原核表达手掌参GcGASA蛋白的光谱学及功能研究
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摘要
赤霉酸诱导的富含半胱氨酸蛋白是近年来发现的一类在N-末端具有一段不同长度的信号肽,C-末端含有12个保守的半胱氨酸残基的蛋白,这类蛋白在多种植物的重要生理过程中发挥着重要作用。本研究旨在利用原核表达获取可溶性的手掌参中赤霉酸诱导的富含半胱氨酸蛋白(GcGASA),研究其体外活性并运用荧光光谱学手段研究其内源荧光。
方法:利用pET-32(a)作为原核表达载体,对手掌参中赤霉酸诱导的富含半胱氨酸蛋白GcGASA进行原核表达。并通过亲和层析、SDS-PAGE、Native-PAGE等对原核表达融合蛋白Trx-GcGASA进行纯化和鉴定。利用琼脂糖扩散法抑菌试验和清除过氧化氢实验验证Trx-GcGASA的体外活性,并利用稳态荧光光谱检测对经过DTT还原,Native形态,氧化剂处理,以及盐酸胍变性的融合蛋白Trx-GcGASA进行荧光光谱学研究。
结果:Native-PAGE检测结果显示经原核表达和纯化得到的融合蛋白以单体和三聚体两种形式存在。融合蛋白的抗微生物活性和抗氧化活性试验结果显示,融合蛋白在10uM浓度时能够使大肠杆菌出现明显聚集,然而没有明显的清除H2O2能力。稳态荧光光谱学研究显示:(1)当激发波长在250nm-280nm之间时,原核表达纯化得到的融合蛋白Trx-GcGASA的荧光发射主要源于酪氨酸残基。(2)DTT还原处理之后,融合蛋白色氨酸的荧光强度显著增加。(3)融合蛋白经过氧化型的谷胱甘肽GSSG或者过氧化氢氧化处理之后,其中酪氨酸和色氨酸的荧光强度都有所降低,而这与文献报道的此类蛋白的抗氧化活性一致。(4)无论经过1 mM DTT还原处理与否,融合蛋白在6M盐酸胍存在的条件下都不能彻底变性,色氨酸的最大荧光发射峰位置小于350nm。(5)变性曲线显示,原核表达纯化得到的融合蛋白Trx-GcGASA在346.2nm处的荧光强度在盐酸胍浓度为2M-3M之间时发生突变,应用蛋白变性两态模型计算得到融合蛋白的Gibbs自由能为3.7 kJ.mol-1。然而,经过1 mM DTT还原的融合蛋白却表现出相反的趋势,盐酸胍浓度从0M增加到1.3M时,荧光强度显著增加了1.8倍,然后在1.3M到2.6M之间降低了26%。
本文的相关研究为进一步深入探讨GcGASA蛋白的结构、生理活性以及后续有关富含半胱氨酸蛋白质的解折叠和复性研究等工作奠定了基础。
Gibberellin-induced cysteine-rich proteins(GIP),which have N-terminal signal peptides with varying length and share 12 conserved cysteine residues at C-terminal domain, play many siginificant roles in plants. However,the function of GIP in vitro and structural information were unknown up to now.
Methods: The heterogeneous gene of Gymnadnia conopsea(designated gcgasa) has been expressed in Escherichia coli BL21(DE3) using pET-32 (a) as the expression plasmid. Following the purification and identification of fusion protein Trx-GcGASA , we investigated its existing state by Native-PAGE, antimicrobial activity and the ability to eliminate peroxide in vitro. Additionally, the intrinsic fluorescence of Trx-GcGASA has also been studied in the absence and presence of oxidant, reductant and GndHCl by using steady-state fluorescence spectroscopic methods.
Results: Native-PAGE results revealed that fusion protein simultaneously exist in the form of monomer and trimer. The studies on physiological functions suggested that Trx-GcGASA might cause the aggregation of Escherichia coli with the concentration of 10 uM, however, it was not able to eliminate H2O2 directly. The intrinsic fluorescence results revealed that (1) Tyrosine made the dominating contribution to the overall fluorescence emission spectra following excitation at different wavelengths varying from 250 nm to 280 nm. (2) The introduction of DTT greatly enhanced the fluorescence emission from Trp (3) The treatment of GSSG or peroxide gave rise to the decrease on fluorescence intensity for both Tyr and Trp. (4) The fusion protein could not be fully unfolded in 6 M GdnHCl withλmax < 350 nm. (5) The unfolding equilibrium curve showed that the expressed and purified fusion protein experienced a sudden increase in fluorescence intensity at 346.2 nm in the concentraion of GdnHCl varying from 2 M to 3 M with the Gibbs free energy of ~ 3.7 kJ.mol-1. In contrast, a contrary tendency was detected for the denatued and reduced protein, where a remarkbale increase was followed by a sudden decrease on fluorescence intensity from 1.3 M to 2.6 M GdnHCl.
In addition, the aforementioned results would be helpful for the further studies on the physiological functions,unfolding, refolding process and strucure for this protein.
方法:利用pET-32(a)作为原核表达载体,对手掌参中赤霉酸诱导的富含半胱氨酸蛋白GcGASA进行原核表达。并通过亲和层析、SDS-PAGE、Native-PAGE等对原核表达融合蛋白Trx-GcGASA进行纯化和鉴定。利用琼脂糖扩散法抑菌试验和清除过氧化氢实验验证Trx-GcGASA的体外活性,并利用稳态荧光光谱检测对经过DTT还原,Native形态,氧化剂处理,以及盐酸胍变性的融合蛋白Trx-GcGASA进行荧光光谱学研究。
结果:Native-PAGE检测结果显示经原核表达和纯化得到的融合蛋白以单体和三聚体两种形式存在。融合蛋白的抗微生物活性和抗氧化活性试验结果显示,融合蛋白在10uM浓度时能够使大肠杆菌出现明显聚集,然而没有明显的清除H2O2能力。稳态荧光光谱学研究显示:(1)当激发波长在250nm-280nm之间时,原核表达纯化得到的融合蛋白Trx-GcGASA的荧光发射主要源于酪氨酸残基。(2)DTT还原处理之后,融合蛋白色氨酸的荧光强度显著增加。(3)融合蛋白经过氧化型的谷胱甘肽GSSG或者过氧化氢氧化处理之后,其中酪氨酸和色氨酸的荧光强度都有所降低,而这与文献报道的此类蛋白的抗氧化活性一致。(4)无论经过1 mM DTT还原处理与否,融合蛋白在6M盐酸胍存在的条件下都不能彻底变性,色氨酸的最大荧光发射峰位置小于350nm。(5)变性曲线显示,原核表达纯化得到的融合蛋白Trx-GcGASA在346.2nm处的荧光强度在盐酸胍浓度为2M-3M之间时发生突变,应用蛋白变性两态模型计算得到融合蛋白的Gibbs自由能为3.7 kJ.mol-1。然而,经过1 mM DTT还原的融合蛋白却表现出相反的趋势,盐酸胍浓度从0M增加到1.3M时,荧光强度显著增加了1.8倍,然后在1.3M到2.6M之间降低了26%。
本文的相关研究为进一步深入探讨GcGASA蛋白的结构、生理活性以及后续有关富含半胱氨酸蛋白质的解折叠和复性研究等工作奠定了基础。
Gibberellin-induced cysteine-rich proteins(GIP),which have N-terminal signal peptides with varying length and share 12 conserved cysteine residues at C-terminal domain, play many siginificant roles in plants. However,the function of GIP in vitro and structural information were unknown up to now.
Methods: The heterogeneous gene of Gymnadnia conopsea(designated gcgasa) has been expressed in Escherichia coli BL21(DE3) using pET-32 (a) as the expression plasmid. Following the purification and identification of fusion protein Trx-GcGASA , we investigated its existing state by Native-PAGE, antimicrobial activity and the ability to eliminate peroxide in vitro. Additionally, the intrinsic fluorescence of Trx-GcGASA has also been studied in the absence and presence of oxidant, reductant and GndHCl by using steady-state fluorescence spectroscopic methods.
Results: Native-PAGE results revealed that fusion protein simultaneously exist in the form of monomer and trimer. The studies on physiological functions suggested that Trx-GcGASA might cause the aggregation of Escherichia coli with the concentration of 10 uM, however, it was not able to eliminate H2O2 directly. The intrinsic fluorescence results revealed that (1) Tyrosine made the dominating contribution to the overall fluorescence emission spectra following excitation at different wavelengths varying from 250 nm to 280 nm. (2) The introduction of DTT greatly enhanced the fluorescence emission from Trp (3) The treatment of GSSG or peroxide gave rise to the decrease on fluorescence intensity for both Tyr and Trp. (4) The fusion protein could not be fully unfolded in 6 M GdnHCl withλmax < 350 nm. (5) The unfolding equilibrium curve showed that the expressed and purified fusion protein experienced a sudden increase in fluorescence intensity at 346.2 nm in the concentraion of GdnHCl varying from 2 M to 3 M with the Gibbs free energy of ~ 3.7 kJ.mol-1. In contrast, a contrary tendency was detected for the denatued and reduced protein, where a remarkbale increase was followed by a sudden decrease on fluorescence intensity from 1.3 M to 2.6 M GdnHCl.
In addition, the aforementioned results would be helpful for the further studies on the physiological functions,unfolding, refolding process and strucure for this protein.
引文
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[3] Segura A, Moreno M, Madueno F, et al.Snakin-1, a peptide from potato that is active against plant pathogens.Mol Plant Microbe Interact, 1999, 12(1):16-23
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[8] de la Fuente J I, Amaya I, Castillejo C, et al.The strawberry gene FaGAST affects plant growth through inhibition of cell elongation.J Exp Bot, 2006, 57(10):2401-2411
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[11] Furukawa T, Sakaguchi N, Shimada H.Two OsGASR genes, rich GAST homologue genes that are abundant in proliferating tissues, show different expression patterns in developing panicles.Genes Genet Syst, 2006, 81:171-180
[12] Taylor B H, Scheuring C F.A molecular marker for lateral root initiation:the rsi-1 gene of tomato (Lycopersicon esculentum Mill) is activated in early lateral root primordia.Mol Gen Genet, 1994, 243:148-157
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[18] Wang L, Wang Z, Xu Y Y, et al.OsGSR1 is involved in crosstalk between gibberellins and brassinosteroids in rice.Plant J, 2009, 57:498-510
[19] Bindschedler L V, Whitelegge J P, Millar D J, et al. A two component chitin-binding protein from French bean association of a proline-rich protein with a cysteine-rich polypeptide. FEBS Lett, 2006, 580:1541-1546
[20] Kovalskaya N, Hammond BW.Expression and funcional characterization of the plant antimicrobial snakin-1 and defensin recombinant proteins.Protein Expr Purif, 2009, 63:12-17
[21] Liu Y, Meng G Q, Zhou J P, et al.Expression, Purification and identification of Gibberellin-induced Cysteine-rich Protein of Gymanadenia conopsea.Chinese Journal of Agricultural Blotechnology, 6(3):271–276
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[24] Qing-Xin Hua, Wenhua Jia, Bruce H Frank, et al.A Protein Caught in a Kinetic Trap: Structures and Stabilities of Insulin Disulfide Isomers.Biochemistry, 2002, 41(3) : 14700-14715
[25] Tse Siang Kang, Zoran Radic, Todd T Talley, et al.Protein Folding Determinants:Structural Features Determining Alternative Disulfide Pairing inαandχ/λ-Conotoxins.Biochemistry, 2007, 46:3338-3355
[26] Qi-Long Huang, Jie Zhao, Yue-Hua Tang, et al.Sequence Determinant ausing Different Folding Behaviors of Insulin and Insulin-like Growth Factor-1. Biochemistry, 2007, 46:218-224
[27]曹佐武.小分子肽的Tricine-SDS-PAGE分离方法.生物学通报,2003,38(3):55-56
[28] A. Hasnain, S Hasan Arif, R Ahmad, et al.Identification of Species-Marker Bands inNative and SDS-PAGE Patterns of Soluble Muscle Proteins of Four Species of Genus Chann (Channidae:Channiformes) with Evidence of Some Intraspecies Heterogeneity Asian Fisheries Science , 2005, 18:49-58
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[31] Bao-Cun Li, Shuang-Quan Zhang, Wen-Bing Dan, et al.Expression in Escherichia coli and purification of bioactive antibacterial peptide ABP-CM4 from the Chinese silkworm. Bombyx mori Biotechnol Lett, 2007, 29:1031-1036
[32] Ying-Fang Yang, Kuo-Chang Cheng, Ping-Hsing Tsai, et al.Alanine substitutions of noncysteineresidues in the cysteine-stabilizedαβmotif.Protein Science,2009,18:1498-1506
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[2] Wigoda N, Ben-Nissan G, Granot D, et al.The gibberellin-induced, cysteine-rich protein GIP2 from Petunia hybrida exhibits in planta antioxidant activity.Plant J, 2006, 48(5):796-805
[3] Segura A, Moreno M, Madueno F, et al.Snakin-1, a peptide from potato that is active against plant pathogens.Mol Plant Microbe Interact, 1999, 12(1):16-23
[4] Shi L, Olszewski N E.Gibberellin and abscisic acid regulate gast1 expression at the level of transcription.Plant Mol Biol, 1998, 38:1053-1060
[5] Shi L, Gast R T, Golparaj M, Olszewski N E.Characterization of a shoot-specific, GA3- and ABA-regulated gene from tomato.Plant J, 1992, 2:623-632
[6] Ben-Nissan G, Weiss D.The petunia homologue of the tomato gast1:transcript accumulation coincides with gibberellin-induced cell elongation.Plant Mol Biol, 1996, 32:1067-1074
[7] Kotilaninen M, Helariutta Y, Metho M, et al.GEG participates in the regulation of cell and organ shape during corolla and carpel development in Gerbera hybrida.Plant Cell, 1999, 11: 1093-1104
[8] de la Fuente J I, Amaya I, Castillejo C, et al.The strawberry gene FaGAST affects plant growth through inhibition of cell elongation.J Exp Bot, 2006, 57(10):2401-2411
[9] Aubert D, Chevillard M C, Dorne A M, et al.Expression patterns of gasa genes in Arabidopsis thaliana: the gasa4 gene is up-regulated by gibberellin in meristematic regions.Plant Mol Biol, 1998, 36:871-883
[10] Ben-Nissan G, Lee J Y, Borohov A, et al.GIP, a Petunia hybrida GA-induced cysteine-rich protein:a possible role in root elongation and transition to folowering.Plant J, 2004, 37:229-238
[11] Furukawa T, Sakaguchi N, Shimada H.Two OsGASR genes, rich GAST homologue genes that are abundant in proliferating tissues, show different expression patterns in developing panicles.Genes Genet Syst, 2006, 81:171-180
[12] Taylor B H, Scheuring C F.A molecular marker for lateral root initiation:the rsi-1 gene of tomato (Lycopersicon esculentum Mill) is activated in early lateral root primordia.Mol Gen Genet, 1994, 243:148-157
[13] Zimmermann R, Sakai H, Hochholdinger F,et al.The Gibberellic acid stimulated-like gene family in maize and its role in lateral root development.Plant Physiol, 2010, 152:356-365
[14] Segura A, Moreno M, Madueno F, et al.Snakin-1, a peptide from potato that is active against plant pathogens.Mol Plant Microbe Interact, 1999, 12:16-23
[15] Berrocal-Lobo M, Segura A, Moreno M, et al.Snakin-2, an antimicrobial peptide from potato whose gene is locally induced by wounding and responds to pathogen infection.Plant Physiol, 2002, 128:951-961
[16] Wigoda N, Ben-Nissan G, Granot D, et al.The gibberellin-induced, cysteine-rich protein GIP2 from Petunia hybrida exhibits in planta antioxidant activity.Plant J, 2006, 48:796-805
[17] Peng J Z, Lai L J, Wang X J.PRGL:A cell wall proline-rich protein containing GASA domain in Gerbera hybrida.Sci China Ser C-Life Sci, 2008, 51(6):520-525
[18] Wang L, Wang Z, Xu Y Y, et al.OsGSR1 is involved in crosstalk between gibberellins and brassinosteroids in rice.Plant J, 2009, 57:498-510
[19] Bindschedler L V, Whitelegge J P, Millar D J, et al. A two component chitin-binding protein from French bean association of a proline-rich protein with a cysteine-rich polypeptide. FEBS Lett, 2006, 580:1541-1546
[20] Kovalskaya N, Hammond BW.Expression and funcional characterization of the plant antimicrobial snakin-1 and defensin recombinant proteins.Protein Expr Purif, 2009, 63:12-17
[21] Liu Y, Meng G Q, Zhou J P, et al.Expression, Purification and identification of Gibberellin-induced Cysteine-rich Protein of Gymanadenia conopsea.Chinese Journal of Agricultural Blotechnology, 6(3):271–276
[22]郭余珍,冯恩民.蛋白质结构研究现状与展望.生物信息学,2007,4(5):183-184
[23]许以民.拉曼光谱及其在结构生物学中的应用.化学工业出版社,2005,3月第1版
[24] Qing-Xin Hua, Wenhua Jia, Bruce H Frank, et al.A Protein Caught in a Kinetic Trap: Structures and Stabilities of Insulin Disulfide Isomers.Biochemistry, 2002, 41(3) : 14700-14715
[25] Tse Siang Kang, Zoran Radic, Todd T Talley, et al.Protein Folding Determinants:Structural Features Determining Alternative Disulfide Pairing inαandχ/λ-Conotoxins.Biochemistry, 2007, 46:3338-3355
[26] Qi-Long Huang, Jie Zhao, Yue-Hua Tang, et al.Sequence Determinant ausing Different Folding Behaviors of Insulin and Insulin-like Growth Factor-1. Biochemistry, 2007, 46:218-224
[27]曹佐武.小分子肽的Tricine-SDS-PAGE分离方法.生物学通报,2003,38(3):55-56
[28] A. Hasnain, S Hasan Arif, R Ahmad, et al.Identification of Species-Marker Bands inNative and SDS-PAGE Patterns of Soluble Muscle Proteins of Four Species of Genus Chann (Channidae:Channiformes) with Evidence of Some Intraspecies Heterogeneity Asian Fisheries Science , 2005, 18:49-58
[29] Bradford MM.Anal Biochem, 1976, 72:248
[30]唐威华,张景六,王宗阳,等.SDS-PAGE法测定His-tag融合蛋白分子量产生偏差的原因.植物生理学报,2000,26(1):64-68
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