化学伴侣对单链抗体在E. coli中可溶性表达的影响以及含硒五肽模拟谷胱甘肽过氧化物酶
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摘要
生物体内存在着一系列的酶催化系统,调节各种生理生化反应,从而保证细胞及组织正常生理功能的发挥。氧化应激是体内一种氧化和抗氧化的不平衡状态,即细胞代谢过程中自由基的代谢紊乱。现代医学认为,过多的活性氧在体内堆积与克山病、再灌注损伤、脑局部缺血、肿瘤、各种炎症和衰老等发病机制密切相关[1,2]。谷胱甘肽过氧化物酶(GPX)是生物机体内重要的抗氧化酶之一,它利用硒代半胱氨酸(Sec)作为催化官能团,通过谷胱甘肽催化还原氢过氧化物,从而保护细胞免遭氧化损伤。由于天然GPX存在分子量大,稳定性差,难于制备(Sec由终止密码子UGA编码,用普通DNA重组技术很难表达含硒蛋白质),来源有限等问题,因此对GPX的人工模拟引起人们的关注。迄今已有很多种GPX模拟物问世,其中以Sies等制备的ebselen最为著名[3]。模拟GPX不仅有助于阐明其催化机理,而且具有重要的实际应用价值。我们应用化学和生物学技术模拟了GPX的酶学行为。
含硒单链抗体(Selenium-containing single-chain Fv fragment ofmonoclonal antibody 2F3,Se-scFv2F3)是我室利用单克隆抗体技术和基因工程技术,结合化学突变法成功制备的一种有效的GPX模拟物。先用E. coli表达单链抗体2F3(scFv2F3),再用化学法引入催化基团Sec,最后获得Se-scFv2F3。为了提高scFv2F3在工程菌中的可溶性表达量,本文研究了各种小分子化学伴侣,包括甘油,葡萄糖和环糊精对其表达水平的影响。研究结果表明,甘油和β-环糊精极大地促进了scFv2F3的可溶性表达,而且β-环糊精的作用要比甘油更明显。关于β-环糊精作为化学伴侣,在细菌培养过程中对重组蛋白可溶性表达的影响的报道还属首次。
同时,我们从酶催化的本质(底物结合和分子内催化)出发,根据GPX活性部位的结构(催化官能团Sec,结合底物GSH的主要氨基酸残基Arg-167, Arg-40,Gln-130),设计构建了一个新的GPX酶模型,含硒五肽(Se-5P),其催化效率分别是Ebselen和Sec的11倍和220倍。经比较得知,该模拟物高GPX活力的原因在于它能识别底物,这也证明只有在催化基团和底物的结合位点相互协同作用的情况下,才能使模拟酶获得较高的催化效率。为了研究该模拟物的抗氧化能力,我们建立了Vc/Fe2+自由基诱生心肌线粒体损伤模型和和H2O2诱导肝细胞损伤体系。通过对多项指标(如线粒体膨胀度、MDA含量、细胞存活率、LDH含量等)的测定,发现该Se-5P模拟物能够清除自由基,有效保护线粒体和肝细胞免遭氧化损伤。
Selenium, as an essential trace element in organism, mainly functionsas the constituent of selenoenzymes. Glutathione peroxidase (GPX) isa well-known selenoenzyme that works as an antioxidant and catalyzesthe reduction of harmful peroxide by glutathione and protects cellsagainst oxidative damage. Because many diseases are related tooxidative stress, GPX is an ancient foe of many diseases. Antioxidantsare very useful for biological bodies, and considerable effort has beenspent to find compounds that could imitate the properties of GPX.Although many GPX mimics have been made, they possess seriousdisadvantages: low activity, low solubility in water, and, in somecases, toxicity. In order to overcome these drawbacks, we have proposeda new strategy of imitating GPX. First, a receptor with a substratebinding site is generated. Next, a catalytic group is incorporated intothe receptor near the substrate binding site, allowing the catalyticgroup access to the functional group of the substrate, and finally ahighly efficient enzyme mimic is obtained.
1. Chemical chaperones increase the expression level ofsoluble single-chain Fv antibody (scFv2F3)
The selenium-containing single chain Fv fragment of monoclonalantibody 2F3 (Se-scFv2F3) is a high efficient GPX mimic prepared withthe technology of preparation monoclonal antibody and recombinant DNA.It has more important prospects for application in clinic than thoseearly developed because of its high activity and low antigenicity.Se-scFv2F3 is generated by chemical modification of the single chainFv fragment of monoclonal antibody 2F3 (scFv2F3) and the scFv2F3 waslargely expressed in inclusion form in E. coli. Because scFv2F3 ininclusion form cannot be used directly to prepare Se-scFv2F3 beforeits renaturation and the efficiency of renaturation is low, we havebegun to express scFv2F3 as a soluble form in the periplasm of hostusing a new expression vector. Although scFv2F3 was found in the solublefraction, allowing purification under native nondenaturing conditions,a significant quantity of scFv2F3 was stored in inclusion body.To overcome the formation of aggregates, and the subsequent packaginginto inclusion bodies during in vitro folding, chemical chaperoneshave been used. Chemical chaperones are low molecular weight compounds,which can stabilize the structure of proteins in vitro. These reagentsinclude the organic solvent dimethyl sulfoxide and cellular osmolytesglycerol, glucose and trimethylamine N-oxide. A lot of reports havebeen shown that glycerol, as a chemical chaperones, can stabilize theprotein conformation, correct its defective folding and facilitate itsswitch from inclusion bodies to the soluble form. Cyclodextrins (CDs)are cyclic oligosaccharides with a hydrophilic outer surface and asomewhat lipophilic central cavity;it has been used to prevent theirreversible aggregation of partially refolded proteins.As mentioned above, glycerol and glucose added to the culture mediumfacilitate the expression of some soluble recombination proteins. Inthis work, we investigated whether they have the similar effects onthe expression of soluble scFv2F3. Besides, the examples concerningCDs mentioned above indicated the effects of CDs on protein refoldingin vitro.
We studied the effects of chemical chaperones, such as glycerol,glucose and β-cyclodextrin added to the culture medium, on expressionof soluble form of scFv2F3. The expression level was evaluated bydetermining the content of scFv2F3 in whole cell soluble proteins. Ourresults suggest that glycerol and β-cyclodextrin greatly increase theexpression, and β-cyclodextrin is more effective compared withglycerol. Glucose has a slight effect on the expression. To ourknowledge, this is the first example that β-cyclodextrin was used asa chemical chaperone during cell culture to improve the expressionlevel of recombinant protein. In addition, glucose and β-cyclodextrincan decrease the toxic effect of IPTG to cells.2. Construction of selenium-containing 5-mer peptidesWe design and obtain a kind of short peptide identifying GSH withsolid-phase synthesis technology based on the active center of nativeGPX. This mimic is characterized with HPLC-MS. Its activity is 11U/μmol according to Wilson's methods and it has the highest activityrelative to its molecular weight compared with other small molecularmimics. The optimal pH and temperature of Se-5P catalyzing thereduction of H2O2 by GSH are found to be 8.94 and 43.2 ℃ respectively.In addition, Se-5P shows similar kinetic behavior as natural GPXs, thatis they both abide the Ping-Pong mechanism. And the values of kcat/KmH2O2and kcat/KmGSH of Se-5P are higher compared with natural enzyme, indicatingthat Se-5P has higher affinity with both substrates. In summary, wehave successfully synthesized a GPX mimic with small size, highactivity and specific binding site for substrates. These advantagesmade the novel mimic have very extensive perspective for use in medicineand drugs.3. The biological effects of of selenium-containing5-mer peptides
We construct the Vc/Fe2+ ROS-induced Mitochondria and H2O2-inducedculture hepatocyte cells damage model systems, demonstrating thedamaged mitochondria and hepatocyte cells have great changes in thetwo systems. The damages include: the extent of mitochondria swellingis increased, lipid peroxidation is occurred with dose-dependent,penetration of the cell membrane decrease, DNA in the cellsfragmentations and the cell viability decreases. We investigate theprotective effects of mitochondria and cultured hepatocyte cells bySe-5P using the two damage systems, indicating that they can inhibitthe swelling of mitochondria, the increase of the MDA content, and thedecrease of the cell viability. They also can prevent the leakage ofLDH to sustain the well penetration in cell membrane. These resultsindicate that Se-5P can scavenge free radicals to sustain the functionof antioxidative defense system in cells.In addition, we found that Se-5P cannot be inactivated by freeradicals induced by H2O2 while the native GPX was inactivated by freeradicals, indicating that Se-5P is superior to the native GPX inthis aspect. H2O2-induced culture hepatocyte cells damage is similar tooxidative stress occurred in the organism, thus, the studies forthe Se-5P using the two systems provide the theory bases for thisGPX mimic to treat the diseases induced by free radicals, such ascataract, angiocardiopathy and cancers.
含硒单链抗体(Selenium-containing single-chain Fv fragment ofmonoclonal antibody 2F3,Se-scFv2F3)是我室利用单克隆抗体技术和基因工程技术,结合化学突变法成功制备的一种有效的GPX模拟物。先用E. coli表达单链抗体2F3(scFv2F3),再用化学法引入催化基团Sec,最后获得Se-scFv2F3。为了提高scFv2F3在工程菌中的可溶性表达量,本文研究了各种小分子化学伴侣,包括甘油,葡萄糖和环糊精对其表达水平的影响。研究结果表明,甘油和β-环糊精极大地促进了scFv2F3的可溶性表达,而且β-环糊精的作用要比甘油更明显。关于β-环糊精作为化学伴侣,在细菌培养过程中对重组蛋白可溶性表达的影响的报道还属首次。
同时,我们从酶催化的本质(底物结合和分子内催化)出发,根据GPX活性部位的结构(催化官能团Sec,结合底物GSH的主要氨基酸残基Arg-167, Arg-40,Gln-130),设计构建了一个新的GPX酶模型,含硒五肽(Se-5P),其催化效率分别是Ebselen和Sec的11倍和220倍。经比较得知,该模拟物高GPX活力的原因在于它能识别底物,这也证明只有在催化基团和底物的结合位点相互协同作用的情况下,才能使模拟酶获得较高的催化效率。为了研究该模拟物的抗氧化能力,我们建立了Vc/Fe2+自由基诱生心肌线粒体损伤模型和和H2O2诱导肝细胞损伤体系。通过对多项指标(如线粒体膨胀度、MDA含量、细胞存活率、LDH含量等)的测定,发现该Se-5P模拟物能够清除自由基,有效保护线粒体和肝细胞免遭氧化损伤。
Selenium, as an essential trace element in organism, mainly functionsas the constituent of selenoenzymes. Glutathione peroxidase (GPX) isa well-known selenoenzyme that works as an antioxidant and catalyzesthe reduction of harmful peroxide by glutathione and protects cellsagainst oxidative damage. Because many diseases are related tooxidative stress, GPX is an ancient foe of many diseases. Antioxidantsare very useful for biological bodies, and considerable effort has beenspent to find compounds that could imitate the properties of GPX.Although many GPX mimics have been made, they possess seriousdisadvantages: low activity, low solubility in water, and, in somecases, toxicity. In order to overcome these drawbacks, we have proposeda new strategy of imitating GPX. First, a receptor with a substratebinding site is generated. Next, a catalytic group is incorporated intothe receptor near the substrate binding site, allowing the catalyticgroup access to the functional group of the substrate, and finally ahighly efficient enzyme mimic is obtained.
1. Chemical chaperones increase the expression level ofsoluble single-chain Fv antibody (scFv2F3)
The selenium-containing single chain Fv fragment of monoclonalantibody 2F3 (Se-scFv2F3) is a high efficient GPX mimic prepared withthe technology of preparation monoclonal antibody and recombinant DNA.It has more important prospects for application in clinic than thoseearly developed because of its high activity and low antigenicity.Se-scFv2F3 is generated by chemical modification of the single chainFv fragment of monoclonal antibody 2F3 (scFv2F3) and the scFv2F3 waslargely expressed in inclusion form in E. coli. Because scFv2F3 ininclusion form cannot be used directly to prepare Se-scFv2F3 beforeits renaturation and the efficiency of renaturation is low, we havebegun to express scFv2F3 as a soluble form in the periplasm of hostusing a new expression vector. Although scFv2F3 was found in the solublefraction, allowing purification under native nondenaturing conditions,a significant quantity of scFv2F3 was stored in inclusion body.To overcome the formation of aggregates, and the subsequent packaginginto inclusion bodies during in vitro folding, chemical chaperoneshave been used. Chemical chaperones are low molecular weight compounds,which can stabilize the structure of proteins in vitro. These reagentsinclude the organic solvent dimethyl sulfoxide and cellular osmolytesglycerol, glucose and trimethylamine N-oxide. A lot of reports havebeen shown that glycerol, as a chemical chaperones, can stabilize theprotein conformation, correct its defective folding and facilitate itsswitch from inclusion bodies to the soluble form. Cyclodextrins (CDs)are cyclic oligosaccharides with a hydrophilic outer surface and asomewhat lipophilic central cavity;it has been used to prevent theirreversible aggregation of partially refolded proteins.As mentioned above, glycerol and glucose added to the culture mediumfacilitate the expression of some soluble recombination proteins. Inthis work, we investigated whether they have the similar effects onthe expression of soluble scFv2F3. Besides, the examples concerningCDs mentioned above indicated the effects of CDs on protein refoldingin vitro.
We studied the effects of chemical chaperones, such as glycerol,glucose and β-cyclodextrin added to the culture medium, on expressionof soluble form of scFv2F3. The expression level was evaluated bydetermining the content of scFv2F3 in whole cell soluble proteins. Ourresults suggest that glycerol and β-cyclodextrin greatly increase theexpression, and β-cyclodextrin is more effective compared withglycerol. Glucose has a slight effect on the expression. To ourknowledge, this is the first example that β-cyclodextrin was used asa chemical chaperone during cell culture to improve the expressionlevel of recombinant protein. In addition, glucose and β-cyclodextrincan decrease the toxic effect of IPTG to cells.2. Construction of selenium-containing 5-mer peptidesWe design and obtain a kind of short peptide identifying GSH withsolid-phase synthesis technology based on the active center of nativeGPX. This mimic is characterized with HPLC-MS. Its activity is 11U/μmol according to Wilson's methods and it has the highest activityrelative to its molecular weight compared with other small molecularmimics. The optimal pH and temperature of Se-5P catalyzing thereduction of H2O2 by GSH are found to be 8.94 and 43.2 ℃ respectively.In addition, Se-5P shows similar kinetic behavior as natural GPXs, thatis they both abide the Ping-Pong mechanism. And the values of kcat/KmH2O2and kcat/KmGSH of Se-5P are higher compared with natural enzyme, indicatingthat Se-5P has higher affinity with both substrates. In summary, wehave successfully synthesized a GPX mimic with small size, highactivity and specific binding site for substrates. These advantagesmade the novel mimic have very extensive perspective for use in medicineand drugs.3. The biological effects of of selenium-containing5-mer peptides
We construct the Vc/Fe2+ ROS-induced Mitochondria and H2O2-inducedculture hepatocyte cells damage model systems, demonstrating thedamaged mitochondria and hepatocyte cells have great changes in thetwo systems. The damages include: the extent of mitochondria swellingis increased, lipid peroxidation is occurred with dose-dependent,penetration of the cell membrane decrease, DNA in the cellsfragmentations and the cell viability decreases. We investigate theprotective effects of mitochondria and cultured hepatocyte cells bySe-5P using the two damage systems, indicating that they can inhibitthe swelling of mitochondria, the increase of the MDA content, and thedecrease of the cell viability. They also can prevent the leakage ofLDH to sustain the well penetration in cell membrane. These resultsindicate that Se-5P can scavenge free radicals to sustain the functionof antioxidative defense system in cells.In addition, we found that Se-5P cannot be inactivated by freeradicals induced by H2O2 while the native GPX was inactivated by freeradicals, indicating that Se-5P is superior to the native GPX inthis aspect. H2O2-induced culture hepatocyte cells damage is similar tooxidative stress occurred in the organism, thus, the studies forthe Se-5P using the two systems provide the theory bases for thisGPX mimic to treat the diseases induced by free radicals, such ascataract, angiocardiopathy and cancers.
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66. Romero F. J., Bosch More M. F., Romero M. J., Jareno E. J., Romero B., Marin N., Roma J. Lipid peroxidation product and antioxidants in human disease. Environ Health Perspect, 1998, 106(Suppl. 5): 1229-1234.
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69. Lahiri D. K., Farlow M. R., Nurnberger J. I. Jr., Greig N. H. Effects of cholinesterase inhibitors on the secretion of beta-amyloid precursor protein in cell cultures. Ann N Y Acad Sci, 1997, 826: 416-421.
70. Liu X.C., Mosbach K. Catalysis of benzisoxazole isomerization by molecularly imprinted polymers. Macromol. Rapid Commun. 1998, 19, 671.
71. Santora B.P., Larsen A.O., Gagne M.R. Molecularly tmprinted Ti-lewis acid catalysts: application to the Diels-Alder reaction. Organometallics 1998, 17, 3138.
72. Polborn K., Severin K.J. Molecular imprinting with an organometallic transition state analogue. Chem. Soc. Chem. Commun. 1999, 2481.
73. Hilvert D., Hill K.W., Narel K.D., Auditor M.T.M. Antibody catalysis of the Diels-Alder reaction. J. Am. Chem. Soc. 1989, 111, 9261.
74. Suh J., Hah S.S. Organic artificial proteinase with active site comprising three salicylate residues. J. Am. Chem. Soc. 1998, 120, 10088.