WO2009152452A1 - Fermentation method for the production of soluble botulinum neurotoxin proteins - Google Patents

Abstract

A method is provided for the production of soluble Clostridium neurotoxins or neurotoxin fragments, for instance, LH_N fragments from botulinum neurotoxins. Proteins made according to the method of the invention are useful, for example, as immunodiagnostic agents and vaccine components.

Description

FERMENTATION METHOD FOR THE PRODUCTION OF SOLUBLE BOTULINUM NEUROTOXIN PROTEINS

DESCRIPTION OF THE INVENTION

Related Applications

[001] This application claims benefit of U.S. provisional application no. 61/060,978, filed June 12, 2008, the disclosure of which is incorporated by reference in its entirety. This application is also related to U.S. Patent Application No. 60/929,125, filed June 13, 2007; US Patent Application No. 60/960,771 , filed October 12, 2008; and US Patent Application No. 61/006,546, filed October 18, 2008, each of which is herein incorporated by reference in its entirety.

Field of the Invention

[002] The present invention relates to methods for producing soluble recombinant botulinum neurotoxin proteins using bacterial fermentation.

Background of the Invention

[003] Bacterial toxins and toxin fragments are importation as immunodiagnostic reagents, therapeutic agents, and vaccine components. One group of bacterial toxins is the clostridial neurotoxins, such as those from Clostridium botulinum and Clostridium butyricum. Botulinum neurotoxin (BoNT) is one of the most potent toxins known to man. Its ingestion or inhalation inhibits neurotransmitter release from synaptic vesicles, resulting in neuroparalysis and death. The use of Clostridium botulinum neurotoxins as vaccine components is disclosed in U.S. Patent No. 5,919,665 to J. A. Williams, which is incorporated by reference into this application. In addition, U.S. Patent Nos. 6,051 ,239 to Simpson er a/., 6,287,566 to M. T. Dertzbaugh, and 6,461 ,617 to Shone et al, each of which is incorporated by reference into this application, disclose the use of fragments of clostridial neurotoxin as vaccine components. [004] The botulinum neurotoxins (BoNTs) are a family of seven antigenically different protein toxins (serotypes A, B, C, D, E, F, and G). Antisera raised against purified neurotoxins show no cross-protection between the neurotoxin serotypes. In addition, various subtypes exist within each serotype (Minton (1995) In: Current Topics in Microbiology and Immunology 195 'Clostridial Neurotoxins' (Montecucco, C, ed.) pp. 161-194, Springer, Berlin).

[005] These neurotoxins are extremely potent neuroparalytic agents that act primarily at the peripheral nervous system where they inhibit the release of acetylcholine at the neuromuscular junction (Niemann (1991) In Sourcebook of Bacterial Protein Toxins (Alouf, J. E. & Freer, J. H. eds.), pp. 303-348, Academic Press, London). This activity is mediated via highly specific zinc-dependent endopeptidase activity directed at small proteins involved in the fusion and release of synaptic vesicles.

[006] The botulinum neurotoxins are structurally similar, with 30-40% sequence homology. Each neurotoxin type shares a common architecture in which a catalytic L-chain (LC, ~ 50 kDa) is disulfide linked to a receptor binding and translocating H-chain (HC, ~ 100 kDa). (Niemann, 1991). The C- terminal 50 kDa fragment (Hc fragment) is responsible for receptor-binding at the presynaptic nerve surface (Halpern & Loftus (1993) J. Biol. Chem. 268, 11188-11192); (Shone et al. (1985) Eur. J. Biochem., 151 , 75-82). The N- terminal 50 kDa portion of the heavy chain (HN fragment) is involved in translocation of the enzymatically active light chain to within the nerve terminal (Shone er a/. (1987) Eur. J. Biochem., 167, 175-180). Removal of the Hc domain from the BoNT leaves a fragment (LH_N, ~ 100 kDa) consisting of the light chain and translocation domain which, although virtually non-toxic, is stable and relatively soluble. Residual toxicity can be eliminated, for instance, by introducing single or double mutations into the enzymatic domain, which yield a non-toxic LHN fragment that can be used in vaccine development. [007] Structure of Botulinum Neurotoxins and the LHN Fragment

Light Chain Heavy Chain

(Enzymatic) (Translocation) (Binding)

Domain Domain Domain

Holotoxin

[008] When they are expressed as recombinant (r) proteins, rLH_N fragments have a tendency to form aggregates that may have a molecular mass ranging up to several million kilodaltons (kD). Although aggregated rLH_N can be recovered from insoluble lysate material by detergent extraction/reductant treatment and further purified (~90%) by anion exchange (Q Sepharose) and gel filtration (Superdex 200) chromatography, the recovered aggregate has undesirable properties. For example, purified, aggregated ΓLHN from serotype E (ΓLHN/E) is recognized in a conformation sensitive ELISA to a much lesser degree (~5-10-fold) compared to the native serotype E neurotoxin (BoNT/E) control, indicating that conformational epitopes are absent and/or buried within the aggregate. Further, animal efficacy data indicate that immunization with aggregated LhWE does not protect animals against BoNT/E toxin challenge.

[009] The relative amount of ΓLHN produced in soluble form versus insoluble form during the fermentation process is a contributing factor to the development of aggregates. Applicants have observed that when the ratio of soluble:insoluble LH_N protein is low (i.e., less of the LH_N protein is in a soluble form) during fermentation, then the greater the tendency of the LH_N to form aggregates, both during the fermentation process itself and during downstream isolation procedures. [010] One method for determining if a protein is expressed in a soluble form during fermentation is to lyse the cultured host cells containing the expressed protein, centrifuge the cell lysate at 25,000xg for 15 minutes to pellet cell debris and aggregated protein material, then analyze the supernatant for presence of the protein. Soluble proteins are found in the supernatant, whereas the pellet contains the insoluble proteins and protein aggregates. SDS-PAGE, followed by a detection method such as Coomassie staining or western blotting, can be used to estimate the amount of protein in each fraction. Thus, it is possible to determine what proportion of a given protein is expressed in soluble form for a given fermentation method.

[011] There is a need for methods of producing neurotoxin fragments, such as LH_N/A, LH_N/B, and LHN/E fragments, that result in an increase in proportion or an increase in total amounts of soluble ΓLHN during fermentation. Methods that increase the proportion or total amount of rLH_N minimize the formation of aggregates, both during the fermentation process and during downstream purification procedures. Accordingly, methods that increase the amount or proportion of soluble protein increase the efficiency and cost- effectiveness of the protein-production process.

SUMMARY OF THE INVENTION

[012] The disclosure provides methods for increasing the proportion or total amount of soluble Clostridium neurotoxin and neurotoxin fragments (for instance, recombinant LH_N) during the fermentation process, thereby reducing or preventing aggregate formation during the production of the neurotoxin. In one embodiment, the fragment is a rl_H_N fragment (a non-toxic derivative of Clostridium botulinum or Clostridium butyricum neurotoxin A, B, C, D, E, F, or G). For instance, in one embodiment, the LH_N fragment is a recombinant fragment chosen from ΓLHN/A, ΓLHN/B, or ΓLHN/E. In another embodiment, the Clostridium neurotoxin fragment comprises LH_N and a portion of the Hc domain as provided in WO 2007/044382, which is herein incorporated by reference in its entirety. In another embodiment, the Clostridium neurotoxin fragment is further modified or fused to one or more additional peptides as disclosed in WO 2004/024909, which is herein incorporated by reference in its entirety.

[013] The invention includes a method for increasing soluble Clostridium neurotoxin or neurotoxin fragment (e.g., rl_H_N) production during fermentation by a novel method of auto-induction. In one embodiment, the invention comprises growing a host cell that has been transformed with a nucleic acid encoding a neurotoxin or neurotoxin fragment (e.g., recombinant LH_N fragment) in a growth-supporting media until the culture reaches a designated OD₆oo or saturation density, and reducing the temperature of the culture below about 25° C.

[014] In some embodiments, the growth-supporting media is a media that supports auto-induction of synthesis of the recombinant LHN fragment. That is, in some embodiments the media is formulated such that it is not necessary to add an exogenous induction agent, such as isopropylthio-β-D- galactoside (IPTG), in order to induce expression of the recombinant LH_N fragment.

[015] In one embodiment of the invention, the Clostridium toxin or toxin fragment is grown in growth-supporting media at about 30° C to 40° C until the culture reaches a designated OD₆oo- In one embodiment, the toxin or toxin fragment is grown in media at about 35° C to 40° C prior to induction. In one embodiment the designated OD_60O is about 15 to 20.

[016] The temperature of the culture is reduced after the culture reaches the designated OD₆oo or saturation density. In one embodiment, the temperature of the culture is reduced to about or less than 20° C for induction. For instance, the invention includes a method for increasing soluble rLH_N production during fermentation by reducing the temperature of the culture to about 10° C , 11⁰ C , 12° C, 13° C, 14° C, 15° C, 16⁰ C, 17° C, 18° C, or 19° C. In another embodiment, the temperature of the culture is reduced to about 15° C to about 20° C.

[017] In one embodiment of the invention, the method for increasing soluble Clostridium toxin or toxin fragment further comprises reducing the dissolved oxygen in the culture after the culture reaches the designated OD_6OO- The invention includes methods of increasing soluble peptide production by reducing the dissolved oxygen in the culture by at least about 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 25 fold, or 25 fold compared to the dissolved oxygen of the culture prior to induction (for instance, at the designated ODβoo or saturation density).

[018] The invention includes reducing the dissolved oxygen of the culture by reducing the agitation of the culture. The reduction of agitation of the culture can occur at or about the same time as the reduction in temperature.

[019] The invention includes, for instance, a method for increasing soluble ΓLHN comprising reducing the dissolved oxygen in the culture to about 0 to 10% DO and reducing the temperature of the culture below about 25° C. In one embodiment, the invention includes a method for increasing soluble ΓLH_N comprising reducing the dissolved oxygen in the culture to about 0 to 5% DO and reducing the temperature of the culture to about 15° C to 17° C.

[020] In one embodiment of the invention, the method for increasing soluble Clostridium toxin and toxin fragment production further comprises, after reducing the temperature of the culture, growing the host cells for an additional period of time under conditions that permit expression of the gene. In one embodiment of the invention, the cells are cultured for an additional about 10 to 25 hours, about 15 to 25 hours or about 15 to 20 hours. In one embodiment of the invention, the cells are cultured an additional about 16 to 20 hours at about 16° C.

[021] Various embodiments of the method further comprise isolating the Clostridium toxin or toxin fragment (e.g., ΓLHN) expressed during the culture. Expressed toxins or toxin fragments can be isolated by methods generally known in the art, for instance, SDS-PAGE. In some embodiments, the Clostridium toxin or toxin fragment (e.g., ΓLHN) resulting from the auto- induction methods of the present invention is purified.

[022] The methods of the invention can be used to produce toxins and toxin fragments that exhibit reduced aggregation as compared to toxins and toxin fragments expressed in the same host cell under conditions that do not result in auto-induction. The amount of aggregation can be determined by methods known in the art, for instance, SDS-PAGE.

[023] Additional objects and advantages of the invention will be set forth in part in the description that follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by the compositions and methods particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[024] Figures 1A and 1B show a Coomassie Blue stained gel and western blot analysis, respectively, of ΓLHN/A fragments in different cellular fractions before and after induction of protein expression.

[025] Figure 2A and 2B also show a Coomassie Blue stained gel and western blot analysis, respectively, of rl_H_N/A fragments expressed in E. coli strain ER2566 following induction of protein expression at different temperatures.

[026] Figure 3 is a graph summarizing the amounts of rl_H_N/A found in the soluble versus insoluble fractions.

[027] Figure 4A and 4B present a Coomassie Blue stained gel and western blot analysis, respectively, of rl_H_N/A fragments expressed in E. coli strain BL21 following induction of protein expression at different temperatures.

[028] Figure 5 is a graph summarizing the amounts of ΓLHN/A found in the soluble versus insoluble fractions.

[029] Figure 6A and 6B are fermentation profiles that plot various culture parameters on the Y-axis against time on the X-axis.

[030] Figure 7A and 7B are Coomassie stained gel and western blot analysis, respectively, of the soluble fraction of cell lysates collected at approximately 20 to 21 hours post-induction in a 5 liter fermentor. [031] Figure 8A and 8B are fermentation profiles that plot various culture parameters on the Y-axis against time on the X-axis for two fermentations of E. coli ER2566 (pET26b-LH_NE (OPT-) in a working volume 10-liter fermentor.

[032] Figure 9A and 9B present a Coomassie Blue stained gel and western blot analysis, respectively, of rl_H_N/B fragments expressed by E. coli ER2566 (pET26b- rl_H_NB-H(-)-L(-) following induction of protein expression at different temperatures.

[033] Figure 10 is a fermentation profile plotting various culture parameters on the Y-axis against time on the X-axis for E. coli ER2566 (pET26b- rl_H_NB-H(-)-L(-) in a 50-liter fermentor in which the induction protocol was applied.

DETAILED DESCRIPTION

[034] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited herein, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated documents or portions of documents defines a term that contradicts that term's definition in the application, the definition that appears in this application controls.

[035] The use of the singular includes the plural unless specifically stated otherwise. The word "a" or "an" means "at least one" unless specifically stated otherwise. The use of "or" means "and/or" unless stated otherwise. The meaning of the phrase "at least one" is equivalent to the meaning of the phrase "one or more." Furthermore, the use of the term "including," as well as other forms, such as "includes" and "included," is not limiting. Also, terms such as "element" or "component" encompass both elements or components comprising one unit and elements or components comprising more than one unit unless specifically stated otherwise. !. Definitions

[036] In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[037] BoNT: botulinum neurotoxin. When a letter follows this designation, the letter indicates the serotype. For example, BoNT/A is botulinum neurotoxin type A.

[038] LHN: a fragment of a clostridial neurotoxin (including, but not limited to, botulinum or tetanus) of approximately 10OkDa which may be a single-chain or di-chain molecule comprising the light chain and the H_N domain. The latter domain represents the N-terminal 5OkDa of the neurotoxin heavy chain and is closely associated with the light chain domain in the fragment. Examples of LHN fragments, including LH_N/A and LH_N/E, are described in United States Provisional Application Nos. 60/929,125, filed June 14, 2007, and 60/960,771 , filed October 12, 2007, the entire disclosure of each of which is relied upon and incorporated by reference. Additional examples include those described in U.S. Patent No. 6,461 ,617 to Shone et al. LH_N fragments may include those fragments possessing, as well as and those fragments lacking, the initial methionine.

[039] LHN fragments include those that have been modified to render them endopeptidase negative. This may be accomplished by mutating the HELIH active site motif found in serotypes A, B, E, F, G, and tetanus neurotoxin to, for example, HQLIY. For botulinum neurotoxin serotype C, the native motif HELNH may be mutated to, for example, HQLNY. For serotype D, the active site motif HELTH may be mutated to, for example, HQLTY.

[040] Endopeptidase negative: displays no endopeptidase activity by conventional assays. In one embodiment, a Clostridium neurotoxin or neurotoxin fragment (e.g., LHN) is made endopeptidase negative by modifying one or two or more amino acids by methods known in the art. Amino acid modifications that confer an endopeptidase negative phenotype, include, but are not limited to modifications at residues E224 and/or H227 for C. botulinum type A neurotoxin or fragments such as LH_N/A, residues E231 and/or H234 for C. botulinum type B neurotoxin or fragments such as LH_N/B, and residues E213 and/or H216 for C. botulinum type E neurotoxin or fragments such as LHN/E. For instance, the invention includes LH_N/A comprising E224Q and/or H227Y modifications, LH_N/B comprising E231Q and/or H234Y modifications and LHN/E comprising E213Q and/or H216Y modifications.

[041] Auto-induction: a fermentation technique in which over- expression of a target protein in a host is induced by reduction of dissolved oxygen (DO) and reduction of temperature. Auto-induction can be performed with small and large culture volumes. Typically, the target protein is expressed from a nucleic acid on a plasmid in a bacterial cell. Typically, cells are grown in a fermentor and induction occurs by (1) by reducing the agitation of cells within the fermentor (which results in a reduction of dissolved oxygen) and (2) reducing the temperature within the fermentor.

[042] Auto-induction does not require the addition of a conventional inducer, such as IPTG, during the culture process. However, auto-induction can be induced by contacting the cells with an inducer such as IPTG and reducing the temperature of the culture.

[043] Soluble: a "soluble" recombinant protein is one that exists in solution in the cytoplasm of the host cell. If the protein contains a signal sequence the soluble protein is exported to the periplasmic space in bacteria hosts and is secreted into the culture medium in eukaryotic cells capable of secretion or by bacterial host possessing the appropriate genes. In contrast, an insoluble protein is one that exists in denatured form inside cytoplasmic granules (called an inclusion bodies) in the host cell. A soluble protein is a protein that is not found in an inclusion body inside the host cell or is found both in the cytoplasm and in inclusion bodies and in this case the protein may be present at high or low levels in the cytoplasm. A soluble protein is distinct from a "solubilized" protein. An insoluble recombinant protein found inside an inclusion body may be solubilized (i.e., rendered into a soluble form) by treating purified inclusion bodies with denaturants such as guanidine hydrochloride, urea or sodium dodecyl sulfate (SDS). These denaturants must then be removed from the solubilized protein preparation to allow the recovered protein to renature (refold). A distinction is also made between proteins that are soluble (i.e., dissolved) in a solution devoid of significant amounts of ionic detergents (e.g., SDS) or denaturants (e.g., urea, guanidine hydrochloride) and proteins that exist as a suspension of insoluble protein molecules dispersed within the solution. A soluble protein will not be removed from a solution containing the protein by centrifugation using conditions sufficient to remove bacteria present in a liquid medium (e.g., centrifugation at 5,00Og for 4-5 minutes). A method of testing whether a protein is soluble or insoluble is described in U.S. Patent No. 5,919,665.

[044] Dissolved Oxygen (DO): refers to the amount of oxygen dissolved in the culture and is typically expressed as percent DO.

[045] At the beginning of fermentation, typically, aeration is set at about 0.5 or 1.0 WM, agitation at about 200 to 400 rpm, and DO calibrated to about 100%. As the culture grows, oxygen is utilized and the DO decreases. Typically, the DO drops to about 30% at which point, agitation is increased to maintain a 30% DO until the culture reaches the designated cell density and/or growth phase (e.g., about 15-20 OD₆oo). At the designated cell density and/or growth phase, agitation is decreased (e.g., to about 200 to 400 rpm) which, in turn, causes the DO to decrease to about 0-5%.

[046] Auto-induction can be achieved by reducing the DO of a culture while also reducing the temperature of the culture. For instance, auto- induction can be achieved in the present invention by reducing the DO of the culture by at least about 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold or 50 fold or more compared to the DO of the culture prior to auto-induction.

II. Methods For Increasing The Proportion Or Total Amount of Soluble Clostridium Toxin or Toxin Fragments (e.g., rLH_N Fragments) [047] The invention provides methods of increasing soluble Clostridium toxin or toxin fragments (e.g., ΓLHN) production during the culture process. In general, the culture method results in the production of more soluble protein than insoluble protein. For instance, in one embodiment, the soluble rLH_N protein is at least 60% of total rl_H_N protein, although it may also be at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or even at least 98% of the total rl_H_N protein produced in that culture. In another embodiment, however, it is the total amount of rl_H_N protein that increases. Increases may be measured relative to a culture process in which the induction temperature remains above 25 ⁰C or above 20 ⁰C. Alternatively, increases may be measured by comparison to a culture process that employs a growth-supporting media that does not comprise both lactose and a carbon source. It is also possible for the method to result in an increase in both the relative percentage of soluble protein and the total amount of soluble protein.

[048] The methods of the present invention can be used to increase or enhance the expression of a variety of Clostridium neurotoxins or neurotoxin fragments, including, but not limited to, BoNT/A, BoNT/B, BoNT/E, BoNT/F, BoNT/G, tetanus, LH_N/A, LH_N/B, LH_N/E, LH_N/F, LH_N/G, LH_N tetanus, Clostridium neurotoxins and fragments disclosed in WO 2007/044382 and WO 2004/024909, and fusion proteins comprising a Clostridium neurotoxin or toxin fragment (e.g., his-tagged LHN).

[049] A. Bacterial Cells and Vectors

[050] Generally, in order to express a Clostridium neurotoxin or toxin fragment, such as an rl_H_N fragment, a suitable cell line is transformed with a DNA sequence encoding the neurotoxin or toxin fragment under the control of known regulatory sequences. Genes encoding botulinum neurotoxins are available with codon bias appropriate for expression in a variety of host cells, and any of a wide variety of host cells are suitable for expression of a Clostridium neurotoxin or toxin fragment (e.g., rl_H_N fragment).

[051] In one embodiment of the present invention, bacterial cells are hosts cells for expression of the Clostridium toxin or toxin fragment (e.g., rLHN). For example, various strains of E. coli (e.g., BL21(DE3), ER2566, HB101 , MC1061) are well-known as host cells in the field of biotechnology. Various strains of B. subtilis, Pseudomonas, other bacilli, and the like may also be used. For expression of a protein in bacterial cells, DNA encoding the propeptide is generally not necessary.

[052] The host cells used in the fermentation method of the present invention are generally a strain of Escherichia coli. In many of those embodiments, the E. coli strain expresses in its chromosome the coding sequence for T7 RNA polymerase under the control of an inducible lacUV5 promoter. Non-limiting examples of E. coli strains that are suitable for use in auto-induction methods include ER2566 and BL21 (DE3).

[053] In another embodiment, suitable host cells or cell lines may be mammalian cells, such as Chinese hamster ovary cells (CHO), the monkey kidney COS-1 cell line, or mammalian CV-1 cells. The selection of suitable mammalian host cells and methods for transformation, culturing, amplification, screening, product production and purification are known in the art. (See, e.g., Gething and Sambrook, Nature, 293:620-625 (1981); Kaufman et al., MoI Cell Biol., 5(7):1750-1759 (1985); Howley et al., U.S. Patent 4,419,446.)) Yeast cells such as Pichia pastoris may also be used.

[054] The expression vector encoding the Clostridium neurotoxin or toxin fragment (e.g., LHN fragment) can vary widely in its components, but they will include those regulatory elements appropriate to the host cell selected. The neurotoxin or neurotoxin fragment (e.g., LHN fragment) may be under the control of a promoter, for instance, a T7 promoter. As can be appreciated by a skilled artisan, various types of vectors can be used to express the Clostridium neurotoxin and fragments using the methods of the invention. For instance, in some embodiments, the expression vector includes, but is not limited to, pET26b (Novagen).

[055] Nucleic acid sequences encoding various botulinum neurotoxins have been cloned and those nucleic acid sequences are known in the art. For example, BoNT/E from C. botulinum is provided in GenBank accession no. AB082519. A nucleic acid sequence of a full length neurotoxin E from C. butyricum is provided in GenBank accession no. AB088207. [056] Methods of manipulating nucleic acids and of expressing the encoded proteins are known in the art, and include those described in Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor) and Current Protocols in Molecular Biology (Eds. Aufubel, Brent, Kingston, More, Feidman, Smith and Stuhl, Greene Publ. Assoc, Wiley-lnterscience, NY, N.Y., 1992). Thus, it is possible to modify a nucleic acid sequence by replacing the codon for one amino acid with a codon for another amino acid. Methods of manipulating botulinum neurotoxin nucleic acid sequences to obtain fragments, including LH_N fragments, are described in U.S. Patent No. 6,461,617 and WO 2007/044382. In addition, WO 2007/044382 describes methods of modifying those nucleic acids to abolish endopeptidase activity.

[057] The invention also encompasses LH_N fragments in which the solubility has been improved by modifications to the amino acid sequence. Examples of solubility-improving modifications are described in WO 2007/044382. By way of example, certain cysteine residues in the LHN fragment may be replaced with another amino acid that does not form a disulfide bond, such as serine. Alternatively, or in addition, solubility may be improved by extending the sequence of an LHN fragment by providing additional sequences from an adjoining segment, such as the Hc fragment.

[058] In some embodiments, the vector encodes an LHN fragment that is expressed as a fusion protein with, for example, a tag to increase the stability of the resulting fusion protein or to simplify purification. Such tags are known in the art. Representative, non-limiting, examples include sequences that encode a series of histidine residues, the epitope tag FLAG, the Herpes simplex glycoprotein D, beta-galactosidase, maltose binding protein, streptavidin tag or glutathione S-transferase.

[059] B. Auto-induction

[060] In some embodiments, the bacterial cells are cultured on a growth-supporting media that supports auto-induction of synthesis of the recombinant LHN fragment from the expression vector. Thus, in these embodiments, the media is formulated such that it is not necessary to add an exogenous induction agent, such as isopropylthio-β-D-galactoside (IPTG), in order to induce expression of the recombinant LH_N fragment. That is, when cultured in a medium containing lactose, there is little expression the LH_N fragment at early stages of the culture, but protein expression is turned on automatically at a later (high-density or saturation) stage without any intervention. The general conditions necessary for auto-induction in high- density bacterial cultures are described in US 2004/0180423 to Studier, which is incorporated by reference in its entirety.

[061] In brief, a strain of E. coli that expresses the coding sequence for T7 RNA polymerase under the control of an inducible promoter, for instance, lacUVδ promoter, and contains a vector containing the recombinant LH_N fragment under the control of the T7 promoter is inoculated into culture medium. The medium can vary in its constituents, but it should include lactose and at least one other carbon source. It should also be buffered such that, even at saturation, the pH of the culture remains near neutral. The cells are cultured without intervention and allowed to reach saturation.

[062] Saturation density can be determined by measuring the optical density (OD) of the culture. Typically, OD monitoring of the culture is performed by taking a culture sample from the fermentor, diluting the sample with Dl water or saline buffer, and measuring the diluted sample at 600 nm wavelength using a UV/Vis or Vis spectrophotometer. Saturation densities often have an OD₆oo range of around 10-20, but may reach as high as 30-50. However, lower saturation densities, such as around 5, may occur if the expressed protein is one that affects cell growth. In one embodiment of the invention, the OD of the culture is allowed to reach about 15-20 OD₆oo before the induction phase is initiated.

[063] The induction phase is initiated by the reduction of temperature of the culture and reduction of the dissolved oxygen in the culture. In one embodiment of the invention, during the induction phase, agitation of the culture is reduced to reduce the dissolved oxygen content of the culture to about 0-5%DO. [064] During the induction period, the reduced temperature and percent dissolved oxygen of the culture are maintained. The induction period typically lasts at least about 10 hours, about 12 hours, or about 15 hours. However, as can be appreciated by a skilled artisan, the induction period can vary considerably based on the desired amount of soluble Clostridium neurotoxin protein or protein fragment (e.g., LHN).

[065] C. Induction Temperature

[066] The incubation temperature of the fermentation culture during the induction phase affects the total amount of soluble Clostridium toxins or toxin fragments (e.g., rl_H_N) that is expressed, as well as the relative proportion of soluble to insoluble ΓLHN fragments. Accordingly, in some embodiments, the methods involve induction at a temperature that is lower than the initial culture temperature. In certain embodiments, induction is via auto-induction. In other embodiments, induction is by conventional methods, such as addition of IPTG.

[067] The methods of the invention generally employ an induction temperature that is between about 15°C and about 35°C. In this regard, the minimum induction temperature is typically less than or about 15⁰C, about 16⁰C, about 17⁰C, about 18⁰C, about 19⁰C, about 2O⁰C, about 25⁰C, or about 3O⁰C. The induction temperature may also be expressed as a range. Thus, some embodiments involve an induction temperature of between about 15 to about 35⁰C, between about 15 to about 3O⁰C, between about 15 to about 25⁰C, between about 15 to about 2O⁰C, between about 15 to about 19⁰C, between about 15 to about 18⁰C, between about 15 to about 17⁰C, and between about 15 to about 16⁰C. In other embodiments, the induction temperature is a range of between about 16 to about 35⁰C, between about 16 to about 3O⁰C, between about 16 to about 25⁰C, between about 16 to about 2O⁰C, between about 16 to about 19⁰C, between about 16 to about 18⁰C, and between about 16 to about 17⁰C. In still other embodiments, the induction temperature is a range of between about 17 to about 35⁰C, between about 17 to about 3O⁰C, between about 17 to about 25⁰C, between about 17 to about 2O⁰C, between about 17 to about 19⁰C, and between about 17 to about 18⁰C. In yet other embodiments, the induction temperature is a range of between about 18 to about 35⁰C, between about 18 to about 3O⁰C, between about 18 to about 25⁰C, between about 18 to about 2O⁰C, and between about 18 to about 19⁰C. Still other embodiments involve an induction temperature that is in a range of between about 19 to about 35⁰C, between about 19 to about 3O⁰C, between about 19 to about 25⁰C, between about 19 to about 2O⁰C, between about 20 to about 3O⁰C, and between about 20 to about 25⁰C. In some embodiments, the induction temperature is about 16⁰C.

[068] D. Induction Reduction of Dissolved Oxygen

[069] The methods of the present invention include reducing the amount of dissolved oxygen to stimulate auto-induction. For instance, the amount of dissolved oxygen can be reduced at or about the time the temperature of the culture is reduced, to increase the ratio of soluble protein to insoluble protein expressed by the host cell.

[070] In one embodiment of the invention, the cell culture is grown at a level of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% dissolved oxygen prior to induction. In one embodiment, for instance, prior to induction, the level of dissolved oxygen decreases from about 100% to 30%. When the dissolved oxygen level gets to about 30%, the dissolved oxygen level is maintained by increasing agitation. Upon induction, agitation is decreased so that the dissolved oxygen level decreases. By decreasing agitation, the dissolved oxygen level can be decreased to as little as about 0-5% DO.

[071] The amount of dissolved oxygen at induction and continuing during the induction period can be about 0-3%, 0-5%, 0-10%, 5-10%, 5-15%, 0-15%, or less than 20% DO. The reduction of dissolved oxygen at induction and continuing during the induction period can be at least about 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 20 fold, 25 fold, 30 fold, 35 fold, 40 fold, 45 fold or even 50 fold less than the amount of dissolved oxygen in the culture immediately prior to induction (for instance, at about the time of the determination of saturation density, e.g., OD₆₀O= about 15-20). IH. Applications

[072] Neurotoxic proteins and fragments of those proteins are important immunodiagnostic reagents, therapeutic agents, and vaccine components. Functional neurotoxins are hazardous to work with, however, so investigators prefer to use recombinant proteins that have been genetically modified to reduce or eliminate their neurotoxicity. Unfortunately, it can be difficult to purify some of the recombinant, non-toxic, proteins because they often form aggregates, which have reduced solubility and are less effective reagents for use in immunodiagnostic, therapeutic, and vaccine applications. For example, although aggregated ΓLHN/E can be purified, it is recognized in a conformation-sensitive ELISA to a much lesser degree (~5-10-fold) than is the native BoNT/E toxin, indicating that conformational epitopes are absent and/or buried within the aggregate. Also, immunization with aggregated LHN/E does not protect animals against BoNT/E toxin challenge. Accordingly, the disclosure provides methods for reducing or preventing the formation of aggregates during the fermentation process.

[073] The toxin fragments of the invention can be used as therapeutic agents and vaccine components. Any animal that is susceptible to the wild- type toxin can be vaccinated with the toxin fragment in an immunostimulatory composition. Accordingly, a vaccine composition comprising a clostridial neurotoxin rl_H_N fragment can be used to protect rabbits, rodents, birds, horses, cattle, and humans, including infant humans, from botulism, or from one or more of the symptoms of botulism, such as diarrheal disease, paralysis (either mild or severe), or death.

[074] The immunogenicity of the recombinant proteins can be tested by immunizing mice with, for example, 10 μg of LH_N protein suspended in an adjuvant emulsion. Control mice are immunized with saline emulsified in adjuvant for use as negative controls. The mice are immunized i.p. four times at 2-week intervals. One week after the last immunization, the mice are bled and the serum is analyzed by immunoblot for the presence of specific antibody. ELISA is used to determine the titer of the antisera. Two weeks after the last immunization, each mouse is challenged i.p. with 2 lethal doses of the corresponding BoNT protein. Four days after challenge, the mice are scored for survivors.

[075] Toxin fragments can also be used to prepare compositions comprising neutralizing antibodies that immunoreact with the wild-type toxin. The resulting antisera can be used for the manufacture of a medicament for treating exposure to clostridial neurotoxin. The antisera, or antibodies purified from the antisera, can also be used as diagnostic agents to detect either the LHN fragment or the native protein.

[076] The invention will be further clarified by the following examples, which are intended to be purely exemplary of the invention and in no way limiting.

IV. Examples

[077] Example 1. Detection of ΓLHN/A in Soluble and Insoluble Fractions

[078] The fraction of rl_H_N/A in soluble and insoluble form was evaluated using E. coli BL21 (DE3) and E. coli ER2566. Each strain was transfected with the rl_H_N/A expression vector pET26b-LHnA-H^"-L^". Cultures were grown in a Fernbach containing 500 ml 2xYT. Cells grew at 37 ⁰C to approximate OD of 1.0, then were cooled to 30 ⁰C for induction. After induction for 3 hours, the cells were collected for processing and analyses.

[079] The total cell lysates from before and after induction, the insoluble fraction, and the soluble fractions were prepared and analyzed by SDS-PAGE, western blotting, and ELISA. Figure 1A shows a 4-12% SDS- PAGE gel in which different fractions are compared for the two cultures. Lanes 1 and 7 are molecular weight markers. Lanes 2-6 contain samples from the BL21 culture, while lanes 8-12 present the corresponding samples from the ER2566 cultures. Lanes 2 and 8 are lysates. The insoluble fractions are shown in lanes 3 and 9, while lanes 4 and 10 are the soluble fractions. The final two lanes in each panel compares whole cells 3 hours after induction (lanes 5 and 11) to whole cells pre-induction (lanes 6 and 12). Figure 1B is the corresponding western blot.

[080] The SDS-PAGE results did not definitively indicate how much rLH_N/A protein was present in the soluble fraction. However, the western blot analysis indicates that, for both E. coli strains tested, the majority of the rl_H_N/A was present in the insoluble fraction, with much less ΓLHN/A in the soluble fraction. There was no detectable western signal for the whole cell samples before induction. From the western blot analysis, it seems that the strain ER2566 (pET26b-LHnA-H (-)-L (-)) was better than BL21 (DE3) (pET26b-LHnA-H (-)-L (-)) for rl_H_N/A expression in 2xYT medium.

[081] Using an ELISA, we were able to determine the concentration of the total target protein (i.e., irrespective of whether it was monomeric or polymeric). The ELISA results for ΓLHN/A protein in the SDS samples are shown in Table 1. The sample ID number corresponds to the lane numbers in Figure 1A. The values are presented in mg/L and are adjusted to the original fermentation culture volume.

[082] Table 1 : ELISA Results

[083] Although strain ER2566 seemed to produce more protein than BL21 by western blot, the results for each strain were similar by ELISA.

[084] Example 2. Effect of Temperature on ΓLHN/A Protein Expression [085] To investigate the effect of temperature on production of rLH_N/A protein following auto-induction, E. coli ER2566(pET26- LHnA-H (-)-L (-)) #1 and E. coli BL21(DE3)(pET 26b-LHnA-H (-)-L (-)) clone2, were cultured and then auto-induced at different temperatures. The yield of rLH_N/A was analyzed by SDS-PAGE, Western blot, and ELISA after the cells were harvested and ruptured by microfluidizer.

[086] For this experiment, both E. coli BL21 (DE3) (pET26b-LHnA-H - L^") and E. coli ER2566 (pET26b-LHnA-H^"-L^~) were grown in a Fernbach containing 500 ml YESPG. Cells grew at 37°C to ~ OD of 4, then were cooled down to experimental temperature for induction using the auto-induction method. After auto-induction for 3 hours, cells were collected for processing and analyses.

[087] Table 2 presents a comparison of the wet culture weights ("WCW") and OD₆oo values observed for each strain at induction temperatures of 15, 20, 25, 30, and 35⁰C.

[088] Table 2. Effect of Induction Temperature on Wet Culture

Weights

[089] Figure 2A shows a 4-12% SDS-PAGE analysis of samples from the E. coli ER2566 (pET 26b-LHnA- L^" H) culture. Samples were also western blotted to detect rLH_N/A protein. Those results are shown in Figure 2B. In Figures 2A and 2B, lane 1 is the marker. Lane 2 is a pre-induction sample. The odd numbered lanes are soluble fractions, while the even numbered lanes are insoluble fractions for each temperature. Lanes 3 and 4: 15°C induction; lanes 5 and 6: 20⁰C induction; lanes 7 and 8: 25⁰C induction; lanes 9 and 10: 3O⁰C induction; and lanes 11 and 12: 35°C induction. The data is presented graphically in Figure 3.

[090] The corresponding SDS-PAGE and western blots for strain BL21 are presented in Figures 4A and 4B, respectively. The lanes are the same as indicated for Figure 2. The results for strain BL21 are summarized graphically in Figure 5.

[091] For strain ER2566, an induction temperature of 20⁰C resulted in the greatest total amount of ΓLHN/A in the soluble fraction. But, the ratio of soluble to insoluble rLH_N/A was slightly higher at 15°C compared to 20°C. When strain BL21 was evaluated, induction temperatures of 2O⁰C and 25⁰C gave similar amounts of rl_H_N/A in the soluble fraction. As the induction temperature decreased from 25⁰C to 20⁰C, the ratio of soluble to insoluble target protein improved. As for strain ER2566, the greatest ratio of soluble:insoluble rl_H_N/A was observed at 15⁰C.

[092] The results indicate that induction temperature affects soluble protein both the total amount and the relative ration of soluble rl_H_N/A production in both strains. The maximum soluble rl_H_N/A protein production was observed when induced at 20-25⁰C. In addition, as the induction temperature decreased, the proportion of soluble rl_H_N/A increased.

[093] Example 3. Effect of Temperature on Large Scale Production of rLHu/A Protein Expression

[094] This section describes the development of a 5 liter fermentation process for the production of LH_N/A, using E. coli ER2566 (pET 26b- LH_NA - Histag (-)-leader (-)). We have shown that LH_N/A cloned on plasmid pET26b into E. coli ER2566 was expressed when this strain was grown and induced by 1 mM IPTG in a shake flask. However, commercial scale production of LH_N/A requires mass cultivation in a fermentor. To become a commercially viable product, it is essential to achieve the optimal expression and production of soluble LH_N/A per unit of volume. We therefore sought ways to enhance expression of the soluble form of LH_N/A when the host cells are cultured using a 5 liter fermentor.

[095] The construction of recombinant E. coli ER2566 (pET 26b- LH_NA -Histag (-)-leader (-)) was similar to that of recombinant E. coli ER2566 (pET 26b- LH_NB -Histag (-)-leader (-)). The growth of the host strain and the production of LHN/A and LH_N/B were comparable. An auto-induction protocol was applied to the production of the LH_NB protein at HPA. Recombinant cells were grown to high cell density and then "auto-induced" to produce the target protein by a lowering of oxygen tension. During this auto-induction phase, the temperature of the fermentation was decreased in order to increase the fraction of the target protein in soluble form. We have confirmed that similar conditions of growth and induction are applicable to the production of LH_NA. [096] 1. Preparation of Enhanced TB Medium [097] For the fermentation studies, the medium used was an enhanced TB medium. The components of Enhanced TB are set out in Table

3.

[098] Table 3. Enhanced TB Medium

[099] To prepare the medium, the glycerol, antifoam, peptone and yeast extract components are added to 2 liters warm de-ionized water and stirred to dissolve. The volume is adjusted to 3 liters, then the medium is sterilized in autoclave at 122.5 ± 1.5°C for 30 minutes. The phosphate salts are dissolved in 800 mL de-ionized water, and the MgSO₄ is dissolved in 80 mL de-ionized water. Both are then sterilized by autoclaving at 122.5 ± 1.5°C for 20 minutes. After each component has cooled to ambient temperature, 80OmL of the phosphate solution and 8OmL of the MgSO₄ solution are added aseptically to the fermentor. The pH of the medium should be 7.2-7.4, and can be adjusted, if needed, with 40% H₃PO₄ / 10N NaOH to maintain the appropriate pH. Antibiotic is added via sterile filtration just prior to use. [0100] 2. General Growth and Auto-induction Method

[0101] Primary seed

[0102] Five μl of a 1/1000 dilution of a sample of E. coli ER2566 (pET 26b- LHuA -Histag (-)-Lead (-)) was inoculated into a 500-ml flask containing 50 ml of enhanced TB medium. The seed was incubated in a 37°C warm- room on an Innova 2100 Platform Shaker at 150 rpm for 14 - 16 hours. The final OD₆Oo was at 4 - 8. ro 103] Second seed

[0104] Five milliliter of the primary seed culture was transferred to a Fernbach flask containing 500 ml of the enhanced TB medium. The seed was incubated in the 37°C warm-room on an Innova 2100 Platform Shaker at 150 rpm in for 6.5 hours. The final OD₆oo was 2 - 4.

[0105] Fermentation in 5-liter fermentor

[0106] Prior to inoculation in the 5 liter fermentor, the fermentation medium in the fermentor was conditioned at 37°C with agitation at 600 rpm and aeration at 2.5 liter/minute for 1 - 2 hours. Dissolved oxygen (DO) was set to 100%. Fermentation was started by transferring 225 ml of the second seed to the fermentor containing 4.5 L medium in order to achieve a starting cell density of 0.1-0.2 at OD₆oo-

[0107] After inoculation, the growth conditions were established as follows: temperature to 37°C; agitation started at 200 rpm and set in cascade- controlled mode; dissolved oxygen set at 50% and controlled by agitation; maximal agitation set at 1000 rpm; Airflow set at 0.5 vvm, and pH set at 7.0 and controlled by 40% H₃PO₄ and 10 N NaOH. When the cells grew to a cell density of 10 to 20 OD₆oo (usually within 4 to 6 hours), the growth temperature was decreased to 16°C or other experimental temperature. At the time the temperature was decreased, agitation was also decreased to 525 rpm to minimize the dissolved oxygen in the growing culture. In the fermentations, dissolved oxygen (DO) was maintained at between 10 and 20%, the lowest achievable levels under these conditions. At the end of the fermentation, cells were harvested by centrifugation at 3800 rpm and 4 - 8 ⁰C in Son/all Centrifuge RC12BP equipped with Sorvall H12000 rotor for 30 minutes. The supernatant solution was disinfected before discard. The cell paste was stored at -70 to -80 °C.

[0108] Two examples of a fermentation profile are shown Figures 6A and 6B. Each graph shows the OD600, %DO, agitation rate, temperature, and LH_N/A (in mg/L) as a function of time. The profiles indicate that the production LH_N/A reached maximal levels when the DO started rising again late in the induction period.

[0109] At different time points during the induction period, 20 ml of culture was centrifuged at 7200 g for 10 min. The cell paste was kept at -20 for later assay by ELISA for LH_N/A titer, BCA protein, SDS-PAGE, and Western blot. To prepare the samples for assays, the cell paste was resuspended in 200 ml of 20 mM Tris and 25 mM EDTA buffer at pH 8.0. The cell lysate was obtained for assay by passing the cell suspension through a microfluidizer at 18000 psi for one passage. The supernatant solution was separated by centrifugation of the lysate sample at 20,800 g for 30 min, followed by removal of the material at the top of the tube.

[0110] Samples collected before induction, at 20.5 hours post induction, and at 21.5 hours post induction were analyzed by SDS-PAGE (Figure 7A) and western blotted using an anti-U-VA antisera (Figure 7B). Most of the material recognized by the anti-LhWA polyclonal antiserum in the western blots was the full-length [-97 kDa] form, but lower molecular weight proteins were also recognized under the assay conditions.

[0111] Using this auto-induction based fermentation method, we have been able to produce 600 to 1200 mg per liter of LHN/A in successive runs in a 5-liter fermentor.

[0112] Effect of Auto-Induction Temperature on Protein Production

[0113] The temperature during the auto-induction phase of small volume cultures greatly affected both the proportion of protein that was expressed in soluble form during culture and the total amount of soluble protein that was produced. Therefore, we evaluated the effect of temperature on protein production in the five liter fermentor. The results are shown in Table 4. Protein titers were determined by ELISA. [0114] Table 4. Effect of Auto-Induction Temperature on Protein Production

[0115] Maintaining the temperature during the auto-induction stage at between 16 and 20 ⁰C, resulted in greater expression of LhWA.

[0116] Effect of Agitation During Auto-Induction

[0117] In an effort to reduce the dissolved oxygen (DO) to zero during the auto-induction phase, we decreased agitation to 400 rpm. However, as shown in Table 5, the DO levels failed to drop to 0 and higher production of ΓLH_N/A was not achieved.

[0118] Table 5. Effect of Reduced Agitation

[0119] Conclusions

[0120] Our results show that it is possible to significantly improve the yield of soluble rl_H_N/A by using a reduced induction-phase culture temperature. Although reducing the induction temperature to around 20⁰C increased the total amount of soluble rl_H_N/A produced, we found that we could achieve a better ratio of soluble:insoluble ΓLHN/A by using an even lower induction temperature, such as 16°C. Our results suggest that this effect is scalable, in that we were able to improve soluble ΓLHN/A production even in a 5 liter fermentation setting.

[0121] Example 4. Production of ΓLHN/E

[0122] For the production of rl_H_N/E by recombinant E. coli ER2566 (pET 26b- LHNE (OPT-), seed cultures were grown in two stages before the second seed culture was transferred to a 10-liter Bioflo 310 fermentor. During the initial growth in the fermentor the temperature was kept at 37 ⁰C. The pH of the culture was maintained at 7.0 by phosphoric acid and sodium hydroxide. The dissolved oxygen (DO) was maintained at 30% by agitation until the agitation rate reached 1200 rpm. After the culture reached an OD₆oo between 10 and 15, the temperature of the culture was decreased to 16 ⁰C, airflow was reduced to 0.5 vvm (medium contains 25 g/L glycerol), and agitation was reduced to 500 rpm in order to maintain the DO at the lowest possible level greater than 0. The culture was maintained at 16⁰C, a sparge of 0.5 vvm, and agitation at 500, for 20 more hours.

[0123] Figures 8A and 8B show two fermentation profiles for E. coli ER2566 (pET 26b- LH_NE (OPT-) in a working volume 10-liter fermentor, Bioflo 310, containing modified Terrific broth and 50 g/L glycerol. The agitation was set at 500 rpm after inoculation and cascade control was used to maintain DO at 30%. The experimental results are summarized in Table 6.

[0124] Table 6. rLH_N/E Fermentation

[0125] Example 5. Effect of Autoinduction Temperature on ΓLHN/B Production

[0126] Using E. coli ER2566 (pET26b- rl_H_NB-H(-)-L(-), we also investigated the effect of induction temperature on the soluble rLH_N/B protein expression. Cells were grown at 37 ⁰C in a shake flask containing 2xYT. When the culture reached an A600 of 0.5 to 1.0, IPTG was added and a culture flask was incubated in an incubator shaker i which temperature had been pre-set. Three hours after induction, cells were collected and analyzed by SDS-PAGE and western blot.

[0127] Figure 9A shows an SDS-PAGE analysis of samples for the production of rl_H_N/B by E. coli ER2566 (pET26b- rLH_NB-H(-)-L(-) after induction at the indicated temperature for approximately 3-5 hours. Samples were also western blotted to detect ΓLHN/B protein. Those results are shown in Figure 9B. In Figures 9A and 9B, lanes 1-6 are soluble fractions of cell lysates in D-PBS+25 mM EDTA after microfluidizer process; lanes 7- 12 are cell paste suspensions in D-PBS+25 mM EDTA. Lanes 1 and 7: 15⁰C induction; lanes 2 and 8: 20⁰C induction; lanes 3 and 9: 25°C induction; lanes 4 and 10: 29°C induction; lanes 5 and 11: 33°C induction; and lanes 6 and 12: 37°C induction. These results indicate very good expression and production of soluble ΓLHN/B protein was observed for all induction temperatures below 33 ⁰C.

[0128] We also prepared 50 liter fermentation cultures for ΓLHN/B production. One tenth milliliter E. coli ER2566 (pET26b- rl_H_NB-H(-)-L(-) obtained from cell bank cryobial culture was inoculated into each of 2 primary seed cultures containing 10 ml_ of Phytone Peptone L-Broth and 30 mg/L kanamycin. After inoculation, these two primary cultures were incubated for 7 hours at 37 °C and 150 rpm. Two milliliter of bulk primary seed was inoculated into each of 5 secondary seed cultures containing 200 mL of Phytone Peptone based production medium, modified Terrific medium. These secondary seed cultures were incubated for 6-8 hours at 37 ⁰C and 150 rpm. The secondary seed culture was then bulk transferred into a 1000 mL sterile Duran bottle ready for fermentor inoculation. [0129] The 1000 ml_ secondary culture of E. coli ER2566 (pET26b- rLH_NB-H(-)-L(-) was inoculated into a 50 L working volume, stirred fermentor containing modified Terrific production medium and grown at 37 ⁰C until OD reached 15-20. The batch was then auto-induced at 16 ⁰C for 20-22 hours. During the induction period, dissolved oxygen (DO) was reduced below 10% by decreasing agitation and airflow. A typical fermentation result is shown in Figure 10 and in Table 7.

[0130] Table 7. Chemical Analyses of the LHN/B 50 L Fermentation

Samples

[0131] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for increasing soluble Clostridium neurotoxin or neurotoxin fragment production during fermentation comprising: a) growing a host cell that has been transformed with a gene encoding a Clostridium neurotoxin or neurotoxin fragment in a growth-supporting media at about 35-40 ⁰C until the culture reaches saturation density; b) reducing the temperature of the culture below about 30° C; and c) reducing the dissolved oxygen in the culture, wherein reduction of the temperature of the culture and reduction of the dissolved oxygen content in the culture increases the production of soluble Clostridium neurotoxin or neurotoxin fragment.

2. The method of claim 1 , wherein said saturation density is determined by measuring an OD₆oo greater than about 5.

3. The method of claim 1 , wherein said saturation density is determined by measuring an OD₆oo about 15-30.

4. The method of claim 1 , wherein said saturation density is determined by measuring an OD₆oo about 15-25.

5. The method of claims 1-4, wherein said temperature of the culture is reduced below or at about 25° C.

6. The method of claims 1-4, wherein said temperature of the culture is reduced below or at about 20° C.

7. The method of claims 1-4, wherein said temperature of the culture is reduced below or at about 18 ° C.

8. The method of claims 1-4, wherein said temperature of the culture is reduced below or at about 17 ° C.

9. The method of claims 1-4, wherein said temperature of the culture is reduced below or at about 16 ⁰ C.

10. The method of claims 1-4, wherein said temperature of the culture is reduced below or at about 15 ° C.

11. The method of claims 1-10, further comprising (d) growing host cells for at least about 5 hours under the conditions of steps (b) and (c).

12. The method of claims 1-10, further comprising (d) growing host cells for at least about 10 hours under the conditions of steps (b) and (c).

13. The method of claims 1-10, further comprising (d) growing host cells for at least about 15 hours under the conditions of steps (b) and (c).

14. The method of claims 1-13, wherein said dissolved oxygen in the culture is reduced by reducing agitation of the culture.

15. The method of claims 1-14, wherein said dissolved oxygen is reduced to about 0-10% DO.

16. The method of claims 1-14, wherein said dissolved oxygen is reduced to about 0-5% DO.

17. The method of claims 1-16, wherein said growth-supporting media comprises lactose and an alternate carbon source.

18. The method of claims 1-17, wherein said host cell is E. coli.

19. The method of claims 1-18, wherein said Clostridium neurotoxin or neurotoxin fragment is endopeptidase-negative.

0. The method of claims 1-19, wherein said Clostridium neurotoxin or neurotoxin fragment is an ΓLHN fragment from neurotoxin A, B, or E.