Molecularly imprinted polymer-assisted refolding of lysozyme

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.     

Mechanism of pH‐sensitive polymer‐assisted protein refolding and its application in TGF‐β1 and KGF‐2

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     

Evaluation of artificial chaperoning behavior of an insoluble cyclodextrin-rich copolymer: solid-phase assisted refolding of carbonic anhydrase

Insoluble beta-cyclodextrin (beta-CD) copolymers have been used for the refolding of thermally and/or chemically denatured carbonic anhydrase with refolding yield of 40% using 300 mg of the copolymer/ml refolding solution containing 0.042 mg/ml protein. In an attempt to enhance the refolding yield with lower quantities of the copolymer, a new beta-CD-rich copolymer with higher beta-CD content was synthesized. Regarding the need for rapid stripping of the detergent molecules from the detergent-protein complexes formed in the capture step of the technique (artificial chaperone-assisted refolding), experimental variables (e.g. copolymer and the protein contents) were optimized to improve the refolding yields along with depressing the aggregate formation. In addition, comparative studies using different ionic detergents and the copolymer were conducted to get a more comprehensive understanding of the detergent's tail length in the stripping step of the process. Our results indicated that under the optimal developed refolding environment, the denatured CA was refolded with a yield of 75% using only 5mg of the copolymer/1.2 ml refolding solution containing 0.0286 mg/ml protein. Taking into account the recycling potential of the copolymer, the new resin, with significant cost-cutting capability, is a suitable candidate for industrial applications.     

Refolding of denatured/reduced lysozyme at high concentrations by artificial molecular chaperone‐ion exchange chromatography

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     

Multi-block poloxamer surfactants suppress aggregation of denatured proteins

On the basis of elastic light scattering, we have compared the capacity of the multi-block, surfactant copolymers Poloxamer 108 (P108), Poloxamer 188 (P188), and Tetronic 1107 (T1107), of average molecular weight 4700, 8400, and 15,000, respectively, with that of polyethylene glycol (PEG, molecular weight 8000) to suppress aggregation of heat-denatured hen egg white lysozyme (HEWL) and bovine serum albumin (BSA). We also compared the capacity of P188 to that of PEG to suppress aggregation of carboxypeptidase A denatured in the presence of trifluoroethanol and to facilitate recovery of catalytic activity. In contrast to the multi-block copolymers, PEG had no effect in inhibiting aggregation of HEWL or of carboxypeptidase A with the recovery of catalytic activity. At very high polymer:protein ratios (>or=10:1), PEG increased aggregation of heat-denatured HEWL and BSA, consistent with its known properties to promote macromolecular crowding and crystallization of proteins. At a polymer:protein ratio of 2:1, the tetra-block copolymer T1107 was the most effective of the three surfactant copolymers, completely suppressing aggregation of heat-denatured HEWL. At a T1107:BSA ratio of 10:1, the poloxamer suppressed aggregation of heat-denatured BSA by 50% compared to that observed in the absence of the polymer. We showed that the extent of suppression of aggregation of heat-denatured proteins by multi-block surfactant copolymers is dependent on the size of the protein and the copolymer:protein molar ratio. We also concluded that at least one of the tertiary nitrogens in the ethylene-1,2-diamine structural core of the T1107 copolymer is protonated, and that this electrostatic factor underlies its capacity to suppress aggregation of denatured proteins more effectively than nonionic, multi-block poloxamers. These results indicate that amphiphilic, surfactant, multi-block copolymers are efficient as additives to suppress aggregation and to facilitate refolding of denatured proteins in solution. Because of these properties, multi-block, surfactant copolymers are suitable for application to a variety of biotechnological and biomedical problems in which refolding of denatured or misfolded proteins and suppression of aggregation are important objectives.     

A metal binding in the polypeptide chain improves the folding efficiency of a denatured and reduced protein

In order to examine the effect of a metal binding to the polypeptide chain on the aggregation of a protein in the refolding process, we prepared a mutant hen lysozyme possessing the same Ca(2+) binding site as in human alpha-lactalbumin by Escherichia coli expression system (Ser(-1) CaB lysozyme). In the presence of 2 mM CaCl(2), the refolding yield of Ser(-1) CaB lysozyme at a low protein concentration (25 microg/mL) was similar to that of the wild-type lysozyme (80%), but that at high protein concentration (200 microg/mL) decreased (15%) due to aggregation comparing to that of the wild-type lysozyme (45%). However, the refolding yield of Ser(-1) CaB lysozyme in the presence of 100 mM CaCl(2) even at a protein concentration of 200 microg/mL was 80% and was higher than that of the wild-type lysozyme. From analysis of chemical shift changes of the cross peaks in the backbone region of total correlated spectroscopy (TOCSY) spectra of a decapeptide possessing the same calcium binding site as in Ser(-1) CaB lysozyme in the presence of various concentrations of Ca(2+), it was suggested that the dissociation constant of Ca(2+)-peptide complex was estimated to be 20-36 mM. Moreover, the solubility of the denatured Ser(-1) CaB lysozyme in the presence of 100 mM CaCl(2) was higher than that in the presence of 2 mM CaCl(2) whereas the solubility of the denatured Ser(-1) lysozyme in the presence of 100 mM CaCl(2) was not higher than that in the presence of 2 mM CaCl(2). Therefore, it was concluded that the reduced lysozyme possessing the Ca(2+) binding site was efficiently folded in the presence of high concentration of Ca(2+) (100 mM) even at high protein concentration due to depression of aggregation by the binding of Ca(2+) to the polypeptide chain in Ser(-1) CaB lysozyme.     

Glutathione Ethylester, a Novel Protein Refolding Reagent, Enhances both the Efficiency of Refolding and Correct Disulfide Formation

Protein refolding constitutes a crucial process for recombinant proteins. We report here on the development of a multifunctional refolding additive, glutathione ethyl ester (GSHEE), prepared from a redox reagent glutathione and an amino acid ethyl ester, an aggregation suppressor. Compared to glutathione, GSHEE showed 3.2-fold higher efficiency for the refolding yield of hen egg lysozyme. More importantly, a low concentration of GSHEE is more effective for refolding than conventional additives, such as amino acid ethyl esters by two orders of magnitude. The high potency of GSHEE makes it a candidate for use as a refolding additive for use in conjunction with reduced and denatured proteins.     

Eudragit S-100对人转化生长因子β1（TGF-β1）复性的促进作用及其机制

本研究主要是考察一种对pH较为敏感的多聚化合物聚丙烯酸树脂Eudragit S-100是否对人转化生长因子β1（Transforwing growth factor,TGF-β1）复性具有促进作用. 将以包涵体形式存在TGF-β1进行变性,并将变性蛋白直接加入到含有不同浓度Eudragit的蛋白复性缓冲液中,采用MTT法、荧光分光光度法、圆二色谱以及高效液相色谱法等方法来比较分析不同浓度Eudragit S-100对变性TGF-β1的复性促进作用.实验结果表明,在Eudragit S-100作用下TGF-β1的复性产率比普通稀释复性法显著增高且最高达到53%,研究还表明Eudragit S-100的促进蛋白复性的作用是基于Eudragit S-100与TGF-β1发生了特异性的离子结合反应.通过这一反应,Eudragit S-100遮蔽了蛋白多肽间的疏水基团,有效的抑制了蛋白的聚集进而发挥其促复性功能.     

Mixed macromolecular crowding accelerates the oxidative refolding of reduced, denatured lysozyme: implications for protein folding in intracellular environments

The oxidative refolding of reduced, denatured hen egg white lysozyme in the presence of a mixed macromolecular crowding agent containing both bovine serum albumin (BSA) and polysaccharide has been studied from a physiological point of view. When the total concentration of the mixed crowding agent is 100 g/liter, in which the weight ratio of BSA to dextran 70 is 1:9, the refolding yield of lysozyme after refolding for 4 h under this condition increases 24% compared with that in the presence of BSA and 16% compared with dextran 70. A remarkable increase in the refolding yield of lysozyme by a mixed crowding agent containing BSA and Ficoll 70 is also observed. Further folding kinetics analyses show that these two mixed crowding agents accelerate the oxidative refolding of lysozyme remarkably, compared with single crowding agents. These results suggest that the stabilization effects of mixed macromolecular crowding agents are stronger than those of single polysaccharide crowding agents such as dextran 70 and Ficoll 70, whereas the excluded volume effects of mixed macromolecular crowding agents are weaker than those of single protein crowding agents such as BSA. Both the refolding yield and the rate of the oxidative refolding of lysozyme in these two mixed crowded solutions with suitable weight ratios are higher than those in single crowded solutions, indicating that mixed macromolecular crowding agents are more favorable to lysozyme folding and can be used to simulate the intracellular environments more accurately than single crowding agents do.     

蛋白质二硫键异构酶相关蛋白A的表达及性质的研究

克隆了Aspergillus niger T21中的蛋白质二硫键异构酶相关蛋白A（PRPA）基因,并将它插入pET23b表达载体。在E. coli中表达时,PRPA占菌体总蛋白的34%。经过超声破细胞、硫酸铵分级沉淀和离子交换层析获得了纯度大于90%的重组蛋白。PRPA有二硫键异构酶活性。在PRPA存在下,变性和还原的溶菌酶复性率和复性速度降低,电泳结果表明溶菌酶聚集增多。荧光结果表明PRPA表面有较多的疏水基团。     

Lysozyme reactivation using immobilized molecular chaperonin GroEL

The molecular chaperonin, GroEL, was immobilized to a porous matrix and used to reactivate denatured lysozyme. The maximum reactivation yield was obtained at 37°C and pH 6–8 and about 90% activity of the denatured lysozyme was restored under the conditions. The coupling density of GroEL had little effect on the chaperoning activity of GroEL up to 48 mg g^–1 gel. The immobilized GroEL was reusable, indicating the possibility of using it on a large scale for the refolding of proteins.     

Critical analysis of lysozyme refolding kinetics

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.     

Cycloamylose as an efficient artificial chaperone for protein refolding

High molecular weight cyclic alpha-1,4-glucan (referred to as cycloamylose) exhibited an artificial chaperone property toward three enzymes in different categories. The inclusion properties of cycloamylose effectively accommodated detergents, which keep the chemically denatured enzymes from aggregation, and promoted proper protein folding. Chemically denatured citrate synthase was refolded and completely recovered it's enzymatic activity after dilution with polyoxyethylenesorbitan buffer followed by cycloamylose treatment. The refolding was completed within 2 h, and the activity of the refolded citrate synthase was quite stable. Cycloamylose also promoted the refolding of denatured carbonic anhydrase B and denatured lysozyme of a reduced form.     

Initial protein concentration and residual denaturant concentration strongly affect the batch refolding of hen egg white lysozyme

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.     

Molecularly imprinted poly β-cyclodextrin polymer: Application in protein refolding

Regarding our previous report on refolding of alkaline phosphatase [Yazdanparast and Khodagholi, 2005 Arch. Biochem. Biophys] it was found that in spite of the anti-aggregatory effect of 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), a zwitteronic detergent, the recovered activity was almost the same as the recovered activity obtained through the unassisted approach. The low recovery yield is probably due to the bulky groups of the detergent that interfere with its entrance into the small cavity of the stripping agent, cyclodextrin, implying that the stripping of detergent molecules from the detergent–protein complexes plays a major role in successful refolding processes. To improve the efficiency of CHAPS stripping, we evaluated, for the first time, the stripping potential of a molecular imprinting polymer designed to replace β-CD. In this approach, CHAPS was used as the template and the refolding of GuHCl denatured alkaline phosphatase was studied. Our results indicated that under the optimally developed refolding environment and similar to stripping by soluble β-CD, a refolding yield of 79% was obtained for denatured alkaline phosphatase using 20 mg/ml of the molecularly imprinted poly (β-CD) polymer. The major advantage of the new stripping agent, besides of its recycling option and ease of separation from the finished product, is its high potential of preventing aggregate formation. Based on these results, it seems that the new stripping strategy can constitute an ideal approach for refolding of proteins at much lower industrial costs compared to stripping with soluble β-cyclodextrin.     

Continuous refolding of lysozyme with fed-batch addition of denatured protein solution

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.     

Statistical optimization and multiple objective programming of lysozyme refolding catalyzed by recombinant DsbA in vitro

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.     

Binding of a denatured heme protein and ATP to erythroid spectrin

Spectrin is a large, worm-like cytoskeletal protein that is abundant in all cell types. The denatured heme enzyme, horseradish peroxidase showed significant decrease in the reactivation yield, after 30 min of refolding, in presence of increasing concentrations of spectrin from that in the absence. This indicated that spectrin could bind denatured HRP and inhibit their refolding. In presence of 1 mM ATP and 10 mM MgCl(2) the spectrin binding of denatured HRP is abolished. This activity of decreasing the reactivation yield was found to be ATP-dependent and the denatured enzyme after 30 min refolding in the presence of spectrin, pretreated with Mg/ATP, showed about 40% increase in the reactivation yield compared to the same in absence of spectrin. Fluorescence spectroscopic studies indicated binding of ATP to native spectrin showing concentration-dependent quenching of tryptophan fluorescence by ATP. The apparent dissociation constant of binding of ATP to spectrin was estimated to be 1.1 mM. A high affinity binding of spectrin with denatured HRP has been characterized (K(d) = 16 nM). Since these properties are similar to those of established molecular chaperone proteins, these data indicate that spectrin might have a chaperone-like function in erythrocytes.     

固定化分子伴侣GroE促进变性溶菌酶复性的研究

利用重组大肠杆菌表达制备了分子伴侣GroE(GroEL和GroES),研究了GroE以及GroEL辅助变性溶菌酶复性的作用。结果表明,不仅游离GroEL单独作用可使溶菌酶复性收率达到90%以上,而且固定化GroEL亦可有效地促进蛋白质复性,最佳复性温度为37℃,最佳pH值范围为6～8,复性酶的活性收率在85%以上。另外,固定化GroEL可反复回收利用,表明固定化GroEL有可能在实际生物下游过程中得到应用。     

Imidazole as a catalyst for in vitro refolding of enhanced green fluorescent protein

Imidazole is a reagent widely used in protein purifying processes. Here, we reveal a novel chaperone-like activity for imidazole using enhanced green fluorescent protein (EGFP) as a model protein. Experimental results showed that imidazole acted as an effective catalyst for refolding of the chemically denatured EGFP and suppressor for the heat-induced aggregation of EGFP. The refolding kinetics was determined in real time. Both the recovering yield and refolding rate of denatured EGFP in the presence of imidazole were increased. The studies on elucidating the mechanism show that imidazole may catalyze the prolyl cis/trans isomerization and the possible mechanism was discussed. To our knowledge, there are no data on the effect of imidazole on protein folding. Considering the prolyl isomerization is the rate-limited step for refolding of most proteins and aggregation is a universal serious problem for biotechnology, imidazole thus represents a previous unknown type of protein-folding catalyst.