RPA optimization

Abstract

An optimized synthetic polynucleotide encoding a Bacillus anthracis protective antigen and an anthrax vaccine based on the encoded protective antigen. Furthermore, heterologous expression in a host Pseudomonas fluorescens bacteria of an optimized polynucleotide sequence encoding a Bacillus anthracis protective antigen.

Description

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 3, 2010, is named 94725201.txt and is 43,572 bytes in size.

FIELD OF THE INVENTION

The present invention relates generally to a Bacillus anthracis protective antigen-based anthrax vaccine. In particular, the invention relates to heterologous expression bacteria of an optimized polynucleotide sequence encoding a Bacillus anthracis protective antigen in a bacterial host.

BACKGROUND OF THE INVENTION

Anthrax is a zoonotic disease whose etiologic agent is a gram-positive sporulating bacterium, B. anthracis. Human beings can acquire it via infected animals or contaminated animal products. The etiologic agent of anthrax (Bacillus anthracis) is a potential threat as an agent of biowarfare or bioterrorism because exposure to aerosolized B. anthracis spores can be lethal to mammals, such as humans. Vaccination is currently thought of as the most effective way to protect individuals and entire populations from an anthrax infection. Virulence of B. anthracis is due to two major antigens: the antiphagocytic capsular antigen and the anthrax toxin. The antiphagocytic capsular antigen does not protect against anthrax infection. However, the anthrax toxin is highly immunogenic and is the basis for successful anthrax vaccines.

The anthrax toxin has three peptide components: the protective antigen (PA; 83 kDa), the lethal factor (LF; 90 kDa), and the edema factor (EF; 89 kDa). The EF is a calcium-calmodulin-dependent adenylate cyclase believed to cause the edema associated with anthrax infection and to prevent immune cells from ingesting and degrading the bacteria. The LF is a cell-type specific metalloprotease that cleaves mitogen-activated protein kinase-kinases and several peptide hormones. It causes macrophage cell death and release of toxic substances (e.g., those associated with septic shock such as TNF-α and IL-1). LF is the major virulence factor associated with anthrax toxicity and is responsible for systemic shock and death. The genes for all three peptide components have been cloned and sequenced. No single anthrax toxin component alone is toxic; however, a combination of PA and either LF or EF leads to infection and pathogenesis. During the B. anthracis infectious process, PA83 binds to a ubiquitous cell surface receptor. One or more proteases including a furin-like protease is present at the exterior of cells and plays a role in the proteolytic activation of receptor bound PA. PA is secreted as an 83 kDa monomeric polypeptide. Monomeric PA binds to a mammalian cell surface receptor and is proteolytically cleaved. The C-terminal 63 kDa fragment (PA63) remains bound to the cell and the N-terminal 20 kDa (PA20) dissociates from PA63. The cleavage generates PA63 and exposes a high affinity site on PA to which LF/EF can bind competitively. PA63 heptamerizes and inserts into the membrane as a pore upon exposure to acidic pH after receptor mediated endocytosis. The PA63 oligomer translocates EF/LF into the cytosol. The fourth domain of PA (PA-D4) is responsible for initial binding of the anthrax toxin to the cellular receptor, and is an attractive target for vaccines.

Some studies have illustrated that both monoclonal and polyclonal antibodies to PA may neutralize the anthrax toxin and function to provide immunity against the pathogen. One such current anthrax vaccine includes an aluminum hydroxide-adsorbed cell-free filtrate of cultures of a noncapsulating nonproteolytic strain of B. anthracis (Anthrax Vaccine Absorbed, AVA) in which PA is the major protective component.

Although these vaccines have proven efficacious, they possess certain limitations. Namely, vaccine quality and efficacy vary among production batches depending on the levels of PA production and the presence of impurities, such as traces of active toxin components LF and EF, which can produce serious side effects in a subject.

Culture supernatants of B. anthracis have been the major source for purifying PA. However, working with B. anthracis cultures requires expensive biosafety level-3 containment facilities. Additionally, PA preparation from B. anthracis is often contaminated with LF or EF. Heterologous expression of PA from other hosts, such as Bacillus subtilis, has been attempted in the past, but with difficulty. PA production from B. subtilis or a protease deficient B. subtilis host yields only limited quantities of PA, thus increasing the costs of additional production batches. Similarly, Baculovirus vectors have also been used to express PA in insect cells; however, purification is not feasible due to low PA yields and the persistence of undesirable impurities. PA has also been expressed in Escherichia coli, however, a low yield was observed and the protein was insoluble when expressed in the cytoplasm. Heterologous expression in E. coli used codon optimized recombinant PA (rPA) and the protein was targeted into the periplasm of the expression host.

BRIEF SUMMARY OF THE INVENTION

The present invention includes a synthetic polynucleotide having a nucleotide sequence encoding a B. anthracis protective antigen protein, wherein the synthetic polynucleotide sequence has been optimized for heterologous expression in a bacterial host cell such as P. fluorescens.

The present invention also provides a method of producing a recombinant Bacillus anthracis protective antigen protein in the cytoplasm and periplasm of the bacterial cell including optimizing a synthetic polynucleotide sequence for heterologous expression in a bacterial host, wherein the synthetic polynucleotide comprises a nucleotide sequence encoding a Bacillus anthracis protective antigen protein. The method also includes ligating the optimized synthetic polynucleotide sequence into an expression vector and transforming the host bacteria with the expression vector. The method additionally including culturing the transformed host bacteria in a suitable culture media appropriate for the expression of the Bacillus anthracis protective antigen protein and isolating the Bacillus anthracis protective antigen protein. The bacteria host selected can be Pseudomonas fluorescens.

Other embodiments of the present invention include methods of optimizing synthetic polynucleotide sequences for heterologous expression in a host cell by identifying and removing rare codons from the synthetic polynucleotide sequence that are rarely used in the host. Furthermore, these methods can include identification and removal of putative internal ribosomal binding site sequences as well as identification and removal extended repeats of G or C nucleotides from the synthetic polynucleotide sequence. The methods can also include identification and minimization of protective antigen protein secondary structures in the RBS and gene coding regions, as well as removing undesirable enzyme-restriction sites from the synthetic polynucleotide sequences.

Embodiments of the present invention also include vaccines comprising a recombinant Bacillus anthracis protective antigen protein, wherein the recombinant Bacillus anthracis protective antigen protein is encoded by a synthetic polypeptide that has been optimized for heterologous expression in a bacterial host. The bacterial host can be Pseudomonas fluorescens.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 illustrates the PA83 protein with indicated domains;

FIG. 2 illustrates a flow diagram showing steps that can be used during optimization of a synthetic polynucleotide sequence;

FIG. 3 illustrates expression of rPA83 containing the native signal in P. fluorescens;

FIG. 4 illustrates expression of rPA83His without the native signal in the cytoplasm of P. fluorescens;

FIG. 5 illustrates expression of rPA83His with the pbp signal in the periplasm of P. fluorescens;

FIG. 6 illustrates expression of rPA83 without the His tag and with the native signal, without a signal and with the pbp signal in soluble and insoluble fractions of P. fluorescens DC454;

FIG. 7 illustrates expression of rPA83 without the His tag and with the native signal, without a signal and with the pbp signal in soluble and insoluble fractions of P. fluorescens DC417;

FIG. 8 illustrates purification scheme of heterologously expressed rPA83 with C-terminal His tag (PA83His);

FIG. 9 illustrates steps in the purification of heterologously expressed rPA83 and final rPA83 sample as shown by 4-12% SDS-PAGE gel; and

FIG. 10 illustrates cleavage of purified heterologously expressed rPA83 into PA63 and PA20 by trypsin as shown by 4-12% SDS-PAGE gel.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The present invention relates to synthetic polynucleotide sequences that encode for a recombinant B. anthracis protective antigen (rPA) protein that can be used for prophylactic immunization against anthrax infections. Embodiments of the present invention also provide for the heterologous expression of a synthetic polynucleotide in a bacterial host. Other embodiments include a heterologous expression of a synthetic polynucleotide in Pseudomonas fluorescens or E. coli. Additional embodiments of the present invention also include optimized polynucleotide sequences encoding a recombinant Bacillus anthracis rPA that can be expressed using a heterologous P. fluorescens-based expression system. Another embodiment of the present invention also includes a heterologous expression of a synthetic polynucleotide in the cytoplasm of Pseudomonas fluorescens. Additional embodiment of the present invention also includes a heterologous expression of a synthetic polynucleotide in the periplasm of Pseudomonas fluorescens. These optimized polynucleotide sequences can provide for a high yield of soluble rPA.

In heterologous expression systems, optimization steps may improve the ability of the host to produce the foreign protein. Protein expression is governed by a host of factors including those that affect transcription, mRNA processing, and stability and initiation of translation. The polynucleotide optimization steps may include steps to improve the ability of the host to produce the foreign protein as well as steps to assist the researcher in efficiently designing expression constructs. Optimization strategies may include, for example, the modification of translation initiation regions, alteration of mRNA structural elements, and the use of different codon biases. The following paragraphs discuss potential problems that may result in reduced heterologous protein expression, and techniques that may overcome these problems.

One area that can result in reduced heterologous protein expression is a rare codon-induced translational pause. A rare codon-induced translational pause includes the presence of codons in the polynucleotide of interest that are rarely used in the host organism may have a negative effect on protein translation due to their scarcity in the available tRNA pool. One method of improving optimal translation in the host organism includes performing codon optimization which can result in rare host codons being removed from the synthetic polynucleotide sequence.

Another area that can result in reduced heterologous protein expression is by alternate translational initiation. Alternate translational initiation can include a synthetic polynucleotide sequence inadvertently containing motifs capable of functioning as a ribosome binding site (RBS). These sites can result in initiating translation of a truncated protein from a gene-internal site. One method of reducing the possibility of producing a truncated protein, which can be difficult to remove during purification, includes eliminating putative internal RBS sequences from an optimized polynucleotide sequence.

Another area that can result in reduced heterologous protein expression is through repeat-induced polymerase slippage. Repeat-induced polymerase slippage involves nucleotide sequence repeats that have been shown to cause slippage or stuttering of DNA polymerase which can result in frameshift mutations. Such repeats can also cause slippage of RNA polymerase. In an organism with a high G+C content bias, there can be a higher degree of repeats composed of G or C nucleotide repeats. Therefore, one method of reducing the possibility of inducing RNA polymerase slippage, includes altering extended repeats of G or C nucleotides.

Another area that can result in reduced heterologous protein expression is through interfering secondary structures. Secondary structures can sequester the RBS sequence or initiation codon and have been correlated to a reduction in protein expression. Stemloop structures can also be involved in transcriptional pausing and attenuation. An optimized polynucleotide sequence can contain minimal secondary structures in the RBS and gene coding regions of the nucleotide sequence to allow for improved transcription and translation.

Another area that can effect heterologous protein expression are restriction sites: By removing restriction sites that could interfere with subsequent sub-cloning of transcription units into host expression vectors a polynucleotide sequence can be optimized.

As illustrated by FIG. 2, the optimization process can begin by identifying the desired amino acid sequence to be heterologously expressed by the host. From the amino acid sequence a candidate polynucleotide or DNA sequence can be designed. During the design of the synthetic DNA sequence, the frequency of codon usage can be compared to the codon usage of the host expression organism and rare host codons can be removed from the synthetic sequence. Additionally, the synthetic candidate DNA sequence can be modified in order to remove undesirable enzyme restriction sites and add or remove any desired signal sequences, linkers or untranslated regions. The synthetic DNA sequence can be analyzed for the presence of secondary structure that may interfere with the translation process, such as G/C repeats and stem-loop structures. Before the candidate DNA sequence is synthesized, the optimized sequence design can be checked to verify that the sequence correctly encodes the desired amino acid sequence. Finally, the candidate DNA sequence can be synthesized using DNA synthesis techniques, such as those known in the art.

In another embodiment of the invention, the general codon usage in a host organism, such as P. fluorescens, can be utilized to optimize the expression of the heterologous polynucleotide sequence. The percentage and distribution of codons that rarely would be considered as preferred for a particular amino acid in the host expression system can be evaluated. Values of 5% and 10% usage can be used as cutoff values for the determination of rare codons. For example, the codons listed in TABLE 1 have a calculated occurrence of less than 5% in the P. fluorescens MB214 genome and would be generally avoided in an optimized gene expressed in a P. fluorescens host.

TABLE 1
Amino Acid(s)	Codon(s) Used	% Occurrence
G Gly	GGA	3.26
I Ile	ATA	3.05
L Leu	CTA	1.78
	CTT	4.57
	TTA	1.89
R Arg	AGA	1.39
	AGG	2.72
	CGA	4.99
S Ser	TCT	4.18

A variety of host cells can be used for expression of a desired heterologous gene product. The host cell can be selected from an appropriate population of E. coli cells or Pseudomonas cells. Pseudomonads and closely related bacteria, as used herein, is co-extensive with the group defined herein as “Gram(−) Proteobacteria Subgroup 1.” “Gram(−) Proteobacteria Subgroup 1” is more specifically defined as the group of Proteobacteria belonging to the families and/or genera described as falling within that taxonomic “Part” named “Gram-Negative Aerobic Rods and Cocci” by R. E. Buchanan and N. E. Gibbons (eds.), Bergey's Manual of Determinative Bacteriology, pp. 217-289 (8th ed., 1974) (The Williams & Wilkins Co., Baltimore, Md., USA) (hereinafter “Bergey (1974)”). The host cell can be selected from Gram-negative Proteobacteria Subgroup 18, which is defined as the group of all subspecies, varieties, strains, and other sub-special units of the species Pseudomonas fluorescens, including those belonging, e.g., to the following (with the ATCC or other deposit numbers of exemplary strain(s) shown in parenthesis): P. fluorescens biotype A, also called biovar 1 or biovar I (ATCC 13525); P. fluorescens biotype B, also called biovar 2 or biovar II (ATCC 17816); P. fluorescens biotype C, also called biovar 3 or biovar III (ATCC 17400); P. fluorescens biotype F, also called biovar 4 or biovar IV (ATCC 12983); P. fluorescens biotype G, also called biovar 5 or biovar V (ATCC 17518); P. fluorescens biovar VI; P. fluorescens Pf0-1; P. fluorescens Pf-5 (ATCC BAA-477); P. fluorescens SBW25; and P. fluorescens subsp. cellulosa (NCIMB 10462).

The host cell can be selected from Gram-negative Proteobacteria Subgroup 19, which is defined as the group of all strains of P. fluorescens biotype A, including P. fluorescens strain MB101, and derivatives thereof.

In one embodiment, the host cell can be any of the Proteobacteria of the order Pseudomonadales. In a particular embodiment, the host cell can be any of the Proteobacteria of the family Pseudomonadaceae. In a particular embodiment, the host cell can be selected from one or more of the following: Gram-negative Proteobacteria Subgroup 1, 2, 3, 5, 7, 12, 15, 17, 18 or 19.

Additional P. fluorescens strains that can be used in the present invention include P. fluorescens Migula and P. fluorescens Loitokitok, having the following ATCC designations: [NCIB 8286]; NRRL B-1244; NCIB 8865 strain COI; NCIB 8866 strain CO2; 1291 [ATCC 17458; IFO 15837; NCIB 8917; LA; NRRL B-1 864; pyrrolidine; PW2 [ICMP 3966; NCPPB 967; NRRL B-899]; 13475; NCTC 10038; NRRL B-1603 [6; IFO 15840]; 52-1C; CCEB 488-A [BU 140]; CCEB 553 [IEM 15/47]; IAM 1008 [AHH-27]; IAM 1055 [AHH-23]; 1 [IFO 15842]; 12 [ATCC 25323; NIH 11; den Dooren de Jong 216]; 18 [IFO 15833; WRRL P-7]; 93 [TR-10]; 108[52-22; IFO 15832]; 143 [IFO 15836; PL]; 149 [2-40-40; IFO 15838]; 182 [IFO 3081; PJ 73]; 184 [IFO 15830]; 185[W2 L-1]; 186 [IFO 15829; PJ 79]; 187 [NCPPB 263]; 188 [NCPPB 316]; 189 [PJ227; 1208]; 191 [IFO 15834; PJ 236; 22/1]; 194 [Klinge R-60; PJ 253]; 196 [PJ 288]; 197 [PJ 290]; 198[PJ 302]; 201 [PJ 368]; 202 [PJ 372]; 203 [PJ 376]; 204 [IFO 15835; PJ 682]; 205[PJ686]; 206 [PJ 692]; 207 [PJ 693]; 208 [PJ 722]; 212 [PJ 832]; 215 [PJ 849]; 216 [PJ885]; 267 [B-9]; 271 [B-1612]; 401 [C71A; IFO 15831; PJ 187]; NRRL B-3178 [4; IFO 15841]; KY8521; 3081; 30-21; [IFO 3081]; N; PYR; PW; D946-B83 [BU 2183; FERM-P 3328]; P-2563 [FERM-P 2894; IFO 13658]; IAM-1126 [43F]; M-1; A506 [A5-06]; A505[A5-05-1]; A526 [A5-26]; B69; 72; NRRL B4290; PMW6 [NCIB 11615]; SC 12936; A1 [IFO 15839]; F 1847 [CDC-EB]; F 1848 [CDC 93]; NCIB 10586; P17; F-12; AmMS 257; PRA25; 6133D02; 6519E01; Ni; SC15208; BNL-WVC; NCTC 2583 [NCIB 8194]; H13; 1013 [ATCC 11251; CCEB 295]; IFO 3903; 1062; or Pf-5.

Transformation of the Pseudomonas host cells with the vector(s) may be performed using any transformation methodology known in the art, and the bacterial host cells may be transformed as intact cells or as protoplasts (i.e. including cytoplasts). Transformation methodologies include poration methodologies, e.g., electroporation, protoplast fusion, bacterial conjugation, and divalent cation treatment, e.g., calcium chloride treatment or CaCl/Mg²⁺treatment, or other well known methods in the art. See, e.g., Morrison, J. Bact., 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology, 101:347-362 (Wu et al., eds, 1983), Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).

As used herein, the term “fermentation” includes both embodiments in which literal fermentation is employed and embodiments in which other, non-fermentative culture modes are employed. Fermentation may be performed at any scale. In embodiments of the present invention the fermentation medium can be selected from among rich media, minimal media, and mineral salts media; a rich medium can also be used. In another embodiment either a minimal medium or a mineral salts medium is selected. In still another embodiment, a minimal medium is selected. In yet another embodiment, a mineral salts medium is selected. Mineral salts media are generally used.

Mineral salts media consists of mineral salts and a carbon source such as, e.g., glucose, sucrose, or glycerol. Examples of mineral salts media include, e.g., M9 medium, Pseudomonas medium (ATCC 179), Davis and Mingioli medium (see, B D Davis & E S Mingioli (1950) in J. Bact. 60:17-28). The mineral salts used to make mineral salts media include those selected from among, e.g., potassium phosphates, ammonium sulfate or chloride, magnesium sulfate or chloride, and trace minerals such as calcium chloride, borate, and sulfates of iron, copper, manganese, and zinc. No organic nitrogen source, such as peptone, tryptone, amino acids, or a yeast extract, is included in a mineral salts medium. Instead, an inorganic nitrogen source is used and this may be selected from among, e.g., ammonium salts, aqueous ammonia, and gaseous ammonia. A mineral salts medium can contain glucose as the carbon source. In comparison to mineral salts media, minimal media can also contain mineral salts and a carbon source, but can be supplemented with, e.g., low levels of amino acids, vitamins, peptones, or other ingredients, though these are added at very minimal levels.

In one embodiment, media can be prepared using the various components listed below. The components can be added in the following order: first (NH₄)HPO₄, KH₂PO₄ and citric acid can be dissolved in approximately 30 liters of distilled water; then a solution of trace elements can be added, followed by the addition of an antifoam agent, such as Ucolub N 115. Then, after heat sterilization (such as at approximately 121. degree. C.), sterile solutions of glucose MgSO₄ and thiamine-HCL can be added. Control of pH at approximately 6.8 can be achieved using aqueous ammonia. Sterile distilled water can then be added to adjust the initial volume to 371 minus the glycerol stock (123 mL). The chemicals are commercially available from various suppliers, such as Merck. This media can allow for a high cell density cultivation (HCDC) for growth of Pseudomonas species and related bacteria. The HCDC can start as a batch process which is followed by a two-phase fed-batch cultivation. After unlimited growth in the batch part, growth can be controlled at a reduced specific growth rate over a period of 3 doubling times in which the biomass concentration can increased several fold. Further details of such cultivation procedures is described by Riesenberg, D.; Schulz, V.; Knorre, W. A.; Pohl, H. D.; Korz, D.; Sanders, E. A.; Ross, A.; Deckwer, W. D. (1991) “High cell density cultivation of. Escherichia coli, at controlled specific growth rate” J Biotechnol: 20(1) 17-27. TABLE-US-00005 TABLE 5 Medium composition Component Initial concentration KH₂PO₄ 13.3 gl⁻¹ (NH₄)₂HPO₄4.0 g l⁻¹ Citric acid 1.7 g l⁻¹ MgSO₄-7H₂O 1.2 g l⁻¹ Trace metal solution 10 mll⁻¹ Thiamin HCl 4.5 mg l⁻¹ Glucose-H₂O 27.3 g l⁻¹ Antifoam Ucolub N115 0.1 ml l⁻¹ Feeding solution MgSO₄-7H₂O 19.7 g l⁻¹ Glucose-H₂O 770 g l⁻¹ NH₃ 23 g Trace metal solution 6 g l⁻¹ Fe (111) citrate 1.5 g l⁻¹ MnCl₂-4H₂O 0.8 g l⁻¹ ZmCH₂COOl₂-2H₂O 0.3 g l⁻¹ H₃BO₃ 0.25 g l⁻¹ Na₂MoO₄-2H₂O 0.25 g l⁻¹ CoCl₂ 6H₂O 0.15 g l⁻¹ CuCl₂ 2H₂O 0.84 g l⁻¹ ethylene diaminetetracetic acid Na₂ salt 2H₂O (Titriplex III, Merck).

The protective antigen precursor PA83 of Bacillus anthracis, strain Sterne (764 aa) has previously been submitted under NCBI Accession Number AAA22637 and contains the following amino acid sequence: mkkrkvlipl malstilvss tgnleviqae vkqenrllne sesssqgllg yyfsdlnfqa pmvvtssttg dlsipssele nipsenqyfq saiwsgfikv kksdeytfat sadnhvtmwv ddqevinkas nsnkirlekg rlyqikiqyq renptekgld fklywtdsqn kkevissdnl qlpelkqkss nsrkkrstsa gptvpdrdnd gipdsleveg ytvdvknkrt flspwisnih ekkgltkyks spekwstasd pysdfekvtg ridknvspea rhplvaaypi vhvdmeniil sknedqstqn tdsetrtisk ntstsrthts evhgnaevha sffdiggsys agfsnsnsst vaidhslsla gertwaetmg lntadtarin aniryvntgt apiynvlptt slvlgknqt1 atikakenql sqilapnnyy psknlapial naqddfsstp itmnynqfle lektkqlrld tdqvygniat ynfengrvry dtgsnwsevl pqiqettari ifngkdlnlv erriaavnps dplettkpdm tlkealkiaf gfnepngnlq yqgkditefd fnfdqqtsqn iknqlaelna tniytvldki klnakmnili rdklplydrn niavgadesv vkeahrevin ssteglllni dkdirkilsg yiveiedteg lkevindryd mlnisslrqd gktfidkky ndklplyisn pnykvnvyav tkentiinps engdtstngi kkilifskkg yeig (SEQ ID NO: 1) (SEQ ID NO:19 is the native signal peptide, including initial methionine, shown in bold; SEQ ID NO:20 is the PA83 protein sequence shown in plain text). Met has been added for translation start and the native signal was removed when protein was expressed in P. fluorescens without the signal peptide. This sequence has been codon optimized for expression utilizing the elements discussed above. SEQ ID NOs: 2 and 10-15 illustrate optimized nucleotide sequences.

When the recombinant molecule takes the form of an expression system, the transformed or transfected cells can be used as a source of the protein or polypeptide specified as amino acid sequence in SEQ ID NO: 1, TPA-PA, MAT-PA, and PA63.

The sequences recited in this application may be homologous (have similar identity). Proteins and/or protein sequences are “homologous” when they are derived, naturally or artificially, from a common ancestral protein or protein sequence. Similarly, nucleic acids and/or nucleic acid sequences are homologous when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. For example, any naturally occurring nucleic acid can be modified by any available mutagenesis method to include one or more selector codon. When expressed, this mutagenized nucleic acid encodes a polypeptide comprising one or more unnatural amino acid. The mutation process can, of course, additionally alter one or more standard codon, thereby changing one or more standard amino acid in the resulting mutant protein as well. Homology is generally inferred from sequence similarity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of similarity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence similarity is routinely used to establish homology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% or more can also be used to establish homology. Methods for determining sequence similarity percentages (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available.

As noted above, polypeptides may comprise a signal (or leader) sequence at the N-terminal end of the protein, which co-translationally or post-translationally directs transfer of the protein. The polypeptide may also be conjugated to a linker or other sequence for ease of synthesis, purification or identification of the polypeptide (e.g., poly-His), or to enhance binding of the polypeptide to a solid support.

When comparing polypeptide sequences, two sequences are said to be “identical” if the sequence of amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345 358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626 645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151 153; Myers, E. W. and Muller W. (1988) CABIOS 4:11 17; Robinson, E. D. (1971) Comb. Theor 11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406 425; Sneath, P. H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA 80:726 730.

Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Waterman (1981) Add. APL. Math 2:482, by the identity alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.

One example of algorithms that can be suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25:3389 3402 and Altschul et al. (1990) J. Mol. Biol. 215:403 410, respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.

In one approach, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

Within other illustrative embodiments, a polypeptide may be a fusion polypeptide that comprises multiple polypeptides as described herein, or that comprises at least one polypeptide as described herein and an unrelated sequence, such as a known tumor protein. A fusion partner may, for example, assist in providing T helper epitopes (an immunological fusion partner), preferably T helper epitopes recognized by humans, or may assist in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners may be selected so as to increase the solubility of the polypeptide or to enable the polypeptide to be targeted to desired intracellular compartments. Still further fusion partners include affinity tags, which facilitate purification of the polypeptide.

Fusion polypeptides may generally be prepared using standard techniques, including chemical conjugation. Preferably, a fusion polypeptide is expressed as a recombinant polypeptide, allowing the production of increased levels, relative to a non-fused polypeptide, in an expression system. Briefly, nucleic acid sequences encoding the polypeptide components may be assembled separately, and ligated into an appropriate expression vector. The 3′ end of the DNA sequence encoding one polypeptide component is ligated, with or without a peptide linker, to the 5′ end of a DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are in phase. This permits translation into a single fusion polypeptide that retains the biological activity of both component polypeptides.

A peptide linker sequence may be employed to separate the first and second polypeptide components by a distance sufficient to ensure that each polypeptide folds into its secondary and tertiary structures. Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques well known in the art. Suitable peptide linker sequences may be chosen based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the lack of hydrophobic or charged residues that might react with the polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala may also be used in the linker sequence. Amino acid sequences which may be usefully employed as linkers include those disclosed in Maratea et al., Gene 40:39 46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are not required when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

The ligated DNA sequences are operably linked to suitable transcriptional or translational regulatory elements. The regulatory elements responsible for expression of DNA are located only 5′ to the DNA sequence encoding the first polypeptides. Similarly, stop codons required to end translation and transcription termination signals are only present 3′ to the DNA sequence encoding the second polypeptide.

In another embodiment, the present-invention relates to an anthrax vaccine comprising one or more of the codon optimized sequences encoding one or more B. anthracis proteins or polypeptides as described throughout. The present invention relates a method for providing immunity against anthrax said method comprising administering one or more of the codon optimized sequences encoding for any combination of the B. anthracis proteins to a subject such that a protective immune reaction is generated.

Administration of the proteins disclosed herein may be carried out by any suitable means, including both parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), or topical application of the virus (typically carried in the pharmaceutical formulation) to an airway surface. Topical application of the virus to an airway surface can be carried out by intranasal administration (e.g. by use of dropper, swab, or inhaler which deposits a pharmaceutical formulation intranasally). Topical application of the virus to an airway surface can also be carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) containing the protein as an aerosol suspension, and then causing the subject to inhale the respirable particles. Methods and apparatus for administering respirable particles of pharmaceutical formulations are well known, and any conventional technique can be employed. An “immunogenic amount” is an amount of the protein sufficient to evoke an immune response in the subject to which the vaccine is administered.

When the nucleic acids are used as a vaccine, the nucleic acids can be administered directly using techniques such as delivery on gold beads (gene gun), delivery by liposomes, or direct injection, among other methods known to people in the art. Any one or more constructs or replicating a nucleic acid can be used in any combination effective to elicit an immunogenic response in a subject. Generally, the nucleic acid vaccine administered may be in an amount of about 1-5 ug of nucleic acid per dose and will depend on the subject to be treated, capacity of the subject's immune system to develop the desired immune response, and the degree of protection desired. Precise amounts of the vaccine to be administered may depend on the judgment of the practitioner and may be peculiar to each subject and antigen.

The vaccine may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or reinforce the immune response, for example, at 1-4 months for a second dose, and if needed, a subsequent dose(s) after several months. Examples of suitable immunization schedules include: (i) 0, 1 months and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient to elicit the desired immune responses expected to confer protective immunity, or reduce disease symptoms, or reduce severity of disease.

The present invention is explained in greater detail in the Examples that follow. These examples are intended as illustrative of the invention and are not to be taken are limiting thereof.

EXAMPLES

Example 1

Expression and Purification of rPA from P. fluorescens

P. fluorescens has been used to express rPA vaccine antigen rPA83 (SEQ ID NO: 1), rPA63, and rPA-D4 (FIG. 1). Codon optimized polynucleotide sequences that contained SpeI and XhoI flanking restriction sites were synthesized. There are a number of alternative sequences for the codon optimized polynucleotide sequence for expression in P. fluorescence that encode the desired rPA including, rPA83His with the native signal and C-terminal His tag (SEQ ID NO: 2), rPA83 with the native signal peptide (SEQ ID NO: 10), rPA83 without the native signal peptide (SEQ ID NO: 11), rPA83 with an N-terminal phosphate binding protein (php) secretion signal (SEQ ID NO: 12), rPA83 with C-terminal His tag and without the native signal peptide (SEQ ID NO: 13), rPA63His with C-terminal His tag (SEQ ID NO: 14), and DrPAd4His Domain 4 with C-terminal His tag. Further examples of alternative polynucleotide sequences encoding for codon optimized rPA83 include the polynucleotide sequences as shown in SEQ ID NOs: 16-18.

To direct the expression into the cytoplasm, the genes were amplified from their corresponding plasmids by PCR, during which the native secretion signal was removed and AUG codon was incorporated to provide the translation start site (i.e., SEQ IDs NO: 11 and 13). To direct the expression into the periplasm, the genes were amplified from their corresponding plasmids by PCR, during which the periplasmic secretion signal derived from the pbp was fused to the coding sequences for the mature protein (i.e., SEQ ID NO:12). PCR products were subcloned into the shuttle plasmid and verified by sequencing. Upon verification, the genes were cloned into the P. fluorescens expression vector containing the tac promoter and pyrF selection marker, and transformed by electroporation into the P. fluorescens DC454 (ΔpyrF lsc::lacIQ1) or DC417 (ΔhslUV::ΔpyrF lsc::lacIQ1) strains. The colonies that grew on the M9+1% Glucose media in the absence of uracil were screened by restriction digest for the presence of inserts. Cells from selected colonies were grown in media at shake-flask scale and induced with IPTG. OD A₆₀₀ readings were taken at various times during fermentation to monitor cell growth. Cells were collected at 24 hrs post induction, and both soluble and insoluble fractions were analyzed by SDS-PAGE gel. PBS buffer, pH 7.4.

When using the native PA leader sequence (i.e., SEQ ID NO:2), the heterologous expression resulted in most of the protein being present in the culture media, which can simplify the purification of the heterologous protein.

Example 2

Cloning and Expression of 83 kDa rPA83His with Native Signal in DC454

Cloning: An rPA insert was excised out of plasmid pJ3:G01196 (DNA 2.0) with SpeI and XhoI. The insert was gel purified on 1% agarose gel and ligated into vector pDow1169, (a medium copy plasmid with RSF1010 origin, pyrF, a ribosome binding site under control of the tac promoter and the rrnBT1T2 terminator from pKK223-3 (PL-Pharmacia)), which had been digested with SpeI, XhoI and treated with Alkaline Phosphatase. The ligation product was transformed by electroporation into P. fluorescens DC454 strain after purification with Micro Bio-spin 6 Chromatography columns. The transformants were plated on M9 Glucose plate after shaking for two hours in LB media at 30° C. The presence of the insert was confirmed by restriction digest and sequencing.

Protein Expression: Single transformants were inoculated into 50 ml M9 Glucose media and grown overnight. P. fluorescens cultures of 3.0-5.0 OD600 were used to inoculate the shake-flask media. Shake flasks were incubated at 30° C with 300 rpm shaking overnight. Overnight cultures of 15.0-20.0 OD600 were induced with 300 μM isopropyl-β-D-thiogalactopyranoside (IPTG). Cultures were harvested at 24 hours post induction. When using the native PA leader sequence (i.e., SEQ ID NO: 2), the heterologous expression resulted in the protein being unexpectedly present in the culture media, which simplifies the purification of the heterologous rPA83 protein (FIG. 3).

Example 3

Cloning and Expression of 83 kDa rPA83His Without Native Signal in P. fluorescens DC454

Cloning: The insert was amplified by PCR with forward primer PA-nosig-SpeI-F 5′-CTACTAGTAGGAGGTAACTTATGGAAGTGAAGCAGGAGAATCG-3′ (SEQ ID NO: 3) and reverse primer PA-Xho-Rev 5′-CCGCTCGAGTCATTAATGATGGTGGTG ATGGTGC CCGATCTC-3′ (SEQ ID NO: 4) with the plasmid pJ3:G01196 as the template. The insert was amplified with the PCR protocol of TABLE 2. The PCR product was purified with Qiaquick PCR purification kit (Qiagen), digested with SpeI and XhoI (NEB) and purified again with Qiaquick kit before ligating into the expression vector with T4 DNA ligase. The ligation product was transformed by electroporation into P. fluorescens strain DC454 after purification with Micro Bio-spin 6 Chromatography columns. The transformants were plated on M9 Glucose plate after two hours shaking in LB media at 30° C. The plates were incubated at 30° C. for 48 hours. The presence of the insert was confirmed by restriction digest and sequencing.

TABLE 2
PCR PROTOCOL
Reaction Mix (100 μL total volume)	Thermocycling Steps
10	μL	10× Pt HiFi buffer *	Step 1	1	Cycle	2	min.	94° C.
4	μL	50 mM MgSO4 *	Step 2	35	Cycles	30	sec.	94° C.
2	μL	10 mM dNTPs *				30	sec.	55° C.
0.25	mg	Each Primer				3	min.	68° C.
1-5	ng	Template DNA	Step 3	1	Cycle	10	min.	70° C.
1	μL	Pt HiFi Taq DNA Polymerase *	Step 4	1	Cycle	Maintain	4° C.
Remainder	Distilled De-ionized H2O (ddH2O)
(* from Invitrogen Corp, Carlsbad, CA, USA, hereinafter “Invitrogen”)

Protein Expression: Single transformants were inoculated into 50 ml M9 Glucose media and grown overnight. P. fluorescens cultures of 3.0-5.0 OD600 were used to inoculate shake-flask media. Shake flasks were incubated at 30° C. with 300 rpm shaking overnight. Overnight cultures of 15.0-20.0 OD600 were induced with 300 μM isopropyl-β-D-thiogalactopyranoside (IPTG). Cultures were harvested at 24 hours post induction. High levels of expression of soluble rPA83 (SEQ ID NO: 13), were detected in the cytoplasmic fractions. The rPA83 was purified from the cell lysate using multiple chromatography steps. The procedure was designed to minimize degradation of the PA protein during purification. The purification scheme is illustrated in FIG. 8 and described in details in Example 8. Purification results are shown in FIGS. 9-10. The purified protein was stored in 5 mM Hepes, 50 mM NaCl, at pH 7.5 at −80° C. or was lyophilized and stored at 4° C. The yields of purified protein were 1.03 g/L. The majority of the expressed protein was expressed in the soluble fraction. (FIG. 4). The arrow illustrates rPA83 with no signal sequence. The expression of soluble PA protein in the cytoplasm of P. fluorescens can eliminate the need for refolding that was previously used for insoluble rPA83 expressed in the cytoplasm of E. coli.

Example 4

Cloning and Expression of 63 kDa rPA63His in DC454

Cloning: The nucleic acid sequence coding the C-term 63 kDa portion of the 83 kDa rPA was amplified out of plasmid pJ3:G01196 (DNA 2.0) with the following PCR primers: A63-For 5′-CTACTAGTAGGAGGTAACTTATGTCGACCTCCGCTGG GCCTACGG-3′ (SEQ ID NO: 5) and PA-Xho-Rev (SEQ ID NO:4), following the PCR protocol shown in TABLE 2.

The PCR product was purified with Qiaquick PCR purification kit (Qiagen), digested with SpeI and XhoI (NEB), and purified again with Qiaquick kit before ligating into an expression vector with T4 DNA ligase. The ligation product was transformed by electroporation into P. fluorescens strain DC454 after purification with Micro Bio-spin 6 Chromatography columns. The transformants were plated on M9 Glucose plate after two hours of shaking in LB media at 30° C. The plates were incubated at 30° C. for 48 hours. The presence of the insert was confirmed by restriction digest and sequencing.

Protein Expression: Single transformants were inoculated into 50 ml M9 Glucose media and grown overnight. P. fluorescens cultures of 3.0-5.0 OD600 were used to inoculate to the shake-flask media. Shake flasks were incubated at 30° C. with 300 rpm shaking overnight. Overnight cultures of 15.0-20.0 OD600 were induced with 300 μM isopropyl-β-D-thiogalactopyranoside (IPTG). Cultures were harvested at 24 hours post induction.

Example 5

Cloning and Expression of rPAd4His Domain 4 in DC454

Cloning: The nucleic acid sequence coding the C-term domain 4 portion of the 83 kDa rPA was amplified out of plasmid pJ3:G01196 with the following primers: PAdomain4-SpeI-For 5′-CTACTAGTAGGAGGTAACTTATGGAGCTGAACGCCACCAAC-3′ (SEQ ID NO: 6) and PA-Xho-Rev (SEQ ID NO: 4).

The PCR protocol and the protein expression was the same as those previously described. The cultures were harvested at 24 hours post induction.

Example 6

Cloning and Expression of 83 kDa rPA83His in the periplasm of DC454

Cloning: A 24 residue phosphate binding protein (pbp) secretion signal was fused to the N-terminus of 83 kDa rPA protein without its native secretion signal and the starting Methionine, Pbp signal was amplified out of pDOW1113 with the following primer pair: pbpF-SpeI 5′-GGACTAGTAGGAGGTAACTTATGAAACTGAAACGTTTGATG-3′ (SEQ ID NO: 7) and pbp-PA-Rev 5′-CAGAACCTTGCGCTTCTTGGCCACCGCGTTGGC-3′ (SEQ ID NO: 8).

83 kDa rPA was amplified by PCR with the following primer pair: pbp-PA-For 5′-GCCAAGCGCGGTGGCCAAGAAGCGCAAGGTTCTG-3′ (SEQ ID NO: 9) and PA-Xho-Rev (SEQ ID NO: 4). The two PCR products were combined in a Splicing by Overlapping Extension PCR (SOE-PCR) using the following primer pair: pbpF-SpeI (SEQ ID NO: 7) and PA-Xho-Rev (SEQ ID NO: 4). The PCR protocol, subcloning procedure, and protein expression were performed in the same manner as those previously described. The cultures were harvested at 24 hours post induction. Unexpectedly high levels of expression of soluble rPA83, were detected in the soluble fractions (FIG. 5).

Example 7

Cloning and expression of 83 kDa rPA83 Without the His tag in the Cytoplasm and Periplasm of DC454 and DC417

The cloning, PCR protocol, subcloning procedure, and protein expression were performed in the same manner as those previously described. The cultures were harvested at 24 hours post induction. Unexpectedly high levels of expression of soluble rPA83 with the native signal (SEQ ID NO: 10), rPA83 without the native signal (SEQ ID NO: 11) and rPA83 with the pbp signal (SEQ ID NO: 12) were detected in the soluble fractions (FIG. 6 and 7).

Example 8

Purification

Purification and analysis of rPA83His: The purification scheme and results are shown in FIGS. 8-10. It was important to work quickly, keep samples at 4° C. and perform the purification without stopping between individual steps to prevent unwanted degradation of rPA83 during purification. The designed protocol described bellow resulted in purification of high quality rPA83 as shown by single band on an SDS-PAGE gel (FIG. 9).

French Press: The lysis buffer consisted of 20 mM Tris, 10 mM Imidizol, 300 mM NaCl at pH 8.0. PMSF was added to the lysis buffer to final concentration of 10 mM. 8 grams of P. fluorescens rPA83His cell pellet was suspended in 40 ml of lysis buffer with PMSF. Addition of PMSF was important to prevent unwanted degradation of rPA83 during purification. The cells were passed through the French press two times at a pressure of 1,280 pounds per square inch. The samples were spun at 10,000 G for 30 minutes to remove the insoluble fraction.

The following purification steps were the provided. First Nickel Chromatography was used. The supernatant was filtered through a 0.45 um glass fiber filter prior to loading onto the nickel column. The sample was loaded onto a 15 ml nickel charged His-Bind resin from Novagen at 37 cm/hr. Nickel binding/wash buffer A was 20 mM Tris, 10 mM Imidizol, 300 mM NaCl, at pH 8.0. Nickel elution buffer B was 20 mM Tris, 1.0M Imidizol, 300 mM NaCl, pH 8.0. The column was washed with 4 column volumes of nickel binding/wash buffer A. Then, the column was washed with 5 column volumes of 1% nickel elution buffer B. The rPA83His was eluted with an 8 column volume gradient from 1% nickel elution buffer B to 20% nickel elution buffer B at 15 cm/hr.

Next Mustang E endotoxin filtration was utilized. The rPA83His eluent from the nickel column was passed through a Mustang E endotoxin filter at 2 ml/minute. Afterwards, 10 KDa Ultrafiltration was performed. The Mustang E filtrate was buffer exchanged 2 times with a 15 ml 10 kDa ultracentrifugation spin column into anion exchange wash buffer A, which was composed of 20 mM Hepes at pH 7.0.

Then a superose Q Fast Flow Anion Exchange (QFF column) was utilized. The sample was loaded onto a prepacked 5 ml superose Q fast flow column at 5 ml/minute. The column was washed with 10 column volumes of 7.5% anion exchange elution buffer B, which consisted of 20 mM Hepes, 1.0M NaCl, at pH 7.0. The rPA83His was eluted with a 20 column volume gradient from 7.5% anion exchange elution buffer B to 50% anion exchange elution buffer B.

The next step of purification involved a Mustang E Endotoxin Filtration. The rPA83His eluent from the QFF column was passed through a Mustang E endotoxin filter at 2 ml/minute. Then under 10 KDa ultrafiltration, the Mustang E filtrate was buffer exchanged 2 times with a 15 ml 10 kDa ultracentrifugation spin column into final buffer, which was composed of 5 mM Hepes, 50 mM NaCl, at pH 7.5. The final sample was sterile filtered using 0.22 μm filter. The samples were then frozen at −20° C., lyophilized overnight, and vacuum sealed the next day.

SDS-PAGE: The whole cell extract, nickel column load, nickel column eluent, the Mustang E elutent, the QFF eluent, second Mustang E eluent, final sample, and Bacillus anthracis PA (Gold Standard) were run on a 4-12% SDS-PAGE gel (FIG. 9). The gel was stained with simply blue safe stain from Invitrogen. A single band of 83 kDa was detected in the final sample.

Trypsin Cleavage: 1 mg/ml rPA83His was incubated with 1:5000, 1:2500, and 1:1000 w/w ratio of trypsin to rPA83His in 25 mM Hepes, 1 mM CaCl2, and 50 mM NaCl, at pH 8.50 for 35 minutes at RT. The reaction was stopped by adding the SDS-PAGE loading buffer and the samples are immediately run on a 4-12% SDS-PAGE gel (FIG. 10) The purified rPA83 cleaved into PA63 and PA20 as expected.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A method of producing a soluble recombinant Bacillus anthracis protective antigen protein (rPA) in a Pseudomonas fluorescens expression system comprising expressing the soluble rPA from a nucleotide sequence encoding the soluble rPA, wherein the nucleotide sequence is the soluble rPA encoding sequence of a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, or wherein the nucleotide sequence encoding the soluble rPA is a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.

2. The method of claim 1, wherein the soluble rPA encoding nucleotide sequence lacks the sequence encoding the starting methionine.

3. The method of claim 1, wherein the soluble rPA is expressed in the cytoplasm or periplasm of a host cell.

4. The method according to claim 1, comprising expressing the soluble rPA to which a secretion signal has been fused, wherein said secretion signal co-translationally or post-translationally directs transfer of the soluble rPA to the periplasm of a host cell.