Comparative analysis of refolding of chemically denatured beta-lactoglobulin types A and B using the dilution additive mode

The kinetic refolding of beta-lactoglobulin (BLG), types A and B, by beta-cyclodextrin, glucose and sorbitol has been investigated in aqueous solution using fluorescence, far UV-CD and UV-spectrophotometric techniques. A new Pd-complex has been used to denature the protein. CD and fluorescence studies indicated that when incubated with sugar, the denatured BLG is refolded into the native-like structure through the dilution additive mode resulting in a higher yield of active protein than without sugar. CD studies show that these sugars can induce a non-native alpha-helical structure in denatured BLG-A and -B, then aid in the refolding of the protein. Based on the present study, these sugars have a different effect on BLG-A than BLG-B because of their differences in protein thermal stability. BLG-A has a higher thermal stability than BLG-B due to differences in the amino acid sequences.     

Artificial chaperone-assisted refolding of chemically denatured alpha-amylase

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.     

Refolding of partially and fully denatured lysozymes

Lysozyme refolding with high yields sometimes results from incomplete denaturation. Dithiothreitol (DTT) is a reductant commonly used to reduce and unfold disulfide-stabilized lysozymes. Through the use of fluorescence spectroscopy to access the extent of denaturation, we found that the rate and extent of denaturation highly depended on the concentration of DTT. Further, the denaturation exhibited a two-phase transition at a high DTT concentration with DTT at >100 mM and long denaturation time (>24 h) being needed for complete denaturation. A low DTT concentration and a short denaturation time resulted in fast refolding with high activity recovery, while a high DTT concentration and a long denaturation time resulted in slow refolding with low activity recovery. Hence, the renaturation of disulfide-containing lysozyme was highly affected by the extent of denaturation.     

Refolding intermediate of guanidine hydrochloride denatured aminoacylase

The refolding of aminoacylase denatured in 6M guanidine hydrochloride (GdnHCl) has been studied by measuring enzyme activity, fluorescence emission spectra, ANS fluorescence spectra and far-UV circular dichroism spectra. The results showed that GdnHCl-denatured aminoacylase could be refolded and reactivated by dilution. A refolding intermediate was observed for low concentrations of GdnHCl (between 0.5 and 1.2M). This refolding intermediate was characterized by an increased fluorescence emission intensity, a blue-shifted emission maximum, and by increased binding of the fluorescence probe 8-anilino-1-naphthalenesulfonate (ANS). The secondary structure of the intermediate was similar to that of the native enzyme, and was therefore quite similar to the molten globule state often found in the protein folding pathway. Combined with the previous evidence of existence of an intermediate during unfolding process, we therefore proposed that the unfolding and refolding of aminoacylase might share the same pathway. A comparison of the Apo-enzyme and Holo-enzyme showed that there was little effect of the zinc ion on the refolding of the aminoacylase. Our study, the first successful report of the refolding of this metalloenzyme, also showed that lowering the concentration and the temperature of the enzyme improved the refolding rate of aminoacylase. The system therefore provides a useful model to study the refolding of proteins with prosthetic groups.     

Refolding of denatured thioredoxin observed by size-exclusion chromatography

Molecular sieve chromatography can resolve interactive systems into populations having different effective hydrodynamic volumes. In this report, the advantages of such resolution to protein folding are illustrated by using moderate pressure to decrease analysis time and lowered temperature to slow down the kinetics of conformational change. A 300-mm Bio-Sil TSK-125 size-exclusion column was equilibrated with a series of different concentrations of guanidine hydrochloride at 2 degrees C in 50 mM phosphate buffer, pH 7.0. Samples of native Escherichia coli thioredoxin, denatured thioredoxin, or thioredoxin equilibrated with the column solvent were injected, and the effluent was monitored at 220 nm. Injection of equilibrated protein samples defined three denaturant concentration zones identical with those observed by spectral measurements: the native base-line zone where only compact protein is observed in the effluent profile; the transition zone in which both compact and denatured forms are observed in slow exchange; and the denatured base-line zone in which only denatured protein is observed. Unfolding was observed by injection of native protein into columns having isocratic denaturant concentrations in the transition and denatured base-line zones. Effluent profiles indicated a dynamic conversion of compact to denatured protein with a time constant which appeared to decrease markedly with increasing denaturant concentration. Refolding was observed by injection of denatured protein into columns having isocratic concentrations in the transition and native base-line zones. As the denaturant concentration was decreased, the effluent profiles evidenced a persistent slow conversion of denatured to compact protein which was suddenly accelerated about midway in the native base-line zone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Refolding of denatured ribonuclease observed by size exclusion chromatography

The unfolding and refolding of pancreatic ribonuclease have been observed by absorbance, fluorescence, and size exclusion chromatographic measurements in solutions of guanidinium chloride continuously maintained at pH 6.0 and 4 degrees C. The spectral measurements were fitted with a minimal number of kinetic phases while the chromatographic measurements were simulated from an explicit mechanism. All of the measurements are consistent with a minimal mechanism involving seven components. The folded components include the native protein and two transiently stable intermediates each having the same hydrodynamic volume. The intermediate having all native peptide isomers has an unfolding midpoint in 3.8 M denaturant while the intermediate having one nonnative peptide isomer has an unfolding midpoint in 1.3 M denaturant. The unfolded protein is distributed among four components having the same hydrodynamic volume but differing peptide isomers. At equilibrium, 10% of the denatured protein has all native isomers, 60% has one nonnative isomer, 5% has a different nonnative isomer, and 25% has both nonnative isomers. In low denaturant concentrations, the dominant component with one nonnative isomer can refold to transiently populate the compact intermediate with the same nonnative isomer.     

Refolding of denatured/reduced lysozyme at high concentration with diafiltration

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.     

Refolding of denatured lysozyme by water-in-oil microemulsions of sucrose fatty acid esters

Water-in-oil (w/o) microemulsion of sucrose fatty acid ester was used to renature denatured hen egg white lysozyme without aggregation. After lysozyme was denatured in 5 M guanidine hydrochloride for 24 h, the resultant denatured lysozyme was held in the microemulsion, overnight at 25°C. Renatured lysozyme was transferred from the microemulsion phase to the recovery aqueous phase by conventional liquid-liquid extraction. The enzymatic activity of the recovered lysozyme was 93%.     

Refolding of reduced, denatured trypsinogen and trypsin immobilized on Agarose beads.

The reoxidation of fully reduced and denatured bovine trypsinogen and the regeneration of the native structure can be accomplished if the protein is initially attached to Agarose beads. Reoxidation was performed under aerobic conditions, in the presence of mercaptoethanol and dehydroascorbate or with a mixture of reduced and oxidized glutathione. In 24 hours, the yields of regenerated trypsinogen were 60 to 70% with 0.2 to 0.6 mg of protein bound/ml of gel but 30% or less if greater than 1.7 mg of protein were bound. Rapid reoxidation, with dehydroascorbate as catalyst, gave molecules which could not be converted to active trypsin. However, if the incorrectly folded structures were placed in a mixture of reduced and oxidized glutathione, the molecules underwent disulfide interchange and could continue to refold. The rapidly reoxidized molecules regained their native structure with the same rate and to the same extent as they did initially in the absence of rapid reoxidation. Therefore, the rate-limiting step in the refolding of trypsinogen was disulfide interchange. The regenerated Agarose-bound trypsinogen displayed the usual properties of the native molecule in (a) its conversion to active trypsin by a process of limited proteolysis, (b) the kinetic constants of the activated product toward typical trypsin substrates, and (c) the limited cleavage of 1 disulfide bond with sodium borohydride. Refoldind of immobilized trypsin was also observed with an overall yield of 50%. Trypsin can fold spontaneously to its native structure even though it lacks the NH2-terminal hexapeptide of its precursor.     

Refolding of a denatured α-chymotrypsin and its smart bioconjugate by three-phase partitioning

-Chymotrypsin inactivated with 8 M urea and 100 mM dithiothreitol could be completely reactivated by subjecting it to three-phase partitioning (TPP). TPP consisted of adding 30% w/v ammonium sulfate and t-butanol (volume equivalent to aqueous solution of denatured -chymotrypsin) at 25°C. The activated -chymotrypsin was recovered as an interfacial precipitate between the upper organic and lower aqueous phase. It was found that this could be extended to a thermally inactivated smart bioconjugate of -chymotrypsin with Eudragit S-100 (a reversibly soluble-insoluble methmethacrylate). The thermally inactivated bioconjugate had to be further subjected to urea and dithiothreitol before refolding by three-phase partitioning. Ninety per cent of the activity of the bioconjugate could be recovered. The free enzyme and its bioconjugate which lost activity in the presence of 90% dioxane recovered 94 and 90% of their activities, respectively, by employing TPP. The refolded free enzyme and its bioconjugate were evaluated in terms of V _max/K _m and their fluorescence emission spectra.     

Refolding from denatured inclusion bodies,purification to homogeneity and simplified assay of MGDG synthases from land plants

In plant cells, the synthesis of monogalactosyldiacylglycerol (MGDG) is catalyzed within plastid envelope membranes by MGD proteins. MGDG synthesis was also reported in apicomplexan parasites, a phylum of protists harbouring a plastid that proved essential for the parasite survival. MGD activity is therefore a potent target for herbicidal and anti-parasitic molecules. In this study, we describe a detailed in vitro refolding protocol for denatured recombinant MGD accumulated in inclusion bodies from transformed Escherichia coli. The refolding process was dependent on CHAPS detergent and lipids, such as diacylglycerol and phosphatidylglycerol, as well as bivalent metals. Owing to this refolding procedure, the recombinant MGD protein from spinach was purified to homogeneity, allowing a definite characterization of its non-processivity and an investigation of its dimerization using cross-linking reagents. Additionally, using the portion of recombinant enzyme that accumulates in an active form in bacterial membranes, we developed a miniature assay for high-throughput screening for inhibitors.     

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     

1H-NMR relaxation studies of native and thermally denatured lysozyme solutions: an isotope dilution experiment

1H-NMR relaxation times are reported for native and thermally denatured lysozyme aqueous solutions measured as the function of the proton mole fraction in the sample. A two-exponential character of proton longitudinal relaxation function was observed for native lysozyme solutions: the fast component was attributed to the non-exchangeable protein protons, the slow one to water protons. Purely exponential decay of longitudinal magnetization was observed for the thermally denatured samples. This has been explained in terms of a fast spin exchange model. The contributions of the protein protons to the water proton relaxation rate in native and thermally denatured samples were determined, too.     

Thioredoxin-catalyzed Refolding of Recombinant Protein: Refolding of Human Pro-urokinase

We have isolated four independent genomic DNA fragments encoding a rice storage protein, glutelin, by using its cDNA as a probe. Restriction mapping and sequencing analyses showed that all four clones encode type II mRNA of glutelin and have very similar nucleotide sequences. However, several differences were found among the four clones with respect to the nucleotide sequences of their coding and 5′-upstream regions, some of which confirmed corresponding changes in the amino acid sequences of the encoded proteins. These results indicate that the genes encoding type II mRNA species of glutelin constitute a multi-gene family of closely related members.