玉米γ-Zein多肽融合增强外源重组蛋白在大豆种子中的高效积累
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摘要
大豆(Glycine max)种子的高水平蛋白合成和积累能力,为外源重组蛋白的高效表达提供了理想的表达宿主。为进一步提高外源蛋白在大豆种子中的表达水平,本研究基于多肽融合策略,研究玉米(Zea mays)醇溶蛋白γ-Zein对外源蛋白在大豆种子中积累水平的影响。采用农杆菌(Agrobacterium tumefaciens)介导法,分别将大豆种子特异性启动子基因β'-伴球蛋白基因启动子(β-conglycininalpha subunit promoter, BCSP)驱动的绿色荧光蛋白(green fluorescent protein, GFP)编码基因和Zein-GFP基因导入栽培大豆品种,共获得Zein-GFP转基因植株41株,GFP转基因植株46株。反转录PCR(reverse transcription PCR, RT-PCR)和Western blot检测表明,外源基因Zein-GFP和GFP在转基因大豆种子中均能够正常转录和翻译。选取外源基因mRNA表达水平相近的T3代转基因大豆株系进行酶联免疫吸附测定(enzyme linked immunosorbent assay, ELISA)分析表明,转基因大豆种子中融合蛋白Zein-GFP平均表达水平为1.51%TSP (1.37%~1.60%TSP)(可溶性蛋白总量, total soluble protein),未融合蛋白GFP平均表达水平为0.14%TSP(0.13%~0.15%TSP)。融合蛋白Zein-GFP在转基因大豆种子中的积累水平较GFP提高10.78倍。激光共聚焦扫描分析进一步表明,融合蛋白Zein-GFP在转基因大豆种子中主要以蛋白体的形式存在,而未融合GFP蛋白则主要分布在胞质中。本研究结果表明,γ-Zein多肽融合促进了重组蛋白在细胞蛋白体中的积累,并显著提高其在大豆种子中的表达水平。本研究为加快大豆种子反应器的应用提供依据。
Soybean(Glycine max) seeds provide an ideal host for the production of foreign recombinant proteins because of their robust capability for biosynthesis and accumulation of the native proteins. To further enhance the expression of foreign proteins in soybean seeds, a polypeptide fusion strategy was utilized to investigate the influence of the maize(Zea mays) γ-Zein fusion tag on accumulation of the foreign recombinant protein. The GFP(green fluorescent protein) and Zein-GFP driven by the soybean seed-specific promoter BCSP were individually introduced into the cultivated soybean genotype by Agrobacterium tumefaciens-mediated transformation, respectively. Totally 41 Zein-GFP and 46 GFP transgenic plants were obtained in this study. Reverse transcription PCR(RT-PCR) and Western blot analysis confirmed transcript and translation of the foreign genes in the transgenic soybean seeds. The transgenic lines with similar expression of Zein-GFP and GFP at mRNA levels in seeds were selected and accumulation of the recombinant proteins was quantified by enzyme linked immunosorbent assay(ELISA). Average accumulation level of the fusion protein Zein-GFP reached to 1.51% TSP(total soluble protein) ranging from 1.37% to 1.60% TSP in the transgenic seeds, increased by 10.78 fold compared with 0.14% TSP of the unfused GFP(0.13%~0.15%TSP). Subcellular targeting analysis by laser scanning confocal microscopy showed that the γ-Zein fused GFP were present in the protein bodies(PBs), while the unfused GFP was mainly distributed in the cytoplasm.Taken together, the results showed that the γ-Zein polypeptide fusion induced the formation of PBs and significantly enhanced accumulation of the foreign recombinant protein in the transgenic soybean seeds, and thus provide the basis for application of the soybean-based bio-reactor.
Soybean(Glycine max) seeds provide an ideal host for the production of foreign recombinant proteins because of their robust capability for biosynthesis and accumulation of the native proteins. To further enhance the expression of foreign proteins in soybean seeds, a polypeptide fusion strategy was utilized to investigate the influence of the maize(Zea mays) γ-Zein fusion tag on accumulation of the foreign recombinant protein. The GFP(green fluorescent protein) and Zein-GFP driven by the soybean seed-specific promoter BCSP were individually introduced into the cultivated soybean genotype by Agrobacterium tumefaciens-mediated transformation, respectively. Totally 41 Zein-GFP and 46 GFP transgenic plants were obtained in this study. Reverse transcription PCR(RT-PCR) and Western blot analysis confirmed transcript and translation of the foreign genes in the transgenic soybean seeds. The transgenic lines with similar expression of Zein-GFP and GFP at mRNA levels in seeds were selected and accumulation of the recombinant proteins was quantified by enzyme linked immunosorbent assay(ELISA). Average accumulation level of the fusion protein Zein-GFP reached to 1.51% TSP(total soluble protein) ranging from 1.37% to 1.60% TSP in the transgenic seeds, increased by 10.78 fold compared with 0.14% TSP of the unfused GFP(0.13%~0.15%TSP). Subcellular targeting analysis by laser scanning confocal microscopy showed that the γ-Zein fused GFP were present in the protein bodies(PBs), while the unfused GFP was mainly distributed in the cytoplasm.Taken together, the results showed that the γ-Zein polypeptide fusion induced the formation of PBs and significantly enhanced accumulation of the foreign recombinant protein in the transgenic soybean seeds, and thus provide the basis for application of the soybean-based bio-reactor.
引文
Boothe J,Nykiforuk C,Shen Y,et al.2010.Seed-based expression systems for plant molecular farming[J].Plant Biotechnology Journal,8(5):588-606.
Ceresoli V,Mainieri D,Del Fabbro M,et al.2016.A fusion between domains of the human bone morphogenetic protein-2 and maize 27 kDγ-Zein accumulates to high levels in the endoplasmic reticulum without forming brotein bodies in transgenic tobacco[J].Fronts in Plant Science,(7):358.
Conley A J,Joensuu J J,Richman A,et al.2011.Protein bodyinducing fusions for high-level production and purification of recombinant proteins in plants[J].Plant Biotechnology Journal,9(4):419-433.
Cunha N B,Murad A M,Cipriano T M,et al.2011a.Expression of a functional recombinant human growth hormone in transgenic soybean seeds[J].Transgenic Research,20(4):811-826.
Cunha N B,Murad A M,Ramos G L,et al.2011b.Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds[J].Transgenic Research,20(4):841-855.
De Jaeger G,Scheffer S,Jacobs A,et al.2002.Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences[J].Nature Biotechnology,20(12):1265-1268.
Droogenbroeck B V,Cao J,Stadlmann J,et al.2007.Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds[J].Proceedings of the National Academy of Sciences,104(4):1430-1435.
Floss D M,Schallau K,Rose-John S,et al.2010.Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application[J].Trends in Biotechnology,28(1):37-45.
Gutiérrez S P,Saberianfar R,Kohalmi S E,et al.2013.Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins[J].BMC Biotechnology,13(1):1-11.
He Y,Ning T,Xie T,et al.2011.Large-scale production of functional human serum albumin from transgenic rice seeds[J].Proceedings of the National Academy of Sciences of the USA,108(47):19078-19083.
Hofbauer A,Peters J,Arcalis E,et al.2014.The induction of recombinant protein bodies in different sub-cellular compartments reveals a cryptic plastid-targeting signal in the 27-kDaγ-zein sequence[J].Frontiers in Bioengineering and Biotechnology,2:67.
Hudson L C,Garg R,Bost K L,et al.2014.Soybean seeds:a practical host for the production of functional subunit vaccines[J].Bio Med Research International,2014(9):340804.
Joensuu J J,Conley A J,Lienemann M,et al.2010.Hydrophobin fusions for high-level transient protein expression and purification in Nicotiana benthamiana[J].Plant Physiology,152(2):622-633.
Huang N.2004.High-level protein expression system uses self-pollinating crops as hosts[J].BioProcess International,2:54-59.
Mainieri D,Rossi M,Archinti M,et al.2004.Zeolin,A new recombinant storage protein constructed using maizeγ-Zein and bean phaseolin[J].Plant Physiology,136(3):3447-3456.
Moravec T,Schmidt M A,Herman E M,et al.2007.Production of Escherichia coli heat labile toxin(LT)B subunit in soybean seed and analysis of its immunogenicity as an oral vaccine[J].Vaccine,25:1647-1657.
O'keefe B R,Murad A M,Vianna G R,et al.2015.Engineering soya bean seeds as a scalable platform to produce cyanovirin-N,a non-ARV microbicide against HIV[J].Plant Biotechnology Journal,13(7):884-892.
Phan H T,Hause B,Hause G,et al.2014.Influence of elastinlike polypeptide and hydrophobin on recombinant hemagglutinin accumulations in transgenic tobacco plants[J].PLoS One,9(6):e99347.
Piller K J,Clemente T E,Jun S M,et al.2005.Expression and immunogenicity of an Escherichia coli K99 fimbriae subunit antigen in soybean[J].Planta,222(1):6-18.
Powell R,Hudson L C,Lambirth K C,et al.2011.Recombinant expression of homodimeric 660 k Da human thyroglobulin in soybean seeds:An alternative source of human thyroglobulin[J].Plant Cell Reports,30(7):1327-1338.
Saberianfar R,Joensuu J J,Conley A J,et al.2015.Protein body formation in leaves of Nicotiana benthamiana:Aconcentration-dependent mechanism influenced by the presence of fusion tags[J].Plant Biotechnology Journal,13(7):927-937.
Saberianfar R,Sattarzadeh A,Joensuu J J,et al.2016.Protein bodies in leaves exchange contents through the endoplasmic reticulum[J].Fronts in Plant Science,(7):693.
Saberianfar R,Menassa R.2017.Protein bodies:How the ERdeals with high accumulation of recombinant proteins[J].Plant Biotechnology Journal,15(6):671-673.
Scheller J,Leps M,Conrad U.2006.Forcing single-chain variable fragment production in tobacco seeds by fusion to elastin-like polypeptides[J].Plant Biotechnology Journal,4(2):243-249.
Torrent M,Llompart B,Lasserre-Ramassamy S,et al.2009.Eukaryotic protein production in designed storage organelles[J].BMC Biology,7(1):5.
Torrent M,Llop T I,Ludevid M D.2009.Protein body induction:A new tool to produce and recover recombinant proteins in plants[J].Methods in Molecular Biology,483:193-208.
Virgilio M D,Marchis F D,Bellucci M,et al.2008.The Human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin[J].Journal of Experimental Botany,59(10):2815-2829.
Yamada Y,Nishizawa K,Yokoo M,et al.2008.Anti-hypertensive activity of genetically modified soybean seeds accumulating novokinin[J].Peptides,29(3):331-337.
Yang X,Niu L,Zhang W,Yang J,et al.2018.RNAi-mediated SMV P3 cistron silencing confers significantly enhanced resistance to multiple Potyvirus strains and isolates in transgenic soybean[J].Plant Cell Reports,37(1):103-114.
Zhang D,Lee H F,Pettit S C,et al.2012.Characterization of transferrin receptor mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice(Oryza sativa L.)[J].BMC Biotechnol,12:92.
Zhou L,He H,Liu R,et al.2014.Overexpression of GmAKT2 potassium channel enhances resistance to soybean mosaic virus[J].BMC Plant Biology,14(1):154.
Ceresoli V,Mainieri D,Del Fabbro M,et al.2016.A fusion between domains of the human bone morphogenetic protein-2 and maize 27 kDγ-Zein accumulates to high levels in the endoplasmic reticulum without forming brotein bodies in transgenic tobacco[J].Fronts in Plant Science,(7):358.
Conley A J,Joensuu J J,Richman A,et al.2011.Protein bodyinducing fusions for high-level production and purification of recombinant proteins in plants[J].Plant Biotechnology Journal,9(4):419-433.
Cunha N B,Murad A M,Cipriano T M,et al.2011a.Expression of a functional recombinant human growth hormone in transgenic soybean seeds[J].Transgenic Research,20(4):811-826.
Cunha N B,Murad A M,Ramos G L,et al.2011b.Accumulation of functional recombinant human coagulation factor IX in transgenic soybean seeds[J].Transgenic Research,20(4):841-855.
De Jaeger G,Scheffer S,Jacobs A,et al.2002.Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences[J].Nature Biotechnology,20(12):1265-1268.
Droogenbroeck B V,Cao J,Stadlmann J,et al.2007.Aberrant localization and underglycosylation of highly accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds[J].Proceedings of the National Academy of Sciences,104(4):1430-1435.
Floss D M,Schallau K,Rose-John S,et al.2010.Elastin-like polypeptides revolutionize recombinant protein expression and their biomedical application[J].Trends in Biotechnology,28(1):37-45.
Gutiérrez S P,Saberianfar R,Kohalmi S E,et al.2013.Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins[J].BMC Biotechnology,13(1):1-11.
He Y,Ning T,Xie T,et al.2011.Large-scale production of functional human serum albumin from transgenic rice seeds[J].Proceedings of the National Academy of Sciences of the USA,108(47):19078-19083.
Hofbauer A,Peters J,Arcalis E,et al.2014.The induction of recombinant protein bodies in different sub-cellular compartments reveals a cryptic plastid-targeting signal in the 27-kDaγ-zein sequence[J].Frontiers in Bioengineering and Biotechnology,2:67.
Hudson L C,Garg R,Bost K L,et al.2014.Soybean seeds:a practical host for the production of functional subunit vaccines[J].Bio Med Research International,2014(9):340804.
Joensuu J J,Conley A J,Lienemann M,et al.2010.Hydrophobin fusions for high-level transient protein expression and purification in Nicotiana benthamiana[J].Plant Physiology,152(2):622-633.
Huang N.2004.High-level protein expression system uses self-pollinating crops as hosts[J].BioProcess International,2:54-59.
Mainieri D,Rossi M,Archinti M,et al.2004.Zeolin,A new recombinant storage protein constructed using maizeγ-Zein and bean phaseolin[J].Plant Physiology,136(3):3447-3456.
Moravec T,Schmidt M A,Herman E M,et al.2007.Production of Escherichia coli heat labile toxin(LT)B subunit in soybean seed and analysis of its immunogenicity as an oral vaccine[J].Vaccine,25:1647-1657.
O'keefe B R,Murad A M,Vianna G R,et al.2015.Engineering soya bean seeds as a scalable platform to produce cyanovirin-N,a non-ARV microbicide against HIV[J].Plant Biotechnology Journal,13(7):884-892.
Phan H T,Hause B,Hause G,et al.2014.Influence of elastinlike polypeptide and hydrophobin on recombinant hemagglutinin accumulations in transgenic tobacco plants[J].PLoS One,9(6):e99347.
Piller K J,Clemente T E,Jun S M,et al.2005.Expression and immunogenicity of an Escherichia coli K99 fimbriae subunit antigen in soybean[J].Planta,222(1):6-18.
Powell R,Hudson L C,Lambirth K C,et al.2011.Recombinant expression of homodimeric 660 k Da human thyroglobulin in soybean seeds:An alternative source of human thyroglobulin[J].Plant Cell Reports,30(7):1327-1338.
Saberianfar R,Joensuu J J,Conley A J,et al.2015.Protein body formation in leaves of Nicotiana benthamiana:Aconcentration-dependent mechanism influenced by the presence of fusion tags[J].Plant Biotechnology Journal,13(7):927-937.
Saberianfar R,Sattarzadeh A,Joensuu J J,et al.2016.Protein bodies in leaves exchange contents through the endoplasmic reticulum[J].Fronts in Plant Science,(7):693.
Saberianfar R,Menassa R.2017.Protein bodies:How the ERdeals with high accumulation of recombinant proteins[J].Plant Biotechnology Journal,15(6):671-673.
Scheller J,Leps M,Conrad U.2006.Forcing single-chain variable fragment production in tobacco seeds by fusion to elastin-like polypeptides[J].Plant Biotechnology Journal,4(2):243-249.
Torrent M,Llompart B,Lasserre-Ramassamy S,et al.2009.Eukaryotic protein production in designed storage organelles[J].BMC Biology,7(1):5.
Torrent M,Llop T I,Ludevid M D.2009.Protein body induction:A new tool to produce and recover recombinant proteins in plants[J].Methods in Molecular Biology,483:193-208.
Virgilio M D,Marchis F D,Bellucci M,et al.2008.The Human immunodeficiency virus antigen Nef forms protein bodies in leaves of transgenic tobacco when fused to zeolin[J].Journal of Experimental Botany,59(10):2815-2829.
Yamada Y,Nishizawa K,Yokoo M,et al.2008.Anti-hypertensive activity of genetically modified soybean seeds accumulating novokinin[J].Peptides,29(3):331-337.
Yang X,Niu L,Zhang W,Yang J,et al.2018.RNAi-mediated SMV P3 cistron silencing confers significantly enhanced resistance to multiple Potyvirus strains and isolates in transgenic soybean[J].Plant Cell Reports,37(1):103-114.
Zhang D,Lee H F,Pettit S C,et al.2012.Characterization of transferrin receptor mediated endocytosis and cellular iron delivery of recombinant human serum transferrin from rice(Oryza sativa L.)[J].BMC Biotechnol,12:92.
Zhou L,He H,Liu R,et al.2014.Overexpression of GmAKT2 potassium channel enhances resistance to soybean mosaic virus[J].BMC Plant Biology,14(1):154.