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1.
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
2.
Mechanism of pH‐sensitive polymer‐assisted protein refolding and its application in TGF‐β1 and KGF‐2
Zhifeng Huang Chunyan Ni Xiangtian Zhou Yanlong Liu Yi Tan Jian Xiao Wenke Feng Xiaokun Li Shulin Yang 《Biotechnology progress》2009,25(5):1387-1395
Refolding of proteins at high concentrations often results in non‐productive aggregation. This study, through a unique combination of spectroscopic and chromatographic analyzes, provides biomolecular evidence to demonstrate the ability of Eudragit S‐100, a pH‐responsive polymer, to enhance refolding of denatured‐reduced lysozyme at high concentrations. The addition of Eudragit in the refolding buffer significantly increases lysozyme refolding yield to 75%, when dilution refolding was conducted at 1 mg/mL lysozyme. This study shows evidence of an electrostatic interaction between oppositely charged lysozyme and the Eudragit polymer during refolding. This ionic complexing of Eudragit and lysozyme appears to shield exposed hydrophobic residues of the lysozyme refolding intermediates, thus minimizing hydrophobic‐driven aggregation of the molecules. Importantly, results from this study show that the Eudragit‐lysozyme bioconjugation does not compromise refolded protein structure, and that the polymer can be readily dissociated from the protein by ion exchange chromatography. The strategy was also applied to refolding of TGF‐β1 and KGF‐2. © 2009 American Institute of Chemical Engineers Biotechnol. Prog. 2009 相似文献
3.
DsbA (disulfide bond formation protein A) is essential for disulfide bond formation directly affecting the nascent peptides folding to the correct conformation in vivo. In this paper, recombinant DsbA protein was employed to catalyze denatured lysozyme refolding and inhibit the aggregation of folding intermediates in vitro. Statistical methods, i.e., Plackett–Burman design and small central composite design, were adopted to screen out important factors affecting the refolding process and correlating these parameters with the refolding efficiency including both protein recovery and specific activity of refolded lysozyme. Four important parameters: initial lysozyme concentration, urea concentration, KCl concentration and GSSG (glutathione disulfide) concentration were picked out and operating conditions were optimized by introducing the effectiveness coefficient method and transforming the multiple objective programming into an ordinary constrained optimization issue. Finally, 99.7% protein recovery and 25,600 U/mg specific activity of lysozyme were achieved when 281.35 μg/mL denatured lysozyme refolding was catalyzed by an equivalent molar of DsbA at the optimal settings. The results indicated that recombinant DsbA protein could effectively catalyze the oxidized formation and reduced isomerization of intramolecular disulfide bonds in the refolding of lysozyme in vitro. 相似文献
4.
Protein refolding is a crucial step for the production of therapeutic proteins expressed in bacteria as inclusion bodies. In vitro protein refolding is severely impeded by the aggregation of folding intermediates during the folding process, so inhibition of the aggregation is the most effective approach to high‐efficiency protein refolding. We have herein found that electrostatic repulsion between like‐charged protein and ion exchange gel beads can greatly suppress the aggregation of folding intermediates, leading to the significant increase of native protein recovery. This finding is extensively demonstrated with three different proteins and four kinds of ion‐exchange resins when the protein and ion‐exchange gel are either positively or negatively charged at the refolding conditions. It is remarkable that the enhancing effect is significant at very high protein concentrations, such as 4 mg/mL lysozyme (positively charged) and 2 mg/mL bovine serum albumin (negatively charged). Moreover, the folding kinetics is not compromised by the presence of the resins, so fast protein refolding is realized at high protein concentrations. It was not realistic by any other approaches. The working mechanism of the like‐charged resin is considered due to the charge repulsion that could induce oriented alignment of protein molecules near the charged surface, leading to the inhibition of protein aggregation. The molecular crowding effect induced by the charge repulsion may also contribute to accelerating protein folding. The refolding method with like‐charged ion exchangers is simple to perform, and the key material is easy to separate for recycling. Moreover, because ion exchangers can work as adsorbents of oppositely charged impurities, an operation of simultaneous protein refolding and purification is possible. All the characters are desirable for preparative refolding of therapeutic proteins expressed in bacteria as inclusion bodies. Bioeng. 2011; 108:1068–1077. © 2010 Wiley Periodicals, Inc. 相似文献
5.
An affinity-based reverse micellar system formulated with nonionic surfactant was applied to the refolding of denatured-reduced lysozyme. The nonionic surfactant of sorbitan trioleate (Span 85) was modified with Cibacron Blue F-3GA (CB) as an affinity surfactant (CB-Span 85) to form affinity-based reverse micelles in n-hexane. The water content of 15 was found optimal for lysozyme refolding in the reverse micellar system of 62.7 mmol/L Span 85 with coupled CB of 0.3 and 0.5 mmol/L. In addition, the operating conditions such as pH and the concentrations of urea and redox reagents were optimized. Under the optimized conditions, complete renaturation of lysozyme at 3-3.5 mg/mL was achieved, whereas dilution refolding in the bulk aqueous phase under the same conditions gave much lower activity recovery. Moreover, the secondary structure of the refolded lysozyme was found to be the same as the native lysozyme. Over 95% of the refolded lysozyme was recovered from CB-Span 85 reverse micelles by a stripping solution of 0.5 mol/L MgCl(2). Thus, the present system is advantageous over the conventional reverse micellar system formed with ionic surfactants in the ease of protein recovery. 相似文献
6.
The kinetics of lysozyme refolding and aggregation is studied using an existing competing first- and third-order reaction scheme. The existing model overestimates yield at high refolding concentrations (>1 mg/mL), thus limiting its use for reactor design at industrially relevant refolding concentrations. This study demonstrates that a pathway exists for the incorporation of refolded native protein into aggregates. Specifically, native lysozyme labeled with fluorescein isothiocyanate was added to the refolding buffer prior to dilution refolding of denatured and reduced lysozyme. Aggregates collected from these experiments showed significant fluorescence, indicating that labeled lysozyme had been incorporated into the aggregates during refolding. Although the precise pathway of incorporation has not been elucidated, it is clear from this work that the existing model for lysozyme refolding is not globally applicable. In particular, previous work has analytically demonstrated that neglect of a pathway from native to aggregate can result in the design of a grossly suboptimal reactor strategy. This study demonstrates that such a pathway can exist experimentally and emphasizes the need to critically assess refolding kinetic models before their use in reactor design equations. 相似文献
7.
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
8.
The effects of several variables on the refolding of hen egg white lysozyme have been studied. Lysozyme was denatured in both
urea, and guanidine hydrochloride (GuHCl), and batch refolded by dilution (100 to 1000 fold) into 0.1M Tris-HCl, pH 8.2, 1
mM EDTA, 3 mM reduced glutathione and 0.3 mM oxidised glutathione. Refolding was found to be sensitive to temperature, with
the highest refolding yield obtained at 50°C. The apparent activation energy for lysozyme refolding was found to be 56 kJ/mol.
Refolding by dilution results in low concentrations of both denaturant and reducing agent species. It was found that the residual
concentrations obtained during dilution (100-fold dilution: [GuHCl]=0.06 mM, [DTT]=0.15 mM) were significant and could inhibit
lysozyme refolding. This study has also shown that the initial protein concentration (1–10 mg/mL) that is refolded is an important
parameter. In the presence of residual GuHCl and DTT, higher refolding yields were obtained when starting from higher initial
lysozyme concentrations. This trend was reversed when residual denaturant components were removed from the refolding buffer. 相似文献
9.
Cyclodextrins (CDs), in the presence or absence of detergents, have been reported to suppress aggregate formation during the refolding of a number of proteins. A structure-activity relationship study between CD chemistry and refolding of lysozyme was performed and compared to carbonic anhydrase, in order to better understand the mechanism of CD-assisted protein refolding and to identify CDs that could function as good protein folding agents. Among the natural CDs, which have only hydroxyl groups, alpha-CD, with a smaller cavity size was more effective than the oligosaccharide with a larger cavity, gamma-CD. Replacement of the hydroxyls with other functional groups did not improve, but could seriously interfere, with the lysozyme refolding ability of alpha-CD. In case of gamma-CD, substitution of its hydroxyls with other groups either enhanced or diminished its refolding capability towards lysozyme. In general, neutral CDs were better refolding agents than the charged sugars. The presence of anionic substituents like carboxyl and phosphate groups actually promoted aggregate formation and completely abolished the sugar's refolding ability. This effect was more pronounced with lysozyme than with carbonic anhydrase. CDs with cationic functional groups did not show any significant effects on lysozyme refolding. The presence of both anionic and cationic substituents on the same CD molecule was found to partially restore its renaturation ability. Electrophoresis data indicate that CDs, which promoted lysozyme refolding, arrested aggregation at the stage of smaller soluble aggregates. Interestingly, the structure-activity relationship observed with lysozyme was quite similar to that reported for a non-disulfide protein, carbonic anhydrase. These results suggest that the effects of CDs on protein refolding are attributed to their ability to suppress aggregation of proteins. CDs may show properties similar to chaotropic agents, which may help explain their anti-aggregation and protein refolding ability. Besides alpha-CD, a number of other neutral CDs were found to be effective protein folding aids. 相似文献
10.
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
11.
Gareth J. Mannall James P. Myers John Liddell Nigel J. Titchener‐Hooker Paul A. Dalby 《Biotechnology and bioengineering》2009,103(2):329-340
Steps for the refolding of proteins from solubilized inclusion bodies or misfolded product often represent bottlenecks in process development, where optimal conditions are typically derived empirically. To expedite refolding optimization, microwell screening may be used to test multiple conditions in parallel. Fast, accurate, and reproducible assays are required for such screening processes, and the results derived must be representative of the process at full scale. This article demonstrates the use of these microscale techniques to evaluate the effects of a number of additives on the refolding of IGF‐1 from denatured inclusion bodies, using an established HPLC assay for this protein. Prior to this, microwell refolding was calibrated for scale‐up using hen egg‐white lysozyme (HEWL) as an initial model protein, allowing us to implement and compare several assays for protein refolding, including turbidity, enzyme activity, and chromatographic methods, and assess their use for microwell‐based experimentation. The impact of various microplate types upon protein binding and loss is also assessed. Solution mixing is a key factor in protein refolding, therefore we have characterized the effects of different methods of mixing in microwells in terms of their impact on protein refolding. Our results confirm the applicability and scalability of microwell screening for the development of protein refolding processes, and its potential for application to new inclusion body‐derived protein products. Biotechnol. Bioeng. 2009;103: 329–340. © 2008 Wiley Periodicals, Inc. 相似文献
12.
For production of active proteins using heterologous expression systems, refolding of proteins from inclusion bodies often creates a bottleneck due to its poor yield. In this study, we show that molecularly imprinted polymer (MIP) toward native lysozyme promotes the folding of chemically denatured lysozyme. The MIP, which was prepared with 1 M acrylamide, 1 M methacrylic acid, 1 M 2-(dimethylamino)ethyl methacrylate, and 5 mg/mL lysozyme, successfully promoted the refolding of lysozyme, whereas the non-imprinted polymer did not. The refolding yield of 90% was achieved when 15 mg of the MIP was added to 0.3 mg of the unfolded lysozyme. The parallel relationship between the refolding yield and the binding capacity of the MIP suggests that MIP promotes refolding through shifting the folding equilibrium toward the native form by binding the refolded protein. 相似文献
13.
Ye C Ilghari D Niu J Xie Y Wang Y Wang C Li X Liu B Huang Z 《Journal of biotechnology》2012,160(3-4):169-175
An in-depth understanding of molecular basis by which smart polymers assist protein refolding can lead us to develop a more effective polymer for protein refolding. In this report, to investigate structure-function relationship of pH-sensitive smart polymers, a series of poly(methylacrylic acid (MAc)-acrylic acid (AA))s with different MAc/AA ratios and molecular weights were synthesized and then their abilities in refolding of denatured lysozyme were compared by measuring the lytic activity of the refolded lysozyme. Based on our analysis, there were optimal MAc/AA ratio (44% MAc), M(w) (1700 Da), and copolymer concentration (0.1%, w/v) at which the highest yield of protein refolding was achieved. Fluorescence, circular dichroism, and RP-HPLC analysis reported in this study demonstrated that the presence of P(MAc-AA)s in the refolding buffer significantly improved the refolding yield of denatured lysozyme without affecting the overall structure of the enzyme. Importantly, our bioseparation analysis, together with the analysis of zeta potential and particle size of the copolymer in refolding buffers with different copolymer concentrations, suggested that the polymer provided a negatively charged surface for an electrostatic interaction with the denatured lysozyme molecules and thereby minimized the hydrophobic-prone aggregation of unfolded proteins during the process of refolding. 相似文献
14.
The influence of operational parameters on lysozyme refolding using size-exclusion chromatography 总被引:1,自引:0,他引:1
The influence of several parameters on the gel filtration refolding of hen egg white lysozyme from a starting concentration of 40 mg/ml was investigated. Refolding was found to be unaffected by temperature between 30 and 50°C, giving 100% recovered specific activity. At 10°C a 20% reduction in refolding yield was observed. Refolding was carried out successfully with both acrylamide (Sephacryl S100)- and dextran (Superdex 75)-based gel media. At the isoelectric pH of lysozyme, aggregation was suppressed in the column method, whereas protein aggregates were formed during dilution-based refolding. A number of compounds (carboxymethyl cellulose, dextran, sucrose) were added to the mobile phase to reduce the relative viscosity between the sample and mobile phase. Only sucrose, up to 20% (wt), was found not to interfere with lysozyme refolding. 相似文献
15.
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
16.
Guillaume Roussel Eric A. Perpète André Matagne Emmanuel Tinti Catherine Michaux 《Biotechnology and bioengineering》2013,110(2):417-423
It has recently been reported that 2‐methyl‐2,4‐pentanediol (MPD) can modulate the protein‐binding properties of sodium dodecyl sulfate (SDS), turning it into a non‐denaturing detergent. Indeed both alpha (the lysozyme) and beta (the carbonic anhydrase II) soluble enzymes, as well as a beta membrane protein (PagP) have been successfully refolded into their native form by using this amphiphatic alcohol. In order to support the universal character of our MPD‐based technique, we have extended its transferability to the Omp2a trimeric membrane porin. The far‐UV circular dichroism signature of Omp2a refolded with our original procedure is identical to that obtained by classical techniques, clearly indicating a proper refolding. Moreover, we show that the optimal SDS/MPD ratio for refolding Omp2a is similar to what has been observed for other types of proteins. While the protocol allows refolding at higher protein concentration (up to 4 mg/mL) and ionic strength (up to 1 M NaCl) than other refolding methods, it is also more efficient at basic pH values and medium temperature (20–40°C). Finally, the key role of the cosolvent was highlighted by a thorough study of the efficiency of MPD analogues, and a high variability was observed, as they can be able or unable to induce refolding at low or high salt concentrations. Biotechnol. Bioeng. 2013; 110: 417–423. © 2012 Wiley Periodicals, Inc. 相似文献
17.
Inclusion body refolding processes play a major role in the production of recombinant proteins. Improvement of the size-exclusion chromatography refolding process was achieved by combining a decreasing urea gradient with an increasing arginine gradient (two gradients) for the refolding of NTA protein (a new thrombolytic agent) in this paper. Different refolding methods and different operating conditions in two gradients gel filtration process were investigated with regard to increasing the NTA protein activity recovery and inhibition of aggregation. The refolding of denatured NTA protein showed this method could significantly increase the activity recovery of protein at high protein concentration. The activity recovery of 37% was obtained from the initial NTA protein concentration up to 20 mg/ml. The conclusions presented in this study could also be applied to the refolding of lysozyme. 相似文献
18.
A surfactant–heme complex which shows peroxidase activity in organic media has been prepared by a method utilizing water‐in‐oil (W/O) emulsions. Both the aqueous phase pH and the type of surfactant appeared to have prominent effect on the catalytic activity of the heme complex in benzene. The catalytic efficiency of the heme complex was enhanced more than ten times by adding histidine to the aqueous phase of W/O emulsions in the preparation process. The enhancement of peroxidase activity was observed only in a nonaqueous medium due to the increase of the effective concentration of histidine as an activator. In the present study, we propose a simple preparation method for an artificial heme enzyme which works in nonaqueous media. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 502–506, 1999. 相似文献
19.
Alexandre Di Paolo Valérie Duval André Matagne Christina Redfield 《Biomolecular NMR assignments》2010,4(1):111-114
Lysozyme from lambda bacteriophage (λ lysozyme) is an 18 kDa globular protein displaying some of the structural features common to all lysozymes; in particular, λ lysozyme consists of two structural domains connected by a helix, and has its catalytic residues located at the interface between these two domains. An interesting feature of λ lysozyme, when compared to the well-characterised hen egg-white lysozyme, is its lack of disulfide bridges; this makes λ lysozyme an interesting system for studies of protein folding. A comparison of the folding properties of λ lysozyme and hen lysozyme will provide important insights into the role that disulfide bonds play in the refolding pathway of the latter protein. Here we report the 1H, 13C and 15N backbone resonance assignments for λ lysozyme by heteronuclear multidimensional NMR spectroscopy. These assignments provide the starting point for detailed investigation of the refolding pathway using pulse-labelling hydrogen/deuterium exchange experiments monitored by NMR. 相似文献
20.
Of the various protein refolding methods, direct dilution is one of the simplest and easiest for scaling up the refolding process. However, it requires a large amount of refolding buffer, often utilizes a number of chemicals, and results in a low final protein concentration. In this report, we demonstrate that reduced dithiothreitol (DTTred), a carryover from denaturation, is a crucial and adverse factor in lysozyme refolding. Accordingly, we proposed a method of using high concentration of oxidized glutathione (GSSG) in the refolding buffer to eliminate excess DTTred and aid in the refolding of lysozyme. The efficiency of this method is 84%, which resulted in a high final refolded protein concentration of 1.5 g/l and required only a low dilution factor (4×). Furthermore, compared with the traditional 50× direct dilution (resulting in a similar yield of 74%), the low dilution factor required much less GSSG and other constituents. 相似文献