oalib

OALib Journal期刊

ISSN: 2333-9721

费用:99美元

投稿

匹配条件: “ Renaturation” ,找到相关结果约30条。
列表显示的所有文章,均可免费获取
第1页/共30条
每页显示
Mechanism of enhancement of prochymosin renaturation by solubilization of inclusion bodies at alkaline pH
Zhizhou Zhang,Yuying Zhang,Kaiyu Yang
Science China Life Sciences , 1997, DOI: 10.1007/BF02882045
Abstract: The renaturation efficiency of recombinant prochymosin depends on not only the renaturation conditions but also the solubilization (denaturation) conditions. Compared with pH 8, solubilization of prochymosin-containing inclusion bodies at pH 11 (8 mol/L urea) results in onefold increase of renaturation efficiency (~40% vs. ~ 20 %). Alkaline pH facilitates the solubilization of inclusion bodies via the breakage of intermolecular disulfide bonds. Moreover, alkaline pH renders prochymosin molecules to be in a more reduced and more unfolded state which undergoes refolding readily.
Structure-activity correlationship and folding of recombinant Escherichia coli dihydro folate reductase (DHFR) enzyme through biochemical and biophysical approaches  [PDF]
Jai Mittal, Gayathri Ravitchandirane, Tapan K. Chaudhuri, Pratima Chaudhuri
Journal of Biophysical Chemistry (JBPC) , 2010, DOI: 10.4236/jbpc.2010.12013
Abstract: The design of any antagonist or inhibitor for any enzyme requires the knowledge of structure- function relationship of the protein and the optimum conformational states for maximum and minimum activities. Furthermore, designing of the inhibitors or drugs against an enzyme becomes easier if there is information available about various well characterized intermediate conformation of the molecule. In vivo folding pathway of any recombinant protein is an important parameter for understanding its ability to fold by itself inside the cell, which always dictates the downstream processing for the purification. In the present manuscript we have discussed about the in vivo and in vitro folding, and structure-function relationship of Dihydrofolate reductase enzyme. This is an important enzyme involved in the cell growth and hence inhibition or inactivation of the enzyme may reduce the cell growth. It was observed that the equilibrium unfolding transition of DHFR proceeds through the formation of intermediates having higher exposed surface hydrophobicity, unchanged enzymatic activity and minimum changes in the secondary structural elements. Because of enhanced surface hydrophobicity, and unchanged enzymatic activity, these intermediates could be a nice target for designing drugs against DHFR.
Restauración en el Inmunoblotting de proteínas de Neisseria meningitidis da?adas por calor y agentes reductores
Estrada,Eric; Ochoa,Rolando; Ferriol,Xenia; García,Ana; Martínez,Juan Carlos; Sotolongo,Franklin;
Vaccimonitor , 2000,
Abstract: five different detergents for restoration of igg antibody binding were studied in the washing steps and in the buffer solution used for the dilution of the conjugate and samples. binding of human serum igg antibodies to outer membrane protein (omp) was detected with class-specific peroxidase-conjugated human antibodies. the positive control reacted with those proteins whose molecular weight corresponded approximately to p1, p3, p4 and p5. proteins of 80 kda, 70 kda and 24 kda and another protein of >150 kda were also recognized. our studies indicated that tween 20 was the best detergent for restoration of omp antigenicity, except for the 150 kda protein. tween 20 achieved a greater number and intensity of bands and a lower background with respect to empigen bb, triton x-100, nonidet np-40 and chaps. the use of detergents affected the antigenicity of the 150 kda protein. washing with tween 20 were the most important steps for restoration of igg antibody binding sites of heat-denatured omps.
Isolation, Purification and Renaturation of Recombinant-DNA-Derived Porcine Sonatotropin
基因工程猪生长激素的分离提纯及变复性技术的研究

LI Zhen,YU Rui-Song LIU Hui-Li WANG Ying ZHANG Ping ZHOU Zhi-Ai ZHANG De-Fu,
李震
,于瑞嵩,张平,王英,刘惠莉

生物工程学报 , 2001,
Abstract: Large scale abstraction and isolation of bacterially synthesized, recombinant-DNA-derived, porcine growth hormone (r-pST) is described. The r-pGH is found in genetic engineering E.coli as the form of inclusion bodies. Pellet fraction which were mainly inclusion bodies, after cell breakage and centrifugation, were collected. Cell envelope components, such as protein, lipid, endotoxin and nucleic acids are selectively removed from the pellet fraction by an EDTA/lysozyme/deoxycholate extraction. Inclusion bodies were dissolved using 6mol/L guanidine/HCl and air oxidation is then carried out in the presence of the guanidine/HCl. The Guanidine/HCl protein mixture were diluted by renaturation solution. Guanidine/HCl were removed by dialysis and then correctly refolded, oxidized r-pGH were obtained. Injection experiment of hypophysectomized rats proved r-pST with high native bioactivity was obtained.
The Probable Structure of “Form IV” (Alkali-Denatured Circular DNA)  [PDF]
Ken Biegeleisen
Open Access Library Journal (OALib Journal) , 2016, DOI: 10.4236/oalib.1103114
Abstract:
A detailed molecular model for alkali-denatured duplex circular DNA (“Form IV”) is proposed. The illustrative biological example used is the replicative form of fx174, a 5 kb duplex circular chromosome. The model explains all of Form IV’s known and peculiar features. In a sedimentation coefficient vs. pH titration, Form IV begins to appear at pH 12.3, at which point it can be persuasively argued that no further supertwists can be added to the already-highly-supertwisted chromosome. Therefore a new structure must appear. The sedimentation coefficient s then undergoes a massive, but initially reversible increase as the pH is raised further, culminating at pH 12.8 with a 250% increase. This degree of compactness can only be explained by a 4-stranded tetraplex structure, consisting of a pair of duplexes whose base pairs are mutually intercalated. Above pH 12.8, the structural changes become irreversible, suggesting a further conformational change. It is proposed that this involves an axial rotation of the component duplex strands, so that the bases now stack on the outside, and the phosphate groups lie in the core, where they bond ionically by means of salt bridges. When the irreversibly denatured compact structure is neutralized at moderate-to-high salt concentrations, a third novel structure appears, which has a sedimentation coefficient midway between the native 21 s and the denatured 50 s. It is proposed that this is a hybrid structure; part tetraplex, part duplex. To return to a fully-duplex form, it is necessary to both neutralize the solution, and also to greatly reduce the ionic strength, i.e., to the range 0.001-0.01 M. Since the DNA, under those conditions, cannot possibly have normal complementary base-pairing, the duplex structure must either be tautomerically base-paired, or else stabilized solely by base-stacking, with no base-pairing at all.
Research on Renaturation of Recombinant Human Pro-urokinase Expressed from Escherichia coli
重组人尿激酶原的体外变复性研究

ZHU Hui,LIU Wei,SHI Wei,XUE Yu,Ming,MA Zhong,
朱慧
,刘伟,史蔚,薛宇鸣,马忠

生物工程学报 , 2000,
Abstract: Recombinant human pro-urokinase forms insoluble inclusion body when overexpressed in Escherichia coli, and it must be denatured and renatured in vitro before it acquires activity. This study aimed to increase the renaturation yield of denatured pro-urokinase. We have evaluated the basic renaturation conditions of pro-urokinase through qualitative and quantitative analysis of pH, temperature, denaturant concentration, protein concentration, the ratio of reduced and oxidized thiol reagents. The effects of nonspecific additives, step-wise dilution and urea gradient dialysis have been also compared. The optimal conditions of pro-urokinase renaturation with the yield about 20%-30% have been obtained.
Renaturation and Purification of Single Chain Fv Fragment of Monoclonal Antibady Against Haemachrome
重组抗血红素ScFV包涵体蛋白的复性及纯化研究

XU Han-Mei TANG Ya-Lan DAI Jim YAO Zhen-Sheng HUA Zi-Chun,
徐寒梅

中国生物工程杂志 , 2005,
Abstract: Most of single chain Fv fragment (ScFv) of monoclonal antibody deposited as insoluble inclusion bodies during expression in E. coli. In order to convert inactive and misfolded inclusion body ScFv into soluble products, the renaturation and purification procedure was developed, recombinant ScFv was redissolved, diluted, and was directly loaded onto a Sephadex G-25 column. The size-exclusion chromatography provided a favorable environment for the proteins to renature, it also spatially constrained partially refolded ScFv from aggregating and diffusing toward each other, promoted renaturation. In the size-exclusion chromatography, the ScFv protein flowed faster and was eluted earlier than the denaturant, this will allow the protein sample to pass through decreasing denaturant concentrations, which promotes ScFv refolding and removes denarurant. During this process, ScFv was simultaneously purified, and at least 150mg/L (with the purity more than 95 %) soluble ScFv was obtained. The strategy provides an economic and novel approach for the production of ScFv from inclusion body proteins.
Comparison of Recombinant Human β-NGF from E. coli and CHO in Biochemical Characters
来源于E.coli和CHO表达系统的重组人β-NGF性质比较

JIANG Jing,JIANG Gui-xiang,
姜静
,姜桂香

中国生物工程杂志 , 2006,
Abstract: 基于原核表达系统极难表达具有正确三级结构的重组人β神经生长因子(rhNGF-β),E.coli是否能作为其工业化生产菌株存在争议。为此,将构建于E.coli体系,经表达体外复性后的rhNGF-β与中国仓鼠卵巢细胞(CHO)表达体系分泌的rhNGF-β,运用HPLC、SDS-PAGE、质谱、N-末端分析等手段以及鸡胚背根神经节(DRG)、PC12细胞体外测活法对制备产物进行分析、测定。结果表明:二者具有一致的理化性质和生物学活性,HPLC纯度均大于95%、生物学比活均不低于1.8ng/U;经添加赋型剂制成冻干粉后,在温度37℃±2℃、相对湿度75%±5%条件下进行3个月加速试验,活性均不变。说明E.coli表达体系可生产质量均一、高生物学活性的rhNGF-β,且具有明显成本优势。
In vitro renaturation of proteins from inclusion bodies
Dorota Porowińska,Ewelina Marsza?ek,Paulina Ward?cka,Micha? Komoszyński
Post?py Higieny i Medycyny Do?wiadczalnej , 2012,
Abstract: Recombinant proteins and enzymes are commonly used in many areas of our life, such as diagnostics, industry and medicine, due to heterologous synthesis in prokaryotic expression systems. However, a high expression level of foreign protein in bacteria cells results in formation of inactive and insoluble aggregates – inclusion bodies.Reactivation of aggregated proteins is a complex and time-consuming process. Every protein requires experimental optimization of the process conditions. The choice of the refolding method depends on the type of recombinant protein and its physical, chemical and biological properties.Recovery of the activity of proteins accumulated in inclusion bodies can be divided into 4 steps: 1) inclusion bodies isolation, 2) solubilization of aggregates, 3) renaturation, 4) purification of catalytically active molecules.Efficiency of the refolding process depends on many physical factors and chemical and biological agents. The above parameters determine the time of the folding and prevent protein aggregation. They also assist the folding and have an influence on the solubility and stability of native molecules.To date, dilution, dialysis and chromatography are the most often used methods for protein refolding.
The Renaturation and Purification of RGD-Staphyloki nase by Gel Filtration
RGD-葡激酶的凝胶过滤层析法复性及其纯化

CHENG Ao,SONG Gang,SU Hua-Bo,YU Min,LI Yu-Yang,SONG Hou-Yan,
程鏖
,宋刚,苏华波,于敏,李育阳,宋后燕

生物工程学报 , 2002,
Abstract: A recombinant RGD-Staphylokinase(RGD-Sak) with thrombolytic and anti-thrombolytic bifunction was expressed in E.coli. The expression product accumulates as inclusion bodies. In order to obtain active molecule, the RGD-Sak in the inclusion body should be denatured and then renatured. The renaturation of RGD-Sak was performed by gel filtration. Comparing with the traditional way of dilution renaturation, gel filtration way is better than the traditional one, since there are some advantages, such as simple processing, high recovery, low cost and higher purity after renaturation. After renaturation, RGD-Sak was purified by Q-Sepharose FF, and the purity was more than 95%. Analysis of CD spectra showed that the final product from the two renaturation ways have similar CD spectra. It was demostrated that RGD-Sak molecules proceeded correct refolding through gel filtration or dilution renaturation process.
第1页/共30条
每页显示


Home
Copyright © 2008-2017 Open Access Library. All rights reserved.