重组E.coli γ-ggt和gs原核表达载体的构建、蛋白诱导表达与应用和Hsp70、Hsp90及TAK1相关真核表达载体的构建及鉴定
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摘要
谷胱甘肽(Glutathione)广泛存在于各种生物体中,主要用于维持胞内正常的氧化还原状态,是生物体内的自由基清除剂,具有抗氧化、解毒、保护细胞等重要的生理作用,在临床医药、食品工业、保健品等方面有着非常广泛的用途。近年来,Glutathione的生产方法主要有溶剂萃取法、发酵法、酶转化法及化学合成法。由于直接从组织中分离提取的成本比较高,化学合成法的分离提纯过程又十分困难,酶转化法合成GSH的产率高、后续的分离提取较简单而倍受关注。
茶氨酸(L-Theanine)学名为N-乙基-γ-氨基-L-谷氨酰胺(N-ethyl-γ-L-glutamine),是天然茶叶中的一种特殊氨基酸,也是茶叶具有鲜爽味的主要成分,于1985年得到FDA认可,并确认合成茶氨酸为GRAS,在使用时不作限制用量的规定,茶氨酸作为一种新型的食品添加剂,不仅易溶于水,还有抑制苦味物质,改善食品风味的作用,在食品工业中得到了广泛的应用。此外,茶氨酸还具有降血压、增强抗癌药物的疗效、提高免疫力、松弛神经紧张等药理作用。但由于直接从茶叶中提取高纯度茶氨酸的成本比较高昂,茶氨酸的人工合成,特别是微生物发酵合成方法一直是人们研究的热点。
γ-谷氨酰转肽酶(γ-glutamyltranspeptidase,γ-GGT,EC 2.3.2.2)是生物体内谷胱甘肽代谢途径中的一个关键酶,广泛分布在活体组织中。在大肠杆菌中,此酶在自身信号肽的介导下被转运至周质空间中发挥其生理作用。它不仅可以催化γ-谷氨酰基化合物的水解反应生成γ-谷氨酸,还可以催化γ-谷氨酰基化合物上的γ-谷氨酰基团转移至氨基酸、多肽、H_2O等受体上。利用其这一特性,可以催化合成谷胱甘肽和茶氨酸。为了提高微生物中γ-谷氨酰转肽酶的产量及可溶组分,本实验从大肠杆菌k-12中克隆出γ-ggt基因片段,转入原核表达载体pET28a(+)、pET32a(+)和pGEX-4T-1中,构建了重组质粒,并将其转化至大肠杆菌BL21(DE3)中,以廉价、无毒的乳糖作为诱导剂,测定目的蛋白的表达特性,并在此基础上利用该酶催化合成谷胱甘肽和茶氨酸。
实验结果表明,构建的重组质粒pGEX-4T-1/γ-ggt转化大肠杆菌BL21(DE3),乳糖诱导后表达产生的γ-谷氨酰转肽酶具有更高的蛋白可溶性和酶活,在反应条件优化基础上,利用重组大肠杆菌γ-谷氨酰转肽酶,茶氨酸最大生成量目前可达到80～90g/L,谷胱甘肽的合成仍在进一步研究中。
已有研究报道,诱生型HSP70与细胞凋亡之间存在密切的联系,凋亡信号调节激酶ASK1(Apoptosis signal regulating kinase)是细胞凋亡途径的一个关键激酶,活化的ASK1通过线粒体途径引起细胞凋亡,诱生型HSP70可以直接与之结合,有效抑制胞内ASK1的激活及由此引起的细胞凋亡。诱生型HSP70在mRNA或蛋白水平上是否也具有调控ASK1作用,因此,本实验从人宫颈癌Hela细胞中扩增并克隆诱生型HSP70基因至真核载体pcDNA3.1(-)中,体外鉴定其表达,为后续探究其在细胞凋亡通路中一些具体的调控作用及机制奠定基础。
热休克蛋白在细胞应激中发挥十分重要的作用,参与多种疾病的发生和发展,热休克蛋白90(HSP90)是近年来研究较多的一类热休克蛋白。目前,国际上对HSP90蛋白参与的细胞功能的研究主要集中在保护细胞,抑制细胞凋亡方面,而对它们是否参与IL-1相关的炎症信号通路的调节的研究尚少有报道。在哺乳动物中,TAK1在天然免疫和获得性免疫等细胞信号通路中具有重要作用。我们研究结果表明HSP90能与TAK1结合并影响了IL-1引起的TAK1泛素化以及稳定性,进而影响IL-1引起的下游信号的激活。细胞内的免疫共沉淀表明,HSP90能与TAK1结合,因此本实验将在前期工作的基础上找到作用于TAK1的HSP90分子的功能区,揭示HSP90作用于TAK1的分子机理。本实验成功构建了HSP90三段缺失突变体,分别为HSP90(1-232aa)、(233-732aa)、(402-732aa)以及TAK1两段缺失突变体,分别为TAKl(1-299aa)、(301-579aa),通过体内免疫共沉淀实验显示,HSP90蛋白中的1-401aa是形成HSP90-TAK1复合物所必需的。本实验证实了HSP90与TAK1相互作用的功能区,为研究炎症反应的信号网络和分子机理提供了实验基础。
Glutathione is a necessary material which maintains the environment of the body. It is the coenzyme of some enzyme in the metabolism.Its reduced thiocytidine makes the body bring about immunity by participating in the important oxidoreduction reaction of the body.Glutathione has wide use in the medicine and it has transparent effect especially in the treatment of toxic disease and liver disease.So it is thought highly of by many researchers in the medical field in the world.But the preparation of glutathione is a very difficult problem in the research and it is a hot spot up to now. From the process of the production of glutathione,there are mainly extraction, chemical synthesis,microorganism zymotechnics and chemical enzymatic synthesis.
Theanine,which was first separated from green tea in 1950,is not only the main free amino acid component of tea,but also the major umami component of tea.It possesses high physiological and medical benefits.The preparation of theanine is a research hot point.It is very difficult and more expensive to extract a high purity theanine directly from tea leaves.The technics of chemosynthesis theanine is complicated and the yield of theanine by tissue culture is low.So,the biosynthesis of theanine is getting more and more attention.
γ-glutamyltranspeptidase(GGT,EC 2.3.2.2)catalyzes not only the hydrolysis of theγ-glutamyl linkages ofγ-glutamyl compounds,but also the transfer of theirγ-glutamyl moieties to other amino acids or peptides.In this paper,we report an effective enzymatic method for synthesizing theanine and glutathione involving Escherichia coliγ-GGT.The E.coliγ-GGT encoding fragment was correctly inserted into the prokaryotic expression vector pET28a(+)、pET32a(+)and pGEX-4T-1,while with lactose as inducer,E.coli BL21(DE3)harboring pGEX-4T- 1/γ-ggt can express the best solubility and specific activity ofγ-glutamyl transpeptidase protein.Over 80～90 g/L theanine was successfully achieved and glutathione synthesis needs to further study.
It has been reported that Hsp72 inhibits both the stress-induced activation of ASK1 and ASKl-involved apoptosis.To better understand the mechanism by which Hsp72 modulates stress-activated sig-naling,we construct the eukaryotic expression vector for human induced heat shock protein 70(HSPT0)gene and investigate the possible effects of Hsp72 on the protein or mRNA level of ASK1.
The 90kDa Heat Shock Protein(HSPg0)is one of the most abundant cytosolic proteins in eukaryotes.It normally functions as a molecular chaperone to participate in folding of newly synthesized proteins,refolding of denatured proteins after stress and regulating of client proteins' stability and activity.Up to now,over 100 HSP90 client proteins have been found,which mainly include transcription factors,protein kinases,polymerases and so on.Transforming growth factor(TGF)-β-activated kinasel(TAK1),which was originally identified as a member of MAP kinase kinase kinase(MAPKKK)family mediating TGF-βsignaling,has been shown to be involved in the IL-1 -induced signal pathway.
We have now demonstrated that HSP90 physically associates with TAK1, thereby affects the proteasome/ubiquitin and protein stability of this kinase induced by IL-1.Furthermore,we construct HSP90 and TAK1 truncation mutants,including HSP90(1-232aa),HSP90(233-732aa),HSP90(402-732aa),TAK1(1-299aa),and TAK1(301-579aa),and also identified that the N-terminal domain(amino acids 1-401)of HsPg0 is required for interaction of HSP90 and TAK1.
茶氨酸(L-Theanine)学名为N-乙基-γ-氨基-L-谷氨酰胺(N-ethyl-γ-L-glutamine),是天然茶叶中的一种特殊氨基酸,也是茶叶具有鲜爽味的主要成分,于1985年得到FDA认可,并确认合成茶氨酸为GRAS,在使用时不作限制用量的规定,茶氨酸作为一种新型的食品添加剂,不仅易溶于水,还有抑制苦味物质,改善食品风味的作用,在食品工业中得到了广泛的应用。此外,茶氨酸还具有降血压、增强抗癌药物的疗效、提高免疫力、松弛神经紧张等药理作用。但由于直接从茶叶中提取高纯度茶氨酸的成本比较高昂,茶氨酸的人工合成,特别是微生物发酵合成方法一直是人们研究的热点。
γ-谷氨酰转肽酶(γ-glutamyltranspeptidase,γ-GGT,EC 2.3.2.2)是生物体内谷胱甘肽代谢途径中的一个关键酶,广泛分布在活体组织中。在大肠杆菌中,此酶在自身信号肽的介导下被转运至周质空间中发挥其生理作用。它不仅可以催化γ-谷氨酰基化合物的水解反应生成γ-谷氨酸,还可以催化γ-谷氨酰基化合物上的γ-谷氨酰基团转移至氨基酸、多肽、H_2O等受体上。利用其这一特性,可以催化合成谷胱甘肽和茶氨酸。为了提高微生物中γ-谷氨酰转肽酶的产量及可溶组分,本实验从大肠杆菌k-12中克隆出γ-ggt基因片段,转入原核表达载体pET28a(+)、pET32a(+)和pGEX-4T-1中,构建了重组质粒,并将其转化至大肠杆菌BL21(DE3)中,以廉价、无毒的乳糖作为诱导剂,测定目的蛋白的表达特性,并在此基础上利用该酶催化合成谷胱甘肽和茶氨酸。
实验结果表明,构建的重组质粒pGEX-4T-1/γ-ggt转化大肠杆菌BL21(DE3),乳糖诱导后表达产生的γ-谷氨酰转肽酶具有更高的蛋白可溶性和酶活,在反应条件优化基础上,利用重组大肠杆菌γ-谷氨酰转肽酶,茶氨酸最大生成量目前可达到80～90g/L,谷胱甘肽的合成仍在进一步研究中。
已有研究报道,诱生型HSP70与细胞凋亡之间存在密切的联系,凋亡信号调节激酶ASK1(Apoptosis signal regulating kinase)是细胞凋亡途径的一个关键激酶,活化的ASK1通过线粒体途径引起细胞凋亡,诱生型HSP70可以直接与之结合,有效抑制胞内ASK1的激活及由此引起的细胞凋亡。诱生型HSP70在mRNA或蛋白水平上是否也具有调控ASK1作用,因此,本实验从人宫颈癌Hela细胞中扩增并克隆诱生型HSP70基因至真核载体pcDNA3.1(-)中,体外鉴定其表达,为后续探究其在细胞凋亡通路中一些具体的调控作用及机制奠定基础。
热休克蛋白在细胞应激中发挥十分重要的作用,参与多种疾病的发生和发展,热休克蛋白90(HSP90)是近年来研究较多的一类热休克蛋白。目前,国际上对HSP90蛋白参与的细胞功能的研究主要集中在保护细胞,抑制细胞凋亡方面,而对它们是否参与IL-1相关的炎症信号通路的调节的研究尚少有报道。在哺乳动物中,TAK1在天然免疫和获得性免疫等细胞信号通路中具有重要作用。我们研究结果表明HSP90能与TAK1结合并影响了IL-1引起的TAK1泛素化以及稳定性,进而影响IL-1引起的下游信号的激活。细胞内的免疫共沉淀表明,HSP90能与TAK1结合,因此本实验将在前期工作的基础上找到作用于TAK1的HSP90分子的功能区,揭示HSP90作用于TAK1的分子机理。本实验成功构建了HSP90三段缺失突变体,分别为HSP90(1-232aa)、(233-732aa)、(402-732aa)以及TAK1两段缺失突变体,分别为TAKl(1-299aa)、(301-579aa),通过体内免疫共沉淀实验显示,HSP90蛋白中的1-401aa是形成HSP90-TAK1复合物所必需的。本实验证实了HSP90与TAK1相互作用的功能区,为研究炎症反应的信号网络和分子机理提供了实验基础。
Glutathione is a necessary material which maintains the environment of the body. It is the coenzyme of some enzyme in the metabolism.Its reduced thiocytidine makes the body bring about immunity by participating in the important oxidoreduction reaction of the body.Glutathione has wide use in the medicine and it has transparent effect especially in the treatment of toxic disease and liver disease.So it is thought highly of by many researchers in the medical field in the world.But the preparation of glutathione is a very difficult problem in the research and it is a hot spot up to now. From the process of the production of glutathione,there are mainly extraction, chemical synthesis,microorganism zymotechnics and chemical enzymatic synthesis.
Theanine,which was first separated from green tea in 1950,is not only the main free amino acid component of tea,but also the major umami component of tea.It possesses high physiological and medical benefits.The preparation of theanine is a research hot point.It is very difficult and more expensive to extract a high purity theanine directly from tea leaves.The technics of chemosynthesis theanine is complicated and the yield of theanine by tissue culture is low.So,the biosynthesis of theanine is getting more and more attention.
γ-glutamyltranspeptidase(GGT,EC 2.3.2.2)catalyzes not only the hydrolysis of theγ-glutamyl linkages ofγ-glutamyl compounds,but also the transfer of theirγ-glutamyl moieties to other amino acids or peptides.In this paper,we report an effective enzymatic method for synthesizing theanine and glutathione involving Escherichia coliγ-GGT.The E.coliγ-GGT encoding fragment was correctly inserted into the prokaryotic expression vector pET28a(+)、pET32a(+)and pGEX-4T-1,while with lactose as inducer,E.coli BL21(DE3)harboring pGEX-4T- 1/γ-ggt can express the best solubility and specific activity ofγ-glutamyl transpeptidase protein.Over 80～90 g/L theanine was successfully achieved and glutathione synthesis needs to further study.
It has been reported that Hsp72 inhibits both the stress-induced activation of ASK1 and ASKl-involved apoptosis.To better understand the mechanism by which Hsp72 modulates stress-activated sig-naling,we construct the eukaryotic expression vector for human induced heat shock protein 70(HSPT0)gene and investigate the possible effects of Hsp72 on the protein or mRNA level of ASK1.
The 90kDa Heat Shock Protein(HSPg0)is one of the most abundant cytosolic proteins in eukaryotes.It normally functions as a molecular chaperone to participate in folding of newly synthesized proteins,refolding of denatured proteins after stress and regulating of client proteins' stability and activity.Up to now,over 100 HSP90 client proteins have been found,which mainly include transcription factors,protein kinases,polymerases and so on.Transforming growth factor(TGF)-β-activated kinasel(TAK1),which was originally identified as a member of MAP kinase kinase kinase(MAPKKK)family mediating TGF-βsignaling,has been shown to be involved in the IL-1 -induced signal pathway.
We have now demonstrated that HSP90 physically associates with TAK1, thereby affects the proteasome/ubiquitin and protein stability of this kinase induced by IL-1.Furthermore,we construct HSP90 and TAK1 truncation mutants,including HSP90(1-232aa),HSP90(233-732aa),HSP90(402-732aa),TAK1(1-299aa),and TAK1(301-579aa),and also identified that the N-terminal domain(amino acids 1-401)of HsPg0 is required for interaction of HSP90 and TAK1.
引文
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