Oxidative refolding chromatography: folding of the scorpion toxin Cn5

We have made an immobilized and reusable molecular chaperone system for oxidative refolding chromatography. Its three components-GroEL minichaperone (191-345), which can prevent protein aggregation; DsbA, which catalyzes the shuffling and oxidative formation of disulfide bonds; and peptidyl-prolyl isomerase-were immobilized on an agarose gel. The gel was applied to the refolding of denatured and reduced scorpion toxin Cn5. The 66-residue toxin, which has four disulfide bridges and a cis peptidyl-proline bond, had not previously been refolded in reasonable yield. We recovered an 87% yield of protein with 100% biological activity.     

Synthesis and characterization of delta-atracotoxin-Ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus)

Delta-atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. (1)H NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.     

An efficient system for small protein expression and refolding

The low expression yield and poor refolding efficiency of small recombinant proteins expressed in Escherichia coli have continued to hinder the large-scale purification of such proteins for structural and biological investigations. A system based on a small fusion partner, the B1 domain of Streptococcal protein G (GB1), was utilized to overcome this problem. We have tested this system on a small cysteine-rich toxin, mutant myotoxin alpha (MyoP20G). The highly expressed fusion protein was refolded using an unfolding/refolding protocol. Due to the small size of GB1, we were able to monitor the unfolding/refolding status by heteronuclear single quantum coherence (HSQC) NMR spectroscopy. The final product yielded well-resolved NMR spectra, with a topology corresponding to the natural product. We conclude that GB1 not only increases the expression level but also enhances the refolding of small proteins.     

Reduction and reoxidation of the neurotoxin II from the scorpion Androctonus australis Hector

Reoxidation of the totally reduced scorpion neurotoxin II from Androctonus australis Hector (four disulfide bridges) has been investigated. The totally reduced toxin was highly insoluble in neutral and alkaline conditions, which prevented the use of the usual air oxidation process for renaturation. We tested a new method in which the reduced molecules were first solubilized in 10% (v/v) acetic acid and then oxidized by air through dialysis against a series of buffers with a slow pH gradient from 2.2 to 7.0 or 8.0. In this system, up to 95% of the protein was recovered in solution. Addition of reduced and oxidized glutathione accelerated refolding and also permitted a better recovery of fully active peptide as measured by both toxicity to mice and ability to displace 125I radiolabeled toxin II from its binding site on rat brain synaptosomal fractions. The reoxidation reaction could also be monitored directly by high pressure liquid chromatography. A strong effect of guanidine hydrochloride concentration as well as the temperature was observed both on the solubility of the reoxidation intermediates and on the refolding pathway. Finally, the method used, i.e. dialysis reoxidation with a pH gradient from 2.2 to 8.0 in 0.1 M sodium phosphate, 0.1 M sodium chloride, 20 mM guanidine hydrochloride, 1 mM oxidized and reduced glutathione allowed regeneration in high yield (70%) of a reoxidized toxin form indistinguishable from the native toxin. A minor stable and inactive molecular species (about 30%) showing a difference in mobility by electrophoresis was also detected.     

Bacterial production and refolding from inclusion bodies of a “Weak” toxin, a disulfide rich protein

The gene for the “weak” toxin of Naja kaouthia venom was expressed in Escherichia coli. “Weak” toxin is a specific inhibitor of nicotine acetylcholine receptor, but mechanisms of interaction of similar neurotoxins with receptors are still unknown. Systems previously elaborated for neurotoxin II from venom of the cobra Naja oxiana were tested for bacterial production of “weak” toxin from N. kaouthia venom. Constructs were designed for cytoplasmic production of N. kaouthia “weak” toxin in the form of a fused polypeptide chain with thioredoxin and for secretion with the leader peptide STII. However, it became possible to obtain “weak” toxin in milligram amounts only within cytoplasmic inclusion bodies. Different approaches for refolding of the toxin were tested, and conditions for optimization of the yield of the target protein during refolding were investigated. The resulting protein was characterized by mass spectrometry and CD and NMR spectroscopy. Experiments on competitive inhibition of ¹²⁵I-labeled α-bungarotoxin binding to the Torpedo californica electric organ membranes containing the muscle-type nicotine acetylcholine receptor (α1₂β1_γδ) showed the presence of biological activity of the recombinant “weak” toxin close to the activity of the natural toxin (IC₅₀ = 4.3 ± 0.3 and 3.0 ± 0.5 µM, respectively). The interaction of the recombinant toxin with α7 type human neuronal acetylcholine receptor transfected in the GH₄C₁ cell line also showed the presence of activity close to that of the natural toxin (IC₅₀ 31 ± 5.0 and 14.8 ± 1.3 µM, respectively). The developed bacterial system for production of N. kaouthia venom “weak” toxin was used to obtain ¹⁵N-labeled analog of the neurotoxin.     

Solid-phase refolding of poly-lysine tagged fusion protein of hEGF and angiogenin

A fusion protein, consisting of a human epidermal growth factor (hEGF) as the recognition domain and human angiogenin as the toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as inclusion body in recombinantE. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation. It was probably because of the opposite electric charge at a neutral pH resulting from the vastly different pI values of each domain. The solidphase refolding process that exploited the ionic interactions between ionic exchanger surface and the fusion protein was tried, but the adsorption yield was also very low, below 30%, regardless of the resins and pH conditions used. Therefore, to provide a higher ionic affinity toward the solid matrix, six lysine residues were tagged to theN-terminus of the hEGF domain. When heparin-Sepharose was used as the matrix, the adsorption capacity increased 2.5–3 times to about 88%. Besides the intrinsic affinity of angiogenin to heparin the poly-lysine tag provided additional ionic affinity. And the subsequent refolding yield increased nearly 13-fold, fromc.a 4.8% in the conventional refolding of the untagged fusion protein to 63.6%. The process was highly reproducible. The refolded protein in the column eluate retained R Nase bioactivity, of angiogenin.     

N-terminal Extension of the Cholera Toxin A1-chain Causes Rapid Degradation after Retrotranslocation from Endoplasmic Reticulum to Cytosol

Cholera toxin travels from the plasma membrane to the endoplasmic reticulum of host cells, where a portion of the toxin, the A1-chain, is unfolded and targeted to a protein-conducting channel for retrotranslocation to the cytosol. Unlike most retrotranslocation substrates, the A1-chain escapes degradation by the proteasome and refolds in the cytosol to induce disease. How this occurs remains poorly understood. Here, we show that an unstructured peptide appended to the N terminus of the A1-chain renders the toxin functionally inactive. Cleavage of the peptide extension prior to cell entry rescues toxin half-life and function. The loss of toxicity is explained by rapid degradation by the proteasome after retrotranslocation to the cytosol. Degradation of the mutant toxin does not follow the N-end rule but depends on the two Lys residues at positions 4 and 17 of the native A1-chain, consistent with polyubiquitination at these sites. Thus, retrotranslocation and refolding of the wild-type A1-chain must proceed in a way that protects these Lys residues from attack by E3 ligases.     

Recombinant expression, purification, and characterization of scorpion toxin BmαTX14

Long-chain and cysteine-rich scorpion toxins exhibit various pharmacological profiles for different voltage-gated sodium channel subtypes. However, the exploration of toxin structure-function relationships has progressed slowly due to the difficulty of obtaining synthetic or recombinant peptides. We now report that we have established an effective expression and purification approach for the novel scorpion toxin BmαTX14. BmαTX14 was over-expressed as inclusion bodies in Escherichia coli. The insoluble pellet was successfully transformed into active peptide by using a refolding procedure. One-step purification by reverse-phase HPLC was sufficient to generate chromatographically pure peptide. The yield of recombinant toxin reached 4mg from 1L LB medium. The pharmacological data further showed that BmαTX14 selectively inhibited the fast inactivation of mNa(v)1.4 (EC(50)=82.3±15.7nM) rather than that of rNa(v)1.2 (EC(50)>30μM), which indicates that BmαTX14 is a new α-like toxin. This work enables further structural, functional, and pharmacological studies of BmαTX14 and similar toxins.     

Co- and Post-translocation Roles for HSP90 in Cholera Intoxication

Cholera toxin (CT) moves from the cell surface to the endoplasmic reticulum (ER) where the catalytic CTA1 subunit separates from the rest of the toxin. CTA1 then unfolds and passes through an ER translocon pore to reach its cytosolic target. Due to its intrinsic instability, cytosolic CTA1 must be refolded to achieve an active conformation. The cytosolic chaperone Hsp90 is involved with the ER to cytosol export of CTA1, but the mechanistic role of Hsp90 in CTA1 translocation remains unknown. Moreover, potential post-translocation roles for Hsp90 in modulating the activity of cytosolic CTA1 have not been explored. Here, we show by isotope-edited Fourier transform infrared spectroscopy that Hsp90 induces a gain-of-structure in disordered CTA1 at physiological temperature. Only the ATP-bound form of Hsp90 interacts with disordered CTA1, and refolding of CTA1 by Hsp90 is dependent upon ATP hydrolysis. In vitro reconstitution of the CTA1 translocation event likewise required ATP hydrolysis by Hsp90. Surface plasmon resonance experiments found that Hsp90 does not release CTA1, even after ATP hydrolysis and the return of CTA1 to a folded conformation. The interaction with Hsp90 allows disordered CTA1 to attain an active state, which is further enhanced by ADP-ribosylation factor 6, a host cofactor for CTA1. Our data indicate CTA1 translocation involves a process that couples the Hsp90-mediated refolding of CTA1 with CTA1 extraction from the ER. The molecular basis for toxin translocation elucidated in this study may also apply to several ADP-ribosylating toxins that move from the endosomes to the cytosol in an Hsp90-dependent process.     

Energetics of diphtheria toxin membrane insertion and translocation: calorimetric characterization of the acid pH induced transition

The pH and temperature stabilities of diphtheria toxin and its fragments have been studied by high-sensitivity differential scanning calorimetry. These studies demonstrate that the pH-induced conformational transition associated with the mechanism of membrane insertion and translocation of the toxin involves a massive unfolding of the toxin molecule. At physiological temperatures (37 degrees C), this process is centered at pH 4.7 at low ionic strength and at pH 5.4 in the presence of 0.2 M NaCl. At pH 8, the thermal unfolding of the nucleotide-bound toxin is centered at 58.2 degrees C whereas that of the nucleotide-free toxin is centered at 51.8 degrees C, indicating that nucleotide binding (ApUp) stabilizes the native conformation of the toxin. The unfolding profile of the toxin is consistent with two transitions most likely corresponding to the A fragment (Tm = 54.5 degrees C) and the B fragment (Tm = 58.4 degrees C), as inferred from experiments using the isolated A fragment. These two transitions are not independent, judging from the fact that the isolated A fragment unfolds at much lower temperatures (Tm = 44.2 degrees C) and that the B fragment is insoluble in aqueous solutions when separated from the A fragment. Interfragment association contributes an extra -2.6 kcal/mol to the free energy of stabilization of the A fragment. Whereas the unfolding of the entire toxin is irreversible, the unfolding of the A fragment is a reversible process. These findings provide a thermodynamic basis for the refolding of the A fragment after reexposure to neutral pH immediately following translocation across the lysosomal membrane.     

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.     

The isolation and characterization of temperature-dependent ricin A chain molecules in Saccharomyces cerevisiae

Ricin is a heterodimeric plant protein that is potently toxic to mammalian cells. Toxicity results from the catalytic depurination of eukaryotic ribosomes by ricin toxin A chain (RTA) that follows toxin endocytosis to, and translocation across, the endoplasmic reticulum membrane. To ultimately identify proteins required for these later steps in the entry process, it will be useful to express the catalytic subunit within the endoplasmic reticulum of yeast cells in a manner that initially permits cell growth. A subsequent switch in conditions to provoke innate toxin action would permit only those strains containing defects in genes normally essential for toxin retro-translocation, refolding or degradation to survive. As a route to such a screen, several RTA mutants with reduced catalytic activity have previously been isolated. Here we report the use of Saccharomyces cerevisiae to isolate temperature-dependent mutants of endoplasmic reticulum-targeted RTA. Two such toxin mutants with opposing phenotypes were isolated. One mutant RTA (RTAF108L/L151P) allowed the yeast cells that express it to grow at 37 degrees C, whereas the same cells did not grow at 23 degrees C. Both mutations were required for temperature-dependent growth. The second toxin mutant (RTAE177D) allowed cells to grow at 23 degrees C but not at 37 degrees C. Interestingly, RTAE177D has been previously reported to have reduced catalytic activity, but this is the first demonstration of a temperature-sensitive phenotype. To provide a more detailed characterization of these mutants we have investigated their N-glycosylation, stability, catalytic activity and, where appropriate, a three-dimensional structure. The potential utility of these mutants is discussed.     

The intestinal zonula occludens toxin (ZOT) receptor recognises non-native ZOT conformers and localises to the intercellular contacts

A preliminary structural analysis of Vibrio cholerae zonula occludens toxin (ZOT) was made by equilibrium denaturation and circular dichroism. ZOT is a structurally unstable protein in aqueous solution (DeltaG((H2O)) 3.82 kcal/mol), the putative intra- and extracellular domains unfold co-operatively, with complete denaturation via observed conformational intermediates. Refolding of denatured ZOT is not dependent on disulphide bridge formation. Partial refolding of a maltose binding protein-ZOT fusion did not prevent its specific binding to the ZOT receptor on Caco-2 cells. Immuno-gold labelling showed that the ZOT receptor localises to the intercellular contacts between cells in a confluent monolayer.     

Refolding of Taiwan cobra neurotoxin: intramolecular cross-link affects its refolding reaction

In order to explore the effect of intramolecular cross-linking in the folding reaction of cobrotoxin from Naja naja atra (Taiwan cobra) venom, the toxin molecule was modified with glutaraldehyde (GA). The monomeric GA-modified cobrotoxin (mGA-cobrotoxin) was separated from the dimeric and trimeric derivatives using gel filtration. The results of electrophoretic and chromatographic analyses revealed that mGA-cobrotoxin comprised two modified derivatives, which contained modified Lys residues at positions 26 and 27 and at positions 26, 27, and 47, respectively. Moreover, an intramolecular cross-linking of loops II and III by Lys residues was noted with the monomeric derivative containing three modified Lys residues. In sharp contrast to cobrotoxin observations, the folding rate of mGA-cobrotoxin decreased in the presence of GSH/ GSSG, but notably increased in the absence of thiol compounds. Particularly, the accelerated effect of GSH/GSSG on the refolding reaction was affected by the presence of the intramolecular cross-link. Comparative analyses on cobrotoxin and mGA-cobrotoxin CD spectra revealed that modification with the GA reagent caused a change in the gross conformation of cobrotoxin. Fluorescence measurement revealed that the stability of the microenvironment around the single Trp-29 in mGA-cobrotoxin and unfolded mGA-cobrotoxin was appreciably higher than in cobrotoxin and unfolded toxin. Moreover, the ordered structure formation around Trp-29 in refolded mGA-cobrotoxin was faster than in refolded cobrotoxin as evidenced by fluorescence quenching studies. Taken together, these results suggest that the structural flexibility of unfolded cobrotoxin should be favorable for the thiol catalyst to exert its action in the refolding reaction after modification with GA.     

Oxidative folding of reduced and denatured huwentoxin-I

Huwentoxin-I, a neurotoxic peptide with 33 ammo acid residues and three disulfide bonds, was used to investigate the pathway of reduction/denaturation and of oxidative folding in small proteins with multiple disulfide bonds. Titration of thiol groups, reversed-phase HPLC, 1D NMR spectroscopy, and biological activity assays were used to monitor the extent of reduction/denaturation and renaturation of the toxin. The reduction and denaturation of huwentoxin-I resulted in a 100% loss of bioactivity as measured in a mouse phrenic nerve-diaphragm preparation. About 90% of full biological activity could be restored under optimized conditions of oxidative refolding of the reduced peptide. Several reaction conditions employing air oxidation, oxidized and reduced glutathione (GSSG and GSH), and cystine/cysteine were investigated in order to find optimal conditions for renaturation of huwentoxin-I. The best renaturation yield was achieved in 0.1 mM GSSG and 1 mM GSH at pH 8.5 and 4°C over 24 hr. High concentrations of glutathione and high temperatures reduced renaturation yields. Oxidative refolding of huwentoxin-I in air requires about 6 days for maximal yields and is inhibited by EDTA.     

Synthesis of cholera toxin B subunit gene: cloning and expression of a functional 6XHis-tagged protein in Escherichia coli

Cholera toxin B subunit (CTB) has been extensively studied as immunogen, adjuvant, and oral tolerance inductor depending on the antigen conjugated or coadministered. It has been already expressed in several bacterial and yeast systems. In this study, we synthesized a versatile gene coding a 6XHis-tagged CTB (359bp). The sequence was designed according to codon usage of Escherichia coli, Lactobacillus casei, and Salmonella typhimurium. The gene assembly was based on a polymerase chain reaction, in which the polymerase extends DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was amplified, cloned, and expressed in E. coli in an insoluble form, reaching levels about 13 mg of purified active pentameric rCTB per liter of induced culture. Western blot and ELISA analyses showed that recombinant CTB is strongly and specifically recognized by polyclonal antibodies against the cholera toxin. The ability to form the functional pentamers was observed in cell culture by the inhibition of cholera toxin activity on Y1 adrenal cells in the presence of recombinant CTB. The 6XHis-tagged CTB provides a simple way to obtain functional CTB through Ni(2+)-charged resin after refolding and also free of possible CTA contaminants as in the case of CTB obtained from Vibrio cholerae cultures.     

Ribosome-mediated folding of partially unfolded ricin A-chain

After endocytic uptake by mammalian cells, the cytotoxic protein ricin is transported to the endoplasmic reticulum, whereupon the A-chain must cross the lumenal membrane to reach its ribosomal substrates. It is assumed that membrane traversal is preceded by unfolding of ricin A-chain, followed by refolding in the cytosol to generate the native, biologically active toxin. Here we describe biochemical and biophysical analyses of the unfolding of ricin A-chain and its refolding in vitro. We show that native ricin A-chain is surprisingly unstable at pH 7.0, unfolding non-cooperatively above 37 degrees C to generate a partially unfolded state. This species has conformational properties typical of a molten globule, and cannot be refolded to the native state by manipulation of the buffer conditions or by the addition of a stem-loop dodecaribonucleotide or deproteinized Escherichia coli ribosomal RNA, both of which are substrates for ricin A-chain. By contrast, in the presence of salt-washed ribosomes, partially unfolded ricin A-chain regains full catalytic activity. The data suggest that the conformational stability of ricin A-chain is ideally poised for translocation from the endoplasmic reticulum. Within the cytosol, ricin A-chain molecules may then refold in the presence of ribosomes, resulting in ribosome depurination and cell death.     

Refolding strategies for ketosteroid isomerase following insoluble expression in Escherichia coli

Dilution and column-based protein refolding techniques are compared for refolding Delta 5-3-ketosteroid isomerase (KSI) with a C-terminus his6-tag. Column refolding was performed by removing the denaturant while the protein was adsorbed in an immobilized metal affinity chromatography column. Both dilution refolding and a single-step column-based refolding strategy were optimized to maximize the recovery of KSI enzyme activity, and achieved refolding yields of 87% and 70% respectively. It was found that the column-based refolding yield was reduced at higher adsorbed protein concentrations. An elution gradient with increasing imidazole concentration was used to selectively elute the biologically active KSI protein following column refolding, with high molecular weight KSI aggregates retained in the column. An iterative column-refolding process was then developed to denature and refold protein retained in the column, which significantly increased the refolding yield at high-adsorbed protein concentrations. Repetition of the column refolding operation increased the refolding yield from 50% to 75% for protein adsorbed at a concentration of 2.9 mg/mL of adsorbent. Although for the KSI protein column-based refolding did not improve the overall refolding yield compared to dilution refolding, it may still be advantageous due to the ease of integration with purification operations, increased control over the refolding conditions, and the ability to segregate refolded protein from inactive aggregates during elution.