Characterization of LC-HCC fusion protein of botulinum neurotoxin type A

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses. The gene for encoding the full length light chain with H(CC) (binding) domain of Clostridium botulinum neurotoxin A was synthesized and cloned into a bacterial expression vector pQE30-UA and produced as an N-terminally six-histidine-tagged fusion protein (rBoNT/A LC-H(CC)). This protein was expressed in two different strains of Escherichia coli namely BL21(DE3) and SG13009. Expression at 37 °C revealed localization of rBoNT/A LC- H(CC) in inclusion body whereas it was expressed in soluble form at 21°C. The recombinant fusion protein was purified by nickel affinity gel column chromatography and identified by monoclonal antibody and peptide mass fingerprinting. The recombinant protein was shown to bind with synaptic vesicles and gangliosides (GT1b) using enzyme-linked immunosorbent assay. The rBoNT/A LC-H(CC) was also found to be highly active on its substrate (SNAP-25) from rat brain, indicating that the expressed and purified rBoNT/A LC-H(CC) protein retains a functionally active conformation. Biologically active recombinant fusion protein was also evaluated for its immunological potential.     

Small-Molecule Quinolinol Inhibitor Identified Provides Protection against BoNT/A in Mice

Botulinum neurotoxins (BoNTs), etiological agents of the life threatening neuroparalytic disease botulism, are the most toxic substances currently known. The potential for the use as bioweapon makes the development of small-molecule inhibitor against these deadly toxins is a top priority. Currently, there are no approved pharmacological treatments for BoNT intoxication. Although an effective vaccine/immunotherapy is available for immuno-prophylaxis but this cannot reverse the effects of toxin inside neurons. A small-molecule pharmacological intervention, especially one that would be effective against the light chain protease, would be highly desirable. Similarity search was carried out from ChemBridge and NSC libraries to the hit (7-(phenyl(8-quinolinylamino)methyl)-8-quinolinol; NSC 84096) to mine its analogs. Several hits obtained were screened for in silico inhibition using AutoDock 4.1 and 19 new molecules selected based on binding energy and Ki. Among these, eleven quinolinol derivatives potently inhibited in vitro endopeptidase activity of botulinum neurotoxin type A light chain (rBoNT/A-LC) on synaptosomes isolated from rat brain which simulate the in vivo system. Five of these inhibitor molecules exhibited IC₅₀ values ranging from 3.0 nM to 10.0 µM. NSC 84087 is the most potent inhibitor reported so far, found to be a promising lead for therapeutic development, as it exhibits no toxicity, and is able to protect animals from pre and post challenge of botulinum neurotoxin type A (BoNT/A).     

Expression, purification and development of neutralizing antibodies from synthetic BoNT/B LC and its application in detection of botulinum toxin serotype B

Botulism is a neuroparalytic disease caused by Clostridium botulinum, which produces seven (A-G) neurotoxins (BoNTs). The mouse bioassay is the gold standard for the detection of botulinum neurotoxins, however it requires at least 3-4 days for completion. Most of the studies were carried out in botulinum toxin A and less on type B. Attempts have been made to develop an ELISA based detection system, which is potentially an easier and more rapid method of botulinum neurotoxin detection. In the present study, the synthetic BoNT/B LC gene was constructed using PCR overlapping primers, cloned in a pET28a+ vector and expressed in E. coli BL21DE3. The maximum yield of recombinant proteins was optimized after 16 hrs of post induction at 21°C and purified the recombinant protein in soluble form. Antibodies were raised in Mice and Rabbit. The IgG antibody titer in the case of Mice was 1: 1,024,000 and Rabbit was 1: 512,000 with alum as adjuvant via intramascular route. The biological activity of the recombinant protein was confirmed by in-vitro studies using PC12 cells by the synaptobrevin cleavage, the rBoNT/B LC protein showed the maximum blockage of acetylcholine release at a concentration of 150nM rBoNT/B LC in comparison to the control cells. When the cells were incubated with rBoNT/B LC neutralized by the antisera raised against it, the acetylcholine release was equivalent to the control. IgG specific to rBoNT/B LC was purified from raised antibodies. The results showed that the developed antibody against rBoNT/B LC protein were able to detect botulinum toxin type B approximately up to 1 ng/ml. These developed high titer antibodies may prove useful for the detection of botulinum neurotoxins in food and clinical samples.     

Factors Affecting Autocatalysis of Botulinum A Neurotoxin Light Chain*

The light chain of botulinum neurotoxin serotype A undergoes autocatalytic fragmentation into two major peptides during purification and storage (Ahmed S. A. et al. 2001, J. Protein Chem. 20:221–231) by both intermolecular and intramolecular mechanisms (Ahmed S. A. etal. 2003, Biochemistry 42:12539–12549). In this study, we investigated the effects of buffers and salts on this autocatalytic reaction in the presence and absence of zinc chloride. In the presence of zinc chloride, the fragmentation reaction was enhanced in each of acetate, MES, HEPES and phosphate buffers with maximum occurring in acetate when compared to those in the absence of zinc chloride. Adding sodium chloride in phosphate buffer in the presence of zinc chloride increased the extent of proteolysis. Irrespective of the presence of zinc chloride, adding sodium chloride or potassium chloride in phosphate buffer elicited an additional proteolytic reaction. Higher concentrations of sodium phosphate buffer enhanced the autocatalytic reaction in the absence of zinc chloride. In contrast, in the presence of zinc chloride, higher concentrations of sodium phosphate decreased the autocatalytic reaction. Optimum pH of autocatalysis was not affected significantly by the absence or presence of zinc chloride. Like zinc chloride, other chlorides of divalent metals, such as magnesium, cobalt, iron and calcium also enhanced the autocatalytic reaction. Polyols such as ethylene glycol protected the light chain from fragmentation. Exposure of light chain to UV radiation led to enhanced fragmentation. In order to avoid fragmentation, the protein should be stored frozen in a low concentration buffer of neutral or higher pH devoid of any metal. Our results provide a choice of buffers and salts for isolation, purification and storage of intact botulinum neurotoxin serotype A light chain.     

Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System

Clostridium botulinum subtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producing C. botulinum strain 657Ba at 100× lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenic C. botulinum expression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided in trans by the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed in Escherichia coli has indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of the in vitro, cellular, and in vivo activities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.     

Autocatalytically fragmented light chain of botulinum a neurotoxin is enzymatically active

The zinc-endopeptidase light chain of botulinum A neurotoxin undergoes autocatalytic fragmentation that is accelerated by the presence of the metal cofactor, zinc [Ahmed, S. A. et al. (2001) J. Protein Chem. 20, 221-231]. We show in this paper that >95% fragmented light chain obtained in the absence of added zinc retained 100% of its original catalytic activity against a SNAP-25-derived synthetic peptide substrate. In the presence of zinc chloride, when >95% of the light chain had undergone autocatalytic fragmentation, the preparation retained 35% of its original catalytic activity. On the other hand, in the presence of glycerol, the light chain did not display autocatalysis and retained 100% of the original activity. These results suggest that the activity loss by incubation with zinc was not a direct consequence of autocatalysis and that the environment of the active site was not affected significantly by the fragmentation. The optimum pH 4.2-4.6 for autocatalysis was different than that (pH 7.3) for intrinsic catalytic activity. Inhibition of autocatalysis at low pH by a competitive inhibitor of catalytic activity rules out the presence of a contaminating protease but suggests a rate-limiting step of low pH-induced conformational change suitable for autocatalysis. Our results of LC concentration dependence of the fragmentation reaction indicate that the autocatalysis occurs by both intramolecular and intermolecular mechanisms.     

Preparation of specifically activatable endopeptidase derivatives of Clostridium botulinum toxins type A, B, and C and their applications

Clostridium botulinum neurotoxins are potently toxic proteins of 150 kDa with specific endopeptidase activity for SNARE proteins involved in vesicle docking and release. Following treatment with trypsin, a fragment of botulinum neurotoxin serotype A that lacks the C-terminal domain responsible for neuronal cell binding, but retains full catalytic activity, can be obtained. Known as the LH(N) fragment, we report the development of a recombinant expression and purification scheme for the isolation of comparable fragments of neurotoxin serotypes B and C. Expressed as maltose-binding protein fusions, both have specific proteolytic sites present between the fusion tag and the light chain to facilitate removal of the fusion, and between the light chain endopeptidase and the H(N) translocation domains to facilitate activation of the single polypeptide. We have also used this approach to prepare a new variant of LH(N)/A with a specific activation site that avoids the need to use trypsin. All three LH(N)s are enzymatically active and are of low toxicity. The production of specifically activatable LH(N)/A, LH(N)/B, and LH(N)/C extends the opportunities for exploitation of neurotoxin fragments. The potential utility of these fragments is discussed.     

Calcein permeability of liposomes mediated by type A botulinum neurotoxin and its light and heavy chains

The mode of botulinum neurotoxin action involves binding of its heavy chain for internalization into the presynaptic end of a nerve cell through endocytosis. The low-pH conditions of endosomes trigger translocation of the light chain across the endosomal membrane to the cytosol, where the light chain cleaves specific target proteins involved in the docking and fusion of synaptic vesicles for acetylcholine release. In an effort to model the interaction of botulinum neurotoxin and its subunit chains with lipid bilayer at low pH during the translocation process, we have examined type A botulinum neurotoxin-mediated calcein release from asolectin liposomes. At equimolar concentration (0.1 M), the neurotoxin and its heavy and light chains evoked 23%, 58%, and 28% calcein release, respectively. Calcein release was observed only when the cis-side (the side to which neurotoxin samples were added) pH was lowered to 4. Calcein release activity of the heavy chain was mostly blocked (76%) by a polyclonal antibody raised against the neurotoxin. Additionally, two peptide-specific polyclonal antibodies derived from the N-terminal and C-terminal halves of the heavy chain were also able to block the calcein release activity by 15–20%. In summary, these results suggest that calcein release from liposomes is specifically mediated by the heavy chain, and the light chain also integrates into the membrane. Implications of these results for the molecular mode of neurotoxin light-chain translocation across the endosomal membrane are discussed.     

Isolated light chains of botulinum neurotoxins inhibit exocytosis. Studies in digitonin-permeabilized chromaffin cells

The effects of botulinum neurotoxins or their light and heavy chain subunits were investigated in digitonin-permeabilized adrenal chromaffin cells. Because these cells are permeable to proteins, the toxin had direct access to the cell interior. Botulinum type A neurotoxin and its light chain subunit inhibited Ca2+-dependent catecholamine secretion in a dose-dependent manner. The heavy chain subunit had no effect. Inhibition required introduction of the neurotoxin or light chain into the cell and was not seen when intact cells were incubated with these proteins. The inhibition of secretion by type A neurotoxin and light chain was incomplete, the maximal response being 65%. The inhibition was not overcome by increasing Ca2+ concentrations. The action of the light chain was irreversible and rapid. Botulinum type E neurotoxin also inhibited secretion in a dose-dependent manner. Its potency was increased 30-fold following mild trypsinization, which nicked the single chain protein to the dichain form. In contrast to the results seen with types A and E, botulinum type B neurotoxin did not inhibit secretion, while its light chain totally abolished secretion. Trypsinization of the neurotoxin produced the dichain form, which did not inhibit secretion. Reduction of the trypsinized neurotoxin with dithiothreitol produced inhibition equivalent to that seen with the purified light chain subunit. Isolated type A heavy chain had no effect on the inhibitory action of type A or B light chains. The data demonstrate that the ability of botulinum neurotoxins to inhibit secretion is confined to the light chain region of these proteins. Furthermore, while the botulinum neurotoxin types A, B, and E have similar macrostructures, they are not identical with respect to their biological activities.     

High level expression,purification and immunogenicity analysis of a protective recombinant protein against botulinum neurotoxin type E

Botulinum neurotoxin type E heavy chain consists of two domains: N-terminal half as a translocation domain and C-terminal half (Hcc) as a binding domain. In this research a synthetic gene fragment encoding the binding domain of botulinum neurotoxin type E (BoNT/E-Hcc) was highly expressed in Escherichia coli by pGEX4T-1 vector. After purification, the recombinant BoNT/E-Hcc was evaluated by SDS-PAGE and western blot (immunoblot) analysis. Average yields obtained in this research were 3.7 mg recombinant BoNT/E-Hcc per liter of bacterial culture. The recombinant protein was injected in mice for study of its protection ability against botulinum neurotoxin type E challenges. The challenge studies showed that, vaccinated mice were fully protected against 10⁴ × minimum lethal dose of botulinum neurotoxin type E.     

Partial amino acid sequences of the heavy and light chains of botulinum neurotoxin type E

The dichain type E botulinum neurotoxin, a product of nicking the single chain protein by trypsin, is composed of a heavy and light chains. Sequence of the first 13 and 20 N-terminal residues of these two chains were determined. Also, proof is provided here that (i) the light chain of the nicked (dichain) is derived from the N-terminal one-third of the parent single chain neurotoxin, and (ii) molecular events leading to the activation, of the single chain neurotoxin cannot involve tryptic cleavage at or very close to the N-terminal of the single chain protein. The partial amino acid sequence of the light chain of botulinum type E and tetanus neurotoxins show significant similarity between the two clostridial neurotoxins.     

Effective expression of fragments of a botulinum neurotoxin type A gene, coding for the L-chain and H-chain in E. coli, with formation of products causing protective immunity to administration of the toxin

Native Clostridium botulinum gene coding for type A neurotoxin has been used to construct recombinant derivatives coding separately for L and H polypeptide chains of the toxin. The gene derivatives have been cloned into an expression vector pET28b in E. coli BL21 (DE3) cells. The recombinant L and H proteins seem to be the major individual proteins after IPTG induction of the recombinant cells. Each of the proteins has been accumulated only in inclusion bodies. The recombinant L chain (but not H chain) has been successfully resolubilized. Each of the proteins contains six His residues on the N terminus which allows purification on Ni-agarose columns with high yield. No toxic effect has been observed for both L and H chains after injection of 10 micrograms of recombinant preparations purified from inclusion bodies. Moreover, the injection resulted in an increase in the titer of specific antibodies which protected mice from 1 DLM of type A native botulinum neurotoxin. Hence, the recombinant neurotoxin protein derivatives which are present in E. coli inclusion bodies can be a source of material for producing diagnostic and therapeutic sera against type A botulinum neurotoxin.     

Light chain of botulinum A neurotoxin expressed as an inclusion body from a synthetic gene is catalytically and functionally active

Botulinum neurotoxins, the most potent of all toxins, induce lethal neuromuscular paralysis by inhibiting exocytosis at the neuromuscular junction. The light chains (LC) of these dichain neurotoxins are a new class of zinc-endopeptidases that specifically cleave the synaptosomal proteins, SNAP-25, VAMP, or syntaxin at discrete sites. To facilitate the structural and functional characterization of these unique endopeptidases, we constructed a synthetic gene for the LC of the botulinum neurotoxin serotype A (BoNT/A), overexpressed it in Escherichia coli, and purified the gene product from inclusion bodies. Our procedure can provide 1.1 g of the LC from 1 L of culture. The LC product was stable in solution at 4°C for at least 6 months. This rBoNT/A LC was proteolytically active, specifically cleaving the Glu-Arg bond in a 17-residue synthetic peptide of SNAP-25, the reported cleavage site of BoNT/A. Its calculated catalytic efficiency k _cat/K _m was higher than that reported for the native BoNT/A dichain. Treating the rBoNT/A LC with mercuric compounds completely abolished its activity, most probably by modifying the cysteine-164 residue located in the vicinity of the active site. About 70% activity of the LC was restored by adding Zn²⁺ to a Zn²⁺-free, apo-LC preparation. The LC was nontoxic to mice and failed to elicit neutralizing epitope(s) when the animals were vaccinated with this protein. In addition, injecting rBoNT/A LC into sea urchin eggs inhibited exocytosis-dependent plasma membrane resealing. For the first time, results of our study make available a large amount of the biologically active toxin fragment in a soluble and stable form.     

Evidence for a vesicle-mediated maintenance of store-operated calcium channels in a human embryonic kidney cell line

Direct microinjection of the clostridial neurotoxins botulinum neurotoxin A light chain or tetanus neurotoxin into cells of a human embryonic kidney cell line significantly reduced calcium entry after depletion of internal calcium stores by cyclopiazonic acid, a reversible inhibitor of the sarcoplasmic-endoplasmic reticular calcium-ATPases. Botulinum neurotoxin A light chain specifically hydrolyzes a synaptosomal-associated protein of 25 kilodaltons (SNAP-25), and tetanus neurotoxin specifically hydrolyzes synaptobrevin-2 (vesicle-associated membrane protein 2, VAMP-2) and cellubrevin (vesicle-associated membrane protein 3, VAMP-3). Since these substrate proteins are required for vesicle docking and fusion, inhibition of store-operated calcium entry by botulinum neurotoxin A light chain and tetanus neurotoxin supports a model in which vesicle fusion is a prerequisite for activation of store-operated calcium entry. Brefeldin A, a fungal metabolite that interferes with vesicle traffic, partially reduced calcium entry following store depletion. The size of the reserve pool of vesicles or parallel vesicle recycling pathways employing brefeldin A-sensitive and brefeldin A-insensitive ADP-ribosylation factors may explain the failure of brefeldin A to completely inhibit store-operated calcium entry.     

Expression, purification, and characterization of Clostridium botulinum type B light chain

A full-length synthetic gene encoding the light chain of botulinum neurotoxin serotype B, approximately 50 kDa (BoNT/B LC), has been cloned into a bacterial expression vector pET24a+. BoNT/B LC was expressed in Escherichia coli BL21.DE3.pLysS and isolated from the soluble fraction. The resultant protein was purified to homogeneity by cation chromatography and was determined to be >98% pure as assessed by SDS-polyacrylamide gel stained with SilverXpress and analyzed by densitometry. Mass spectroscopic analysis indicated the protein to be 50.8 kDa, which equaled the theoretically expected mass. N-terminal sequencing of the purified protein showed the sequence corresponded to the known reported sequence. The recombinant BoNT/B light chain was found to be highly stable, catalytically active, and has been used to prepare antisera that neutralizes against BoNT/B challenge. Characterization of the protein including pH, temperature, and the stability of the protein in the presence or absence of zinc is described within. The influence of pH differences, buffer, and added zinc on secondary and tertiary structure of BoNT/B light chain was analyzed by circular dichroism and tryptophan fluorescence measurements. Optimal conditions for obtaining maximum metalloprotease activity and stabilizing the protein for long term storage were determined. We further analyzed the thermal denaturation of BoNT/B LC as a function of temperature to probe the pH and added zinc effects on light chain stability. The synthetic BoNT/B LC has been found to be highly active on its substrate (vesicle associated membrane protein-2) and, therefore, can serve as a useful reagent for BoNT/B research.     

Partial amino acid sequence of the heavy and light chains of botulinum neurotoxin type A

The dichain (nicked) type A botulinum neurotoxin is a protein (mol. wt. 145,000) composed of a heavy and a light chain (mol. wt. 97,000 and 53,000, respectively) that are held together by disulfide bond(s). We report here the sequence of the first 17 amino acid residues of the light chain, and the first 10 residues of the heavy chain. The heavy chain was isolated from the neurotoxin by two different methods, while the light chain was isolated by the only available method. The identical amino acid sequence was found in both preparations of heavy chain. Two samples of the light chain isolated from two separately prepared batches of the neurotoxin also had identical sequences.     

Synthesis and activity of isoleucine sulfonamide derivatives as novel botulinum neurotoxin serotype A light chain inhibitors

The botulinum neurotoxin (BoNT) is the most lethal protein known to man causing the deadly disease botulinum. The neurotoxin, composed of a heavy (HC) and light (LC) chain, work in concert to cause muscle paralysis. A therapeutic strategy to treat individuals infected with the neurotoxin is inhibiting the catalytic activity of the BoNT LC. We report the synthesis, inhibition study and computational docking analysis of novel small molecule BoNT/A LC inhibitors. A structure activity relationship study resulted in the discovery of d-isoleucine functionalized with a hydroxamic acid on the C-terminal and a biphenyl with chlorine at C- 2 connected by a sulfonamide linker at the N-terminus. This compound has a measured IC₅₀ of 0.587 µM for the BoNT/A LC. Computational docking analysis indicates the sulfonamide linker adopts a geometry that is advantageous for binding to the BoNT LC active site. In addition, Arg363 is predicted to be involved in key binding interactions with the scaffold in this study.     

Catalytic features of the botulinum neurotoxin A light chain revealed by high resolution structure of an inhibitory peptide complex

The Clostridium botulinum neurotoxin serotype A light chain (BoNT/A-LC) is a Zn(II)-dependent metalloprotease that blocks the release of acetylcholine at the neuromuscular junction by cleaving SNAP-25, one of the SNARE proteins required for exocytosis. Because of the potential for use of the toxin in bioterrorism and the increasingly widespread application of the toxin in the medical field, there is significant interest in the development of small-molecule inhibitors of the metalloprotease. Efforts to design such inhibitors have not benefited from knowledge of how peptides bind to the active site since the enzyme-peptide structures available previously either were not occupied in the vicinity of the catalytic Zn(II) ion or did not represent the product of SNAP-25 substrate cleavage. Herein we report the 1.4 A-resolution X-ray crystal structure of a complex between the BoNT/A-LC and the inhibitory peptide N-Ac-CRATKML, the first structure of the light chain with an inhibitory peptide bound at the catalytic Zn(II) ion. The peptide is bound with the Cys S gamma atom coordinating the metal ion. Surprisingly, the cysteine sulfur is oxidized to the sulfenic acid form. Given the unstable nature of this species in solution, is it likely that oxidation occurs on the enzyme. In addition to the peptide-bound structure, we report two structures of the unliganded light chain with and without the Zn(II) cofactor bound at 1.25 and 1.20 A resolution, respectively. The two structures are nearly identical, confirming that the Zn(II) ion plays a purely catalytic role. Additionally, the structure of the Zn(II)-bound uncomplexed enzyme allows identification of the catalytic water molecule and a second water molecule that occupies the same position as the peptidic oxygen in the tetrahedral intermediate. This observation suggests that the enzyme active site is prearranged to stabilize the tetrahedral intermediate of the protease reaction.     

Structural and biochemical studies of botulinum neurotoxin serotype C1 light chain protease: implications for dual substrate specificity

Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote alpha-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the alpha-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.     

High-level expression, purification, and characterization of recombinant type A botulinum neurotoxin light chain

Botulinum neurotoxin light chain (BoNT LC, 50 kDa) is responsible for the zinc endopeptidase activity specific for proteins of neuroexocytosis apparatus. We describe the expression of recombinant type A BoNT LC in Escherichia coli as well as the purification and characterization of the recombinant protein. A high level of expression of BoNT/A LC was obtained by an extended postinduction time of 15 h at 30 degrees C. Recombinant BoNT/A LC was isolated from an Ni(2+) column. Due to its high pI ( approximately 8.7), purification was achieved by a single step of passing the protein through anion-exchange chromatography at pH 8.0 without the need of elution. The purified recombinant BoNT/A LC retained proteolytic activity and had a secondary structure similar to that of native LC determined by CD measurement.