R, Gj P, Ge X, et al. A viral suppressor of gene
silencing in plants［J］. Proc Natl Acad Sci USA, 1998, 95(22):
13079-84.
［25］Desai PN, Shrivastava N, Padh H. Production of heterologous
proteins in plants: strategies for optimal expression［J］. Biotechnol
Adv, 2010, 28(4): 427-35.
［26］Gleba Y, Klimyuk V, Marillonnet S. Viral vectors for the expression
of proteins in plants［J］. Curr Opin Biotechnol, 2007, 18(2): 134-41.
［27］Daskalova SM, Radder JE, Cichacz ZA, et al. Engineering of N.
benthamiana L. plants for production of N-acetylgalactosamineglycosylated
proteins--towards development of a plant-based
platform for production of protein therapeutics with mucin type
O-glycosylation［J］. BMC Biotechnol, 2010, 10(4): 62.
［28］Yang Z, Drew DP, JØrgensen B, et al. Engineering mammalian
mucin-type O-glycosylation in plants［J］. J Biol Chem, 2012, 287
(15): 11911-23.
［29］Wösten HA. Hydrophobins: multipurpose proteins［J］. Annu Rev
Microbiol, 2001, 55(9): 625-46.
［30］Scholtmeijer K, Wessels JG, Wösten HA. Fungal hydrophobins in
medical and technical applications［J］. Appl Microbiol Biotechnol,
2001, 56(1/2): 1-8.