The dodecameric vanadium-dependent haloperoxidase from the marine algae Corallina officinalis: cloning, expression, and refolding of the recombinant enzyme

The dodecameric vanadium-dependent bromoperoxidase from Corallina officinalis has been cloned and over-expressed in Escherichia coli. However, the enzyme was found to be predominantly in the form of inclusion bodies. This protein presents a challenging target for refolding, both due to the size (768 kDa) and quaternary structure (12 × 64 kDa). Successful refolding conditions have been established which result in an increase in the final yield of active bromoperoxidase from 0.5 mg to 40 mg per litre of culture. The refolded protein has been characterised and compared to the native enzyme and was shown to be stable at temperatures of 80 °C, over a pH range 5.5–10 and in organic solvents such as ethanol, acetonitrile, methanol, and acetone. The novel refolding approach reported in this paper opens up the full potential of this versatile enzyme for use in large scale biotransformation studies.     

The effect of temperature on protein refolding at high pressure: Enhanced green fluorescent protein as a model

The application of high hydrostatic pressure (HHP) impairs electrostatic and hydrophobic intermolecular interactions, promoting the dissociation of recombinant inclusion bodies (IBs) under mild conditions that favor subsequent protein refolding. We demonstrated that IBs of a mutant version of green fluorescent protein (eGFP F64L/S65T), produced at 37 °C, present native-like secondary and tertiary structures that are progressively lost with an increase in bacterial cultivation temperature. The IBs produced at 37 °C are more efficiently dissociated at 2.4 kbar than those produced at 47 °C, yielding 25 times more soluble, functional eGFP after the lower pressure (0.69 kbar) refolding step. The association of a negative temperature (−9 °C) with HHP enhances the efficiency of solubilization of IBs and of eGFP refolding. The rate of refolding of eGFP as temperature increases from 10 °C to 50 °C is proportional to the temperature, and a higher yield was obtained at 20 °C. High level refolding yield (92%) was obtained by adjusting the temperatures of expression of IBs (37 °C), of their dissociation at HHP (−9 °C) and of eGFP refolding (20 °C). Our data highlight new prospects for the refolding of proteins, a process of fundamental interest in modern biotechnology.     

Thermal-aggregation suppression of proteins by a structured PEG analogue: Importance of denaturation temperature for effective aggregation suppression

Development of protein stabilizing reagents, that suppress aggregation and assist refolding, is an important issue in biochemical technology related with the synthesis and preservation of therapeutic or other functional proteins. In the precedent research, we have developed a structured poly(ethylene glycol) (PEG) analogue with triangular geometry, which turns into a dehydrated state above ca. 60 °C. Focusing on this rather lower dehydration temperature than that of conventional linear PEGs, a capability of the triangle-PEG to stabilize proteins under thermal stimuli was studied for citrate synthase, carbonic anhydrase, lysozyme and phospholipase. Variable temperature high-tension voltage and circular dichroism spectroscopic studies on the mixtures of these proteins and the triangle-PEG showed that the triangle-PEG stabilizes carbonic anhydrase, lysozyme and phospholipase that exhibit denaturation temperatures higher than 60 °C, while substantially no stabilization was observed for citrate synthase that denatures below 60 °C. Hence, the dehydrated triangle-PEG likely interacts with partially unfolded proteins through the hydrophobic interaction to suppress protein aggregation.     

Refolding by High Pressure of a Toxin Containing Seven Disulfide Bonds: Bothropstoxin-1 from <Emphasis Type="Italic">Bothrops jararacussu</Emphasis>

Aggregation is a serious obstacle for recovery of biologically active heterologous proteins from inclusion bodies (IBs) produced by recombinant bacteria. E. coli transformed with a vector containing the cDNA for Bothropstoxin-1 (BthTx-1) expressed the recombinant product as IBs. In order to obtain the native toxin, insoluble and aggregated protein was refolded using high hydrostatic pressure (HHP). IBs were dissolved and refolded (2 kbar, 16 h), and the effects of protein concentration, as well as changes in ratio and concentration of oxido-shuffling reagents, guanidine hydrochloride (GdnHCl), and pH in the refolding buffer, were assayed. A 32% yield (7.6 mg per liter of bacterial culture) in refolding of the native BthTx-1 was obtained using optimal conditions of the refolding buffer (Tris–HCl buffer, pH 7.5, containing 3 mM of a 2:3 ratio of GSH/GSSG, and 1 M GdnHCl). Scanning electron microscopy (SEM) showed that that disaggregation of part of IBs particles occurred upon compression and that the morphology of the remaining IBs, spherical particles, was not substantially altered. Dose-dependent cytotoxic activity of high-pressure refolded BthTx-1 was shown in C2C12 muscle cells.     

Refolding of recombinant human interferon gamma inclusion bodies in vitro assisted by colloidal thermo-sensitive poly(N-isopropylacrylamide) brushes grafted onto the surface of uniform polystyrene cores

Recombinant human interferon gamma (rhIFN-γ) is a protein with great potential for clinical therapy, but rhIFN-γ expressed in Escherichia coli is usually in the form of insoluble inclusion bodies which should be refolded in vitro. A novel type of hairy particles (PNIPAM-grafted-PS) consisted of submicron polystyrene cores and brushes of thermo-sensitive poly(N-isopropylacrylamide) grafted onto the cores was prepared and then applied to assist the refolding of rhIFN-γ in vitro. Two kinds of PNIPAM-grafted-PS particles with different thickness of brush layer (55 nm and 110 nm) were synthesized, which were spherical shape with good dispersion properties and the LCST was about 33 °C. The effect of thickness of brush layer, particle concentration and temperature on the refolding process was investigated, it was shown that particles with larger thickness of brush layer were more effective and the final rhIFN-γ activity could be up to more than 21 times of that in dilution refolding when initial rhIFN-γ concentration was 50 μg/mL. The optimal refolding condition was the concentration ratio of particle to rhIFN-γ 1:1 and refolding temperature of 15 °C. All results above demonstrated that PNIPAM-grafted-PS particles could assist rhIFN-γ refolding which presented an alternative way to facilitate recombinant protein refolding in vitro.     

Kinetically robust monomeric protein from a hyperthermophile

Equilibrium and kinetic studies were carried out under denaturation conditions to clarify the energetic features of the high stability of a monomeric protein, ribonuclease HII, from a hyperthermophile, Thermococcus kodakaraensis (Tk-RNase HII). Guanidine hydrochloride (GdnHCl)-induced unfolding and refolding were measured with circular dichroism at 220 nm, and heat-induced denaturation was studied with differential scanning calorimetry. Both GdnHCl- and heat-induced denaturation are very reversible. It was difficult to obtain the equilibrated unfolding curve of Tk-RNase HII below 40 degrees C, because of the remarkably slow unfolding. The two-state unfolding and refolding reactions attained equilibrium at 50 degrees C after 2 weeks. The Gibbs energy change of GdnHCl-induced unfolding (DeltaG(H(2)O)) at 50 degrees C was 43.6 kJ mol(-1). The denaturation temperature in the DSC measurement shifted as a function of the scan rate; the denaturation temperature at a scan rate of 90 degrees C h(-1) was higher than at a scan rate of 5 degrees C h(-1). The unfolding and refolding kinetics of Tk-RNase HII were approximated as a first-order reaction. The ln k(u) and ln k(r) values depended linearly on the denaturant concentration between 10 and 50 degrees C. The DeltaG(H(2)O) value obtained from the rate constant in water using the two-state model at 50 degrees C, 44.5 kJ mol(-1), was coincident with that from the equilibrium study, 43.6 kJ mol(-1), suggesting the two-state folding of Tk-RNase HII. The values for the rate constant in water of the unfolding for Tk-RNase HII were much smaller than those of E. coli RNase HI and Thermus thermophilus RNase HI, which has a denaturation temperature similar to that of Tk-RNase HII. In contrast, little difference was observed in the refolding rates among these proteins. These results indicate that the stabilization mechanism of monomeric protein from a hyperthermophile, Tk-RNase HII, with reversible two-state folding is characterized by remarkably slow unfolding.     

Reversible and non-reversible thermal denaturation of lysozyme with varying pH at low ionic strength

DSC analysis has been used to quantify the reversibility of unfolding following thermal denaturation of lysozyme. Since the temperature at which protein unfolding occurs, T_m, varies with different solution conditions, the effect on the melting temperature and the degree of refolding after thermal denaturation in low ionic strength sodium phosphate buffers (5–1000 mM) over a range of pH (5–9) in the presence/absence of disaccharides is examined. This study compares the enthalpies of unfolding during successive heating cycles to quantify reversibility following thermal denaturation. The disaccharides, trehalose and maltose were used to assess if the disaccharide induced increase in T_m is reflected in the reversibility of thermally induced denaturation. There was extensive overlap between the T_m values where non-reversible and reversible thermal denaturation occurred. Indeed, for pH 6, at the highest and lowest T_m, no refolding was observed whereas refolding was observed for intermediate values, but with similar T_m values having different proportions of refolded protein. We established a method to measure the degree of reversible unfolding following thermal denaturation and hence indirectly, the degree to which protein is lost to irreversible aggregation, and show that solution conditions which increase melt transition temperatures do not automatically confer an increase in reversibility. This type of analysis may prove useful in assessing the stability of proteins in both the biopharmaceutical and food industries.     

α-Helical burst on the folding pathway of FHA domains from Rad53 and Ki67

We investigated refolding processes of β-sheeted protein FHA domains (FHA1 domain of Rad53 and Ki67 FHA domain) by cryo-stopped-flow (SF) method combined with far-ultraviolet (far-UV) circular dichroism (CD, the average secondary structure content) and small angle X-ray scattering (SAXS, measuring the radius of gyration). In case of FHA1 domain of Rad53, no detectable time course was observed except the initial burst on its refolding process at 4 °C, suggesting that the FHA1 domain of Rad53 was already refolded to its native state within the dead time of the SF apparatus and the rate of the refolding is too fast to be observed at this temperature. In contrast, there was an observable α-helical burst at −15 °C and −20 °C in the presence of 45% ethylene glycol (EGOH) by CD-SF. Besides, the radius of gyration (Rg) of the burst phase intermediate at −20 °C shows the intermediate is already compact, and the compaction process was accompanied with the decrease of α-helical content at the same temperature.     

Distinct Characteristics of Single Starch-Binding Domain SBD1 Derived from Tandem Domains SBD1-SBD2 of Halophilic <Emphasis Type="Italic">Kocuria varians</Emphasis> Alpha-Amylase

Kocuria varians alpha-amylase contains tandem starch-binding domains SBD1-SBD2 (SBD12) that possess typical halophilic characteristics. Recombinant tandem domains SBD12 and single domain SBD1, both with amino-terminal hexa-His tag, were expressed in and purified to homogeneity from Escherichia coli. The circular dichroism (CD) spectrum of His-SBD12 was characterized by a positive peak at 233 nm ascribed to the aromatic stacking. Although the signal occurred in the far UV region, it is an indication of tertiary structure folding. CD spectrum of single domain His-SBD1 exhibited the same peak position, signal intensity and spectral shape as those of His-SBD12, suggesting that the aromatic stacking must occur within the domain, and that two SBD domains in SBD12 and SBD1 has a similar folded structure. This structural observation was consistent with the biological activity that His-SBD1 showed binding activity against raw starch granules and amylose resin with 70–80% efficiency compared with binding of equimolar His-SBD12. Although the thermal unfolding rate of SBD12 and SBD1 were similar, the refolding rates of SBD12 and SBD1 from thermal melting were greatly different: His-SBD12 refolded slowly (T_1/2 = ~84 min), while refolding of single domain His-SBD1 was found to be 20-fold faster (T_1/2 = 4.2 min). The possible mechanism of this large difference in refolding rate was discussed. Maltose at 20 mM showed 5–6 °C increase in thermal melting of both His-SBD12 and His-SBD1, while its effects on the time course of unfolding and refolding were insignificant.     

Efficient lysozyme refolding at a high final concentration and a low dilution factor

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 (DTT^red), 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 DTT^red 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.     

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.     

Ternary System of Solution Additives with Arginine and Salt for Refolding of Beta-Galactosidase

l-Arginine hydrochloride (Arg HCl) has been used for protein refolding as a universal aggregation suppressor for monomeric proteins. This paper presents an investigation of the refolding of tetrameric beta-galactosidase (β-gal) using Arg HCl and other salts. In a binary system using only Arg HCl, the refolding yield of β-gal increased with increasing concentration up to 0.2 M. However, the refolding yield sharply decreased above this concentration, reaching the level below the control yield of 5% at 0.5 M and near zero above 0.75 M, an observation unexpected from monomeric proteins. In a ternary system using both 0.2 M Arg HCl and another salt, the refolding yield increased up to 1.5-fold higher than that in the binary system. These data indicate that aggregation suppressive effects of protein increase with Arg HCl concentration, but also are deleterious to self-association of the protein. This dual nature of Arg HCl effects may have to be taken into account in its application for refolding of oligomeric proteins.     

Designing a Highly Efficient Chemical Chaperone System Using Chitosan-Coated Alginate

In the present work we prepared chitosan-coated alginate beads, to use as a chemical chaperone based on the electrostatic interaction between the carboxylate groups of alginate and the ammonium groups of chitosan. This procedure was an attempt for designing a highly efficient chemical chaperone to improve protein stability and refolding. Based on enzyme recovered activity, turbidity, far-UV CD and fluorescence data, alkaline phosphatase can be stabilized and refolded to a higher degree in the presence of alginate capsules compared with unassisted form and was further improved by including chitosan. Finally the maximum yield was obtained when the refolding process was achieved under a well worked out temperature program: incubation of the captured-enzyme for 20 min at 4 °C followed by overnight incubation at 22 °C, which showed that aggregation is a major limitation to refolding.     

Impact of disulfide bonds on the folding and refolding capability of a novel thermostable GH45 cellulase

A new cellulase (TaCel45) of glycoside hydrolase family 45 was identified in the thermophilic fungus Thielavia arenaria XZ7 and was successfully expressed in Pichia pastoris. The specific activities of TaCel45 towards lichenin, sodium carboxymethylcellulose (CMC-Na), and barley β-glucan were 769, 498, and 486 U/mg protein, respectively, which are higher than the values for all other reported GH45 cellulases. TaCel45 had maximum activity at pH 5.0–6.0 and 60–65 °C with barley β-glucan and CMC-Na as substrates and had a melting temperature (T_m) of 68.4 °C. However, TaCel45 exhibited extraordinary thermostability at 90 and 100 °C, retaining more than 70 and 45% of its activity after a 1-h incubation, respectively. Seven mutants (C11S, C12S, C16S, C31S, C171S, C193S, and C203S) were then constructed to investigate the effects of each disulfide bond on the structure, activity, and stability of TaCel45. As a result, six disulfide bonds (C11-C136, C16-C87, C31-C57, C88-C203, C90-C193, and C160-Cy171) were found to be indispensable for the folding, secretion, and activity of TaCel45, while C12-C48 was critical for thermal adaptation and refolding. The mutant C12S showed decreased optimal temperature and T_m values of 50 and 60.2 °C, respectively, and retained less than 50% of the thermal refolding ability of the wild type. Overall, this study demonstrated that disulfide bonds play a vital role in the folding and refolding capability and thermostability of this GH45 cellulase.

     

Serum amyloid A 2.2 refolds into a octameric oligomer that slowly converts to a more stable hexamer

Serum amyloid A (SAA) is an inflammatory protein predominantly bound to high-density lipoprotein in plasma and presumed to play various biological and pathological roles. We previously found that the murine isoform SAA2.2 exists in aqueous solution as a marginally stable hexamer at 4–20 °C, but becomes an intrinsically disordered protein at 37 °C. Here we show that when urea-denatured SAA2.2 is dialyzed into buffer (pH 8.0, 4 °C), it refolds mostly into an octameric species. The octamer transitions to the hexameric structure upon incubation from days to weeks at 4 °C, depending on the SAA2.2 concentration. Thermal denaturation of the octamer and hexamer monitored by circular dichroism showed that the octamer is ∼10 °C less stable, with a denaturation mid point of ∼22 °C. Thus, SAA2.2 becomes kinetically trapped by refolding into a less stable, but more kinetically accessible octameric species. The ability of SAA2.2 to form different oligomeric species in vitro along with its marginal stability, suggest that the structure of SAA might be modulated in vivo to form different biologically relevant species.     

Purification of the Caenorhabditis elegans Transposase Tc1A Refolded during Gel Filtration Chromatography

Full-length recombinant transposase Tc1A from Caenorhabditis elegans (343 amino acids) expressed in Escherichia coli BL21 in inclusion bodies has been purified in a high yield in a soluble form. The procedure includes denaturation of the inclusion bodies followed by refolding of the Tc1A protein by gel filtration. This last step is absolutely crucial to give a high yield of soluble and active protein since it allows the physical separation of the aggregates from intermediates that give rise to correctly refolded protein. This step is very sensitive to the concentration of protein. Good yields of refolded protein are obtained by refolding 2 to 12 mg of denatured protein. The other purification steps involve the initial use of gel filtration under denaturing conditions and a final step of ion-exchange chromatography. Biological activity of the purified protein was confirmed in an in vitro transposon excision assay and its DNA-binding capacity by UV crosslinking. This new Tc1A purification procedure gives a yield of 12–16 mg/liter E. coli culture, in a form suitable for crystallization studies.     

Salt dependent resistance against chemical denaturation of alkaline protease from a newly isolated haloalkaliphilic Bacillus sp

Only few enzymes from haloalkaliphiles are biochemically characterized for their kinetic behaviour and stability. In view of this realization, an alkaline protease from Bacillus sp. AH-6, displaying salt-dependent resistance against chemical denaturation by Urea and Guanidium hydrochloride was investigated for denaturation and in vitro protein folding. The crude enzyme was highly resistant against urea (8 M) denaturation up to 72 h; however, on purification, it turned sensitive and got denatured within 2 h. Interestingly, the purified enzyme regained the resistance in the presence of NaCl. Effective refolding of the purified enzyme was achieved with glycerol; however, other approaches such as lower protein concentrations, rapid dilution and slow removal of the denaturant did not further add to refolding. The results are important from the viewpoint that only few enzymes from haloalkaliphilic bacteria are characterized. Since the resistance against chemical denaturation is a rare phenomenon, the findings would enrich the knowledge on protein stability and denaturation. Besides, such biocatalysts would definitely have novel applications under harsh chemical environments.     

An efficient in vitro refolding of recombinant bacterial laccase in Escherichia coli

Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are important multicopper enzymes that are used in many biotechnological processes. A recombinant form of laccase from Bacillus sp. HR03 was overexpressed in Escherichia coli BL-21(DE3). Inclusion body (IB) formation happens quite often during recombinant protein production. Hence, developing a protocol for efficient refolding of proteins from inclusion bodies to provide large amounts of active protein could be advantageous for structural and functional studies. Here, we have tried to find an efficient method of refolding for this bacterial enzyme. Solubilization of inclusion bodies was carried out in phosphate buffer pH 7, containing 8 M urea and 4 mM β-mercaptoethanol and refolding was performed using the dilution method. The effect of different additives was investigated on the refolding procedure of denaturated laccase. Mix buffer (phosphate buffer and citrate buffer, 100 mM) containing 4 mM ZnSO₄ and 100 mM sorbitol was selected as an optimized refolding buffer. Also Kinetic parameters of soluble and refolded laccase were analyzed.     

Unfolding and refolding of leucoagglutinin (PHA-L), an oligomeric lectin from kidney beans (Phaseolus vulgaris)

The unfolding and refolding of Phaseolus vulgaris Leucoagglutinin, a homotetrameric legume lectin, was studied at pH 2.5 and 7.2 using fluorescence, far- and near-UV circular dichroism (CD) spectroscopy, 8-anilino-1-naphthalene sulfonate (ANS) binding and FPLC techniques. This protein was found to refold even at pH 2.5 and also exhibited high refolding yield around 60% at pH 2.5 and 85% at pH 7.2. The refolding at pH 2.5 takes place with the formation of a dimeric intermediate. Although the hydrodynamic radius of the completely renatured protein and the dimer at pH 2.5 was found to be same, the ANS binding as well as far-UV CD spectra of the two were different. The denaturation kinetics at pH 2.5 followed single exponential pattern with the rate of denaturation being independent of protein concentration. The renaturation kinetics on the other hand was dependent on the protein concentration providing further evidence of an intermediate state during refolding. From these experiments the folding pathway of the protein at pH 2.5 was proposed.     

Construction,Expression and Refolding of a Bifunctional Fusion Protein Consisting of C-Terminal 12-Residue of Hirudin-PA and Reteplase

To obtain a bifunctional protein simultaneously showing bioactivity of anticoagulant and fibrinolytic for use in the treatment of thrombotic diseases, we constructed a fusion protein (HV12p–rPA) containing C-terminal 12-residue of hirudin-PA (HV12p) and reteplase (rPA). The fusion protein, in which HV12p was linked to rPA via Gly–Gly–Gly, was successfully expressed in an inactive form of inclusion bodies in Escherichia coli. HV12p–rPA was identified by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The expression level of HV12p–rPA was optimized by an orthogonal method and finally enhanced from 12 % to approximate 30 %. We also deeply investigated the condition of renaturation of HV12p–rPA, and the inactive protein was partly renatured through various conditions. The refolding efficacy of HV12p–rPA estimated by the recovery of fibrinolytic activity varied from 0.03 % to 16.6 % and the anticoagulant activity fluctuated in the range from 41 to 2,297 ATU/mg. Bioassays indicated that the resulted fusion protein, as expected, exhibited both fibrinolytic and anticoagulant activities. These works laid a foundation for further characterization of HV12p–rPA.