共查询到20条相似文献,搜索用时 15 毫秒
1.
Etsushi Yamamoto Satoshi Yamaguchi Naoki Sasaki Haeng-Boo Kim Takehiko Kitamori Teruyuki Nagamune 《Bioprocess and biosystems engineering》2010,33(1):171-177
Protein refolding using a simple dilution method in a microchannel often led to the formation of protein aggregates, which
bound to the microchannel wall, resulting in low refolding yields. To inhibit aggregation and improve refolding yields, an
artificial chaperone-assisted (ACA) refolding, which employed detergents and β-cyclodextrin was used. Model proteins, hen
egg white lysozyme and yeast α-glucosidase, were successfully refolded in a microchannel. The microscopic observation showed
that the ACA method suppressed protein aggregation and facilitated the refolding of lysozyme, whereas significant aggregation
was observed when a simple dilution method was employed. The ACA method increased the lysozyme refolding yield by 40% over
the simple dilution approach. Similarly, for α-glucosidase, the refolding yield using the ACA method (ca. 50%) was approximately
three times compared with the simple dilution method. The ACA refolding method is a suitable approach to use in the refolding
of proteins using a microfluidic system. 相似文献
2.
Predictions of matrix‐assisted refolding of α‐lactalbumin: Process efficiency versus batch dilution method
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Protein refolding is an important technique to produce active recombinant proteins from inclusion bodies. Because of the complexity of the refolding process, a trial‐and‐error method is usually used for its design, which is ineffective and time consuming. Therefore, an efficient method for the process prediction is indispensable to optimize the operating conditions. In this article, we suggest a design procedure for matrix‐assisted protein refolding. Three different chromatographic techniques were considered exploiting hydrophobic interaction chromatography, ion‐exchange chromatography, and SEC media. The procedure consisted of quantification of refolding kinetics, analysis of the retention behavior of all protein forms involved in refolding, construction of a dynamic model, and the process simulation. Denatured bovine α‐lactalbumin was used as model protein. The refolding rate was measured for different protein concentration using the batch dilution method. A kinetic scheme for the protein refolding was suggested and incorporated into a dynamic model of chromatographic column and used for predicting the refolding performance. The productivity, yield, and buffer consumption were used as performance indicators for the refolding techniques considered. The matrix‐assisted protein refolding process outperformed batch dilution method with respect to all indicators provided that efficient method for the process design was used. 相似文献
3.
Feng Y Liu L Wang J Liu J Hu W Wang X Yang Z 《Protein expression and purification》2012,84(2):181-187
The full-length cDNA of MTH1in Schistosoma japonicum was previously isolated. However, insoluble protein expression in Escherichia coli is the biggest bottleneck limiting biological and biophysical studies. Protein aggregation could not be significantly prevented using solubilization or refolding techniques, and denatured MTH1 protein could not be refolded to the native monomer form. Hence, integrating several refolding techniques within the protein refolding process of MTH1, a large amount of active MTH1 was obtained for protein crystallization. We primarily utilized the two-step-denaturing and refolding method and the protein refolding screening technique, as well as the continuous dialysis method. First, we identified the refolding buffer composition that allowed for successful refolding to overcome protein precipitation. Next, we used the two-step-denaturing and refolding method and the continuous dialysis method to suppress protein aggregation. In the end, we obtained 15 mg of active MTH1 monomer with 95% purity from 0.5l medium. Integrated refolding techniques proved to be excellent for obtaining the native monomer of S. japonicum MTH1 from inclusion bodies, paving the way for future biological and biophysical studies. 相似文献
4.
Investigation of protein refolding using a fractional factorial screen: a study of reagent effects and interactions 总被引:1,自引:0,他引:1
Willis MS Hogan JK Prabhakar P Liu X Tsai K Wei Y Fox T 《Protein science : a publication of the Protein Society》2005,14(7):1818-1826
A recurring obstacle for structural genomics is the expression of insoluble, aggregated proteins. In these cases, the use of alternative salvage strategies, like in vitro refolding, is hindered by the lack of a universal refolding method. To overcome this obstacle, fractional factorial screens have been introduced as a systematic and rapid method to identify refolding conditions. However, methodical analyses of the effectiveness of refolding reagents on large sets of proteins remain limited. In this study, we address this void by designing a fractional factorial screen to rapidly explore the effect of 14 different reagents on the refolding of 33 structurally and functionally diverse proteins. The refolding data was analyzed using statistical methods to determine the effect of each refolding additive. The screen has been miniaturized for automation resulting in reduced protein requirements and increased throughput. Our results show that the choice of pH and reducing agent had the largest impact on protein refolding. Bis-mercaptoacetamide cyclohexane (BMC) and tris (2-carboxyethylphosphine) (TCEP) were superior reductants when compared to others in the screen. BMC was particularly effective in refolding disulfide-containing proteins, while TCEP was better for nondisulfide-containing proteins. From the screen, we successfully identified a positive synergistic interaction between nondetergent sulfobetaine 201 (NDSB 201) and BMC on Cdc25A refolding. The soluble protein resulting from this interaction crystallized and yielded a 2.2 Angstroms structure. Our method, which combines a fractional factorial screen with statistical analysis of the data, provides a powerful approach for the identification of optimal refolding reagents in a general refolding screen. 相似文献
5.
Development of high efficiency and low cost protein refolding methods is a highlighted research focus in biotechnology. Artificial molecular chaperone (AMC) and protein folding liquid chromatography (PFLC) are two attractive refolding methods developed in recent years. In the present work, AMC and one branch of PFLC, ion exchange chromatography (IEC), are integrated to form a new refolding method, artificial molecular chaperone‐ion exchange chromatography (AMC‐IEC). This new method is applied to the refolding of a widely used model protein, urea‐denatured/dithiothreitol‐reduced lysozyme. Many factors influencing the refolding of lysozyme, such as urea concentration, β‐cyclodextrin concentration, molar ratio of detergent to protein, mobile phase flow rate, and type of detergent, were investigated, respectively, to optimize the conditions for lysozyme refolding by AMC‐IEC. Compared with normal IEC refolding method, the activity recoveries of lysozyme obtained by AMC‐IEC were much higher in the investigated range of initial protein concentrations. Moreover, the activity recoveries obtained by using this newly developed refolding method were still quite high for denatured/reduced lysozyme at high initial concentrations. When the initial protein concentration was 200 mg mL?1, the activity recovery was over 60%. In addition, the lifetime of the chromatographic column during AMC‐IEC was much longer than that during protein refolding by normal IEC. Therefore, AMC‐IEC is a high efficient and low cost protein refolding method. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 相似文献
6.
Dr. Satoshi Yamaguchi Etsushi Yamamoto Teruhisa Mannen Teruyuki Nagamune Prof. Teruyuki Nagamune 《Biotechnology journal》2013,8(1):17-31
In laboratories and manufacturing settings, a rapid and inexpensive method for the preparation of a target protein is crucial for promoting resesrach in protein science and engineering. Inclusion-body-based protein production is a promising method because high yields are achieved in the upstream process, although the refolding of solubilized, unfolded proteins in downstream processes often leads to significantly lower yields. The most challenging problem is that the effective condition for refolding is protein dependent and is therefore difficult to select in a rational manner. Accordingly, considerable time and expense using trial-and-error approaches are often needed to increase the final protein yield. Furthermore, for certain target proteins, finding suitable conditions to achieve an adequate yield cannot be obtained by existing methods. Therefore, to convert such a troublesome refolding process into a routine one, a wide array of methods based on novel technologies and materials have been developed. These methods select refolding conditions where productive refolding dominates over unproductive aggregation in competitive refolding reactions. This review focuses on synthetic refolding additives and describes the concepts underlying the development of reported chemical additives or chemical-additive-b 相似文献
7.
Refolding of horseradish peroxidase is enhanced in presence of metal cofactors and ionic liquids
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The effects of various refolding additives, including metal cofactors, organic co‐solvents, and ionic liquids, on the refolding of horseradish peroxidase (HRP), a well‐known hemoprotein containing four disulfide bonds and two different types of metal centers, a ferrous ion‐containing heme group and two calcium atoms, which provide a stabilizing effect on protein structure and function, were investigated. Both metal cofactors (Ca2+ and hemin) and ionic liquids have positive impact on the refolding of HRP. For instance, the HRP refolding yield remarkably increased by over 3‐fold upon addition of hemin and calcium chloride to the refolding buffer as compared to that in the conventional urea‐containing refolding buffer. Moreover, the addition of ionic liquids [EMIM][Cl] to the hemin and calcium cofactor‐containing refolding buffer further enhanced the HRP refolding yield up to 80% as compared to 12% in conventional refolding buffer at relatively high initial protein concentration (5 mg/ml). These results indicated that refolding method utilizing metal cofactors and ionic liquids could enhance the yield and efficiency for metalloprotein. 相似文献
8.
包涵体中的重组蛋白抽提后可以在变性状态下纯化,而纯化后的体外折叠(即复性)是基因工程下游处埋中的重要环节。荧光光谱研究表明,IL-2分子折叠过程中荧光强度逐渐减小,最大发射峰由316nm红移到348nm。以Trp残基的暴露程度反映分子的折叠状态。GM-CSF在折叠过程中的荧光强度有类似变化;凝胶排阻HPLC可以检测折叠过程中的聚合体;而反相HPLC可以将IL-2分成三个相互独立的异构体色谱峰。据此可以计算出IL-2分子的正确折叠率。常用的稀释复性方法,随着IL-2浓度的增高,它的正确折叠率逐渐降低,蛋白浓度的对数与正确折叠率之间大致呈线性关系。当IL-2浓度为1mg/mL时,其正确折叠率仅为30%,而采取较低的蛋白浓度进行复性会因大量的样品体积导致后期纯化的困难。 相似文献
9.
Continuous refolding of lysozyme with fed-batch addition of denatured protein solution 总被引:3,自引:0,他引:3
A continuous refolding method with addition of denatured protein solution in a fed-batch manner through a ceramic membrane tube was developed. Denatured and fully reduced lysozyme was continuously refolded with high refolding efficiencies. In this method, a denatured lysozyme solution was gradually added from the outer surface of the membrane tube into a refolding buffer flowing continuously inside the tube under controlled mixing conditions. The refolding efficiencies of lysozyme in this continuous refolding were higher than those in a batch dilution method. This method has applicability to large-scale downstream processes and can attain a high efficiency and protein concentration in refolding. Refolded proteins can be supplied continuously following purification steps. 相似文献
10.
The "artificial chaperone method" for protein refolding developed by Rozema et al. (Rozema, D.; Gellman, S. H. J. Am. Chem. Soc. 1995, 117 (8), 2373-2374) involves the sequential dilution of denatured protein into a buffer containing detergent (cetyltrimethylammonium bromide, CTAB) and then into a refolding buffer containing cyclodextrin (CD). In this paper a simplified one-step artificial chaperone method is reported, whereby CTAB is added directly to the denatured solution, which is then diluted directly into a refolding buffer containing beta-cyclodextrin (beta-CD). This new method can be applied at high protein concentrations, resulting in smaller processing volumes and a more concentrated protein solution following refolding. The increase in achievable protein concentration results from the enhanced solubility of CTAB at elevated temperatures in concentrated denaturant. The refolding yields obtained for the new method were significantly higher than for control experiments lacking additives and were comparable to the yields obtained with the classical two-step approach. A study of the effect of beta-CD and CTAB concentrations on refolding yield suggested two operational regimes: slow stripping (beta-CD/CTAB approximately 1), most suited for higher protein concentrations, and fast stripping (beta-CD/CTAB approximately 2.7), best suited for lower protein concentrations. An increased chaotrope concentration resulted in higher refolding yields and an enlarged operational regime. 相似文献
11.
Vincent Dechavanne Nicolas Barrillat Frederic Borlat Aurélie Hermant Laurent Magnenat Mikael Paquet Bruno Antonsson Laurent Chevalet 《Protein expression and purification》2011,75(2):192-203
Production of correctly folded and biologically active proteins in Escherichia coli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions. After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen. The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS–PAGE. The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives. The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests, which confirmed the reliability of the predictions. Finally, the refolded protein was purified and its biological activity was tested in vitro. The screen was applied for the refolding of Interleukin 17F (IL-17F), stromal-cell-derived factor-1 (SDF-1α/CXCL12), B cell-attracting chemokine 1 (BCA-1/CXCL13), granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a. This procedure identified refolding conditions for all the tested proteins. For the proteins where refolding conditions were already available, the optimized conditions identified in the screening process increased the yields between 50% and 100%. Thus, the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein. 相似文献
12.
Bernd Anselment Danae Baerend Elisabeth Mey Johannes Buchner Dirk Weuster-Botz Martin Haslbeck 《Protein science : a publication of the Protein Society》2010,19(11):2085-2095
Refolding of proteins from solubilized inclusion bodies still represents a major challenge for many recombinantly expressed proteins and often constitutes a major bottleneck. As in vitro refolding is a complex reaction with a variety of critical parameters, suitable refolding conditions are typically derived empirically in extensive screening experiments. Here, we introduce a new strategy that combines screening and optimization of refolding yields with a genetic algorithm (GA). The experimental setup was designed to achieve a robust and universal method that should allow optimizing the folding of a variety of proteins with the same routine procedure guided by the GA. In the screen, we incorporated a large number of common refolding additives and conditions. Using this design, the refolding of four structurally and functionally different model proteins was optimized experimentally, achieving 74–100% refolding yield for all of them. Interestingly, our results show that this new strategy provides optimum conditions not only for refolding but also for the activity of the native enzyme. It is designed to be generally applicable and seems to be eligible for all enzymes. 相似文献
13.
Yazdanparast R Khodagholi F Khodarahmi R 《International journal of biological macromolecules》2005,35(5):257-263
It is now well established that alpha-cyclodextrin (alpha-CD) is a valuable folding agent in refolding processes of several denatured enzyme solutions. The refolding of Gu-HCl denatured alpha-amylase in the dilution-additive mode revealed that alpha-CD enhanced the refolding yield by 20-30% depending upon alpha-CD concentration. However, the refolding efficiency of the Gu-HCl denatured alpha-amylase through the artificial chaperone-assisted method indicated that alpha-CD enhanced the activity recovery of denatured alpha-amylase by almost 50% and also increased the reactivation rate constant relative to the unassisted control sample. The higher refolding efficiency should be due to different mechanism played by alpha-CD in this technique. In addition, our data indicated that higher refolding yields are obtained when the residual Gu-HCl concentration is low in the refolding environment and when the capture agent is removed not in a stepwise manner from the protein-detergent complexes in the stripping step of the whole process. Collectively, the results of this investigation expand the range of procedural variations used to refold different denatured proteins through artificial chaperone-assisted method. 相似文献
14.
Yoshii H Furuta T Yonehara T Ito D Linko YY Linko P 《Bioscience, biotechnology, and biochemistry》2000,64(6):1159-1165
Refolding of reduced and denatured protein in vitro has been an important issue for both basic research and applied biotechnology. Refolding at low protein concentration requires large volumes of refolding buffer. Among various refolding methods, diafiltration is very useful to control the denaturant and red/ox reagents in a refolding solution. We constructed a refolding procedure of high lysozyme concentration (0.5-10 mg/ml) based on the linear reduction of the urea concentration during diafiltration under oxygen pressure. When the urea concentration in the refolding vessel was decreased from 4 M with a rate of 0.167 M/h, the refolding yields were 85% and 63% at protein concentrations, 5 mg/ml and 10 mg/ml, respectively, after 11 h. This method gave a high productivity of 40.1,microM/h of the refolding lysozyme. The change in refolding yields during the diafiltration could be simulated using the model of Hevehan and Clark. 相似文献
15.
蛋白质的排阻色谱复性的新进展 总被引:3,自引:0,他引:3
外源蛋白在大肠杆菌中高效表达时 ,常常形成不溶的、无活性的包涵体 ,包涵体蛋白的复性是重组蛋白生产过程中的一个技术难题。排阻色谱 (sizeexclusionchromatography ,SEC)用于蛋白复性是一种较新的、适用于任何一种蛋白的方法 ,与常用的稀释复性法相比 ,它能在高的起始蛋白浓度下对蛋白进行复性 ,活性回收率较高 ,同时又能使目标蛋白得到一定程度的纯化。对使用SEC复性的进展进行了评述 ,其内容包括SEC复性的原理及其复性过程中的影响因素 ,并对其未来发展进行了展望。 相似文献
16.
Over-expression of heterologous proteins in Escherichia coli is commonly hindered by the formation of inclusion bodies. Nevertheless, refolding of proteins in vitro has become an essential requirement in the development of structural genomics (proteomics) and as a means of recovering functional proteins from inclusion bodies. Many distinct methods for protein refolding are now in use. However, regardless of method used, developing a reliable protein refolding protocol still requires significant optimization through trial and error. Many proteins fall into the category of "Challenging" or "Difficult to Express" and are problematic to refold using traditional chaotrope-based refolding techniques. This review discusses new methods for improving protein refolding, such as implementing high hydrostatic pressure, using small molecule additives to enhance traditional protein refolding strategies, as well as developing practical methods for performing refolding studies to maximize their reliability and utility. The strategies examined here focus on high-throughput, automated refolding screens, which can be applied to structural genomic projects. 相似文献
17.
Yanye Feng Ming Zhang Linlin Zhang Ting Zhang Jianfeng Ding Yingping Zhuang Xiaoning Wang Zhong Yang 《PloS one》2012,7(9)
Protein refolding is an important process to recover active recombinant proteins from inclusion bodies. Refolding by simple dilution, dialysis and on-column refolding methods are the most common techniques reported in the literature. However, the refolding process is time-consuming and laborious due to the variability of the behavior of each protein and requires a great deal of trial-and-error to achieve success. Hence, there is a need for automation to make the whole process as convenient as possible. In this study, we invented an automatic apparatus that integrated three refolding techniques: varying dilution, dialysis and on-column refolding. We demonstrated the effectiveness of this technology by varying the flow rates of the dilution buffer into the denatured protein and testing different refolding methods. We carried out different refolding methods on this apparatus: a combination of dilution and dialysis for human stromal cell-derived factor 1 (SDF-1/CXCL12) and thioredoxin fused-human artemin protein (Trx-ARTN); dilution refolding for thioredoxin fused-human insulin-like growth factor I protein (Trx-IGF1) and enhanced fluorescent protein (EGFP); and on-column refolding for bovine serum albumin (BSA). The protein refolding processes of these five proteins were preliminarily optimized using the slowly descending denaturants (or additives) method. Using this strategy of decreasing denaturants concentration, the efficiency of protein refolding was found to produce higher quantities of native protein. The standard refolding apparatus configuration can support different operations for different applications; it is not limited to simple dilution, dialysis and on-column refolding techniques. Refolding by slowly decreasing denaturants concentration, followed by concentration or purification on-column, may be a useful strategy for rapid and efficient recovery of active proteins from inclusion bodies. An automatic refolding apparatus employing this flexible strategy may provide a powerful tool for preparative scale protein production. 相似文献
18.
Sung-Mi Hwang Hye-Jin Kang Sang-Woo Bae Woo-Jin Chang Yoon-Mo Koo 《Biotechnology and Bioprocess Engineering》2010,15(2):213-219
The effects of salt concentration in mobile phase, elution strategy, and hydrophobicity of stationary phase on lysozyme refolding
in hydrophobic interaction chromatography (HIC) were investigated. Butyl Sepharose 4 Fast Flow, the least hydrophobic HIC
resin among the tested adsorbent, showed the best refolding yield. The binding efficiency of unfolded lysozyme on the adsorbent
was maximized when 1 and 0.4 M of initial and final concentration of ammonium sulfate was used in mobile phase, respectively.
The optimum gradient strategy for refolding and elution of lysozyme was determined as linear increase of urea concentration
to 4M. The optimized condition suggests the less hydrophobic environment than conventionally used salt solutions and HIC resins.
Consequently, total refolding yield was improved 1.6 times comparing with optimized dilution-based batch refolding method. 相似文献
19.
Effect of rate of chemical or thermal renaturation on refolding and aggregation of a simple lattice protein 总被引:1,自引:0,他引:1
We used dynamic Monte Carlo simulation to investigate how changing the rate of chemical or thermal renaturation affects the folding and aggregation behavior of a system of simple, two-dimensional lattice protein molecules. Four renaturation methods were simulated: infinitely slow cooling; slow but finite cooling; quenching; and pulse renaturation. The infinitely slow cooling method, which is equivalent to dialysis or diafiltration, provides refolding yields that are relatively high and aggregates that are relatively small (mostly dimers or trimers). The slow but finite cooling method, which is equivalent to multiple-step dilution, provides refolding yields that are almost as high as those observed in the infinitely slow cooling case, but in a relatively short period of time. Quenching, which is equivalent to one-step dilution or quick quenching, is extremely slow and has low re- folding yields. A maximum appears in the refolding yield as a function of denaturant concentration in the simulation but disappears after a very long duration. Finally, the pulse renaturation method provides refolding yields that are substantially higher than those observed in the other three methods, even at high packing fractions. As in the early stages of quenching, there is a maximum in the refolding yield as a function of denaturant concentration when relatively large numbers of denatured chains are added to the refolding solution at each step. 相似文献
20.
To get a better understanding of the molecular aspects of protein folding, the refolding kinetic behavior of guanidine hydrochloride-denatured alkaline phosphatase (ALP) was studied in the presence of alpha-cyclodextrin (alpha-CD) through two different approaches: the dilution additive and the artificial chaperone-assisted methods. It was found that alpha-CD enhanced the recovered activity more than 50% via both approaches while decreased the refolding rate, perhaps due to engaging the hydrophobic patches of the protein in a rigid conformation. In contrast, detergents used in the artificial chaperone method increased the refolding rate significantly. A comparison of the rate constants for the refolding and the activity recovery of denatured ALP in the presence of various concentrations of CD and different kinds of detergents showed that they do not progress in a synchronized pattern. This may be attributed to continuous structural rearrangements in the protein long after the return of enzyme activity. These observations are discussed in terms of kinetic and structural aspects of the refolding pathway. 相似文献