WO1990009444A1

WO1990009444A1 - A method for producing pertussis toxin subunits

Info

Publication number: WO1990009444A1
Application number: PCT/FI1990/000040
Authority: WO
Inventors: Kate Runeberg-Nyman; Per Saris; Ilkka Palva; Matti Sarvas
Original assignee: Genesit Oy
Priority date: 1989-02-10
Filing date: 1990-02-08
Publication date: 1990-08-23
Also published as: AU5027890A

Abstract

A method for the production of a pertussis toxin subunit and a recombinant DNA molecule for the same capable of expression in a Bacillus host comprising: a part of the regulation and secretory protein signal sequence of a gene encoding a secretory protein of a Bacillus strain, operatively linked to a DNA sequence encoding a pertussis toxin subunit; DNA vectors containing said recombinant DNA molecule, bacillary hosts transformed with said DNA vector, a method for producing a recombinant DNA molecule in a Bacillus host employing said DNA vector and a polypeptide and composition of the same. By means of the invention several milligrams of the product are produced per liter of culture.

Description

Title of the Invention

A METHOD FOR PRODUCING PERTUSSIS TOXIN SUBUNITS

This is a continuation-in-part of co-pending application

Serial Number 063,831, filed June^' 19, 1987, which is a continuation-in-part of co-pending application Serial Number

129,357, filed November 30, 1987, which is a continuation of application Serial Number 939,244, filed December 5, 1986, and abandoned, which is a continuation of application Serial

Number 336,405, filed December 31, 1981, and abandoned.

Field of the Invention

The present invention relates to a method for the production of pertussis toxin subunits in a Bacillus hbst, to a novel recombinant DNA molecule capable of efficient expres¬ sion and secretion of pertussis toxin subunits in a Bacflliis host, as well as a novel rDNA molecule useful for prσduclng pertussis toxin subunits within a Bacillus host, and to a method for producing the same. The present invention' further relates to polypeptides and compositions comprising one or more pertussis toxin subunits made by the process and host of the present invention. ^{' "} ' Background of the Invention

Bordetella pertussis is the causative agent of whooping cough. This disease is potentially life threatening for young infants. A vaccine against whooping cough was developed as early as 1936. The vaccine which consists of killed whole organisms has been shown to be effective in preventing the disease but unfortunately it frequently causes adverse reactions. These reactions are usually transient local and systemic reactions, e.g., rise of temperature, but in rare cases irreparable brain damage and death have followed the vaccination.

Because of the association of whooping cough with encephalopathy there has been a decrease in the use of the conventional vaccine and a concomitant increase in the number of disease cases. This has led to a search for a new, less reactogenic vaccine, which would contain only those components of the bacteria that stimulate the immune response, without causing toxic side effects. The first steps towards such an acellular vaccine have already been achieved by isolating from the supernatant of Bordetella ^pertussis culture a fraction consisting of two main components: pertussis toxin (PT) and filamentous hemag- glutinin (FHA) (Sato et al .. Lancet. 1:122-126 (Jan. 21, 1984)).

The seroresponse to the acellular vaccine has been excellent and there have been significantly fewer side effects than by using the conventional cellular vaccine. As the PT and the FHA are purified from the supernatant of a Bordetella ^pertussis culture they are, however, always contaminated with other components such as LPS, a potent endotoxin, and agglutinogens. ^■3 -

The reasons for the rare but dangerous side effects of pertussis toxin produced by Bordetella pertussis are not known yet. Thus, there still remains a need for an even purer product than the acellular toxin of the prior art. Experiments in mice have shown that immunization ith PT alone will be sufficient to protect against experimental infections caused by pertussis toxin (Sato et al .. Infection and Immunity 46:415-421 (1984)). The successful production of pertussis toxin in some organism other than Bordetell pertussis would therefore be of great importance in the development of a detoxified vaccine.

The complete nucleotide sequence of pertussis toxin^' gene has been described previously (Locht et al .. Science 232:1258- , 1263 (1986); Nicosia et al.. PNAS 83:4631-4635 (1986)). In British Patent No. 2091 268, the production of a multitude of antigenic proteins in a Bacillus strain is suggested, includ¬ ing those produced by Bordetella pertussis. Pertussis toxin subunits have also been expressed in E. coli as intracellular aggregates (Burnette et aJL, Bio/Technology 6: ^' 69 -708 (1988)).

OBJECTS OF THE INVENTION

I

It is an object of the invention to provide a method for the production of a safe and effective vaccine against whooping cough by producing pertussis toxin subunifs in a

Bacillus host.

Another object of the invention is to provide a nlfthod for reducing Bordetella pertussis derived and potentially brain toxic impurities. In addition, because a Gram positive bacterium rather than a Gram-negative bacterium (i.e., E. coll) is used, the purified component is not contaminated by endotoxins produced by Gram-negative bacteria. Another object of the invention is to provide an i munostimulant that would have the same benefits as the vaccine.

Yet another object of the invention is to provide a diagnostic antigen for the identification of pertussis toxin infection. The antigen produced by the method of the inven¬ tion has the advantage that it does not cause cross reactions that are possible when the antigens produced in Gram-negative bacteria are used. Using the method of the invention, large amounts of product (from 100 mg/1 to 1 g/1) can be produced and either secreted into the culture medium or retained within the cytoplasm of the host cell, depending upon the PT subunit selected.

SUMMARY OF THE INVENTION

The invention comprises a method for the production of pertussis toxin subunits by cultivating a Bacillus host containing a recombinant DNA molecule comprising at least a part of the regulation and the secretion signal sequence of a gene encoding a secretory protein of a Bacillus strain, preferably the regulation and secretion signals of the α- amylase gene from Bacillus amyloliouefaciens. operatively linked to a DNA sequence encoding the pertussis toxin subunits Si to S5 or at least one part of any of said subunits or any combination thereof, optionally linked to at least a part of any of the signal sequences of subunits Si to S5. In parti¬ cular, the production of the pertussis subunit Sj is dis- closed.

In another preferred method of the present invention, the signal sequence of the gene encoding a secretory protein of a Bacillus strain, preferably the α-amylase gene from J amyloliquefaciens, is deleted (omitted) from the recombinant DNA molecule introduced into the Bacillus' host. In this method, good yields of pertussis toxin are produced within the Bacillus host. The toxin can be easily recovered by ordinary methods.

According to the invention the recombinant DNA molecule may be introduced to the Bacillus host by transforming the host by a vector being capable of replicating in several copies in a Bacillus strain. Another possibility is that the recombinant DNA molecule is integrated in the chromosome of the Bacillus host.

The invention also discloses the Bacillus host and the recombinant DNA molecule used in the expression and secretion as well as the preparation of the DNA molecule. Also the pertussis toxin subunits and closely related polypeptides prepared by the method of the invention and compositions comprising said polypeptides are disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the Western blot analysis of Si producing Bacillus subtilis IH 6540 culture supernatant.

Figure 2 shows the structure of the expression vectors with an α-amylase promoter (Palva et al. , (1981) and a truncated α-amylase signal sequence, used in intracellular production of S1-S5 subunits.

Figure 3 shows the predicted N-terminal sequences of S_j- S5 subunits produced in B. subtilis.

Figure 4 shows the Western blot analysis of S1-S5 producing B. subtilis BRBl cell fractions. B. subtilis strain

BRBl harboring plasmids determining production of subunits were grown in P-broth (Tryptone 12 g, yeast extract 24 g, glycerol 4 ml, KH₂P0₄ 2.3 g, K₂HP0₄ '3H0 16.4 g, in a total volume of 1000 ml) supplemented with 5% glucose and 10 g/ml kanamycin. For analysis, 1 μl was subjected to 12.5% SDS-PAGE followed by Western blotting. Lane 1: pKTH1785; Lane 2: pKTH1777; Lane 3: pKTH1782; Lane 4: pKTH229; Lane 5: pKTH1778; Lane 6: pKTH39; Lane 7: 300 ng PT.

DETAILED DESCRIPTION OF THE INVENTION

By "gene encoding a secretory protein" is meant a recombinant DNA molecule capable of efficient expression and secretion of a pertussis toxin subunit.

By "deleted nonfunctional signal sequence of a gene encoding a secretory protein" is meant to include at least the initiation methionine of the signal sequence or a larger N- terminal part of it.

By "regulation and secretion signal sequence" it is meant to include within the scope of the instant invention the entire sequence or any biologically active fragment thereof. By "DNA sequence encoding a pertussis toxin subunit" it is meant to include within the scope of the instant invention the entire sequence or a biologically active fragment thereof to effectively encode pertussis toxin subunit Sj, S2. S3, S4, or S₅.

By "pertussis toxin subunits" it is meant to include within the scope of the invention pertussis toxin subunits S_j, S_2> S3, S4, and S5, and polypeptides which differ from those subunits by one or more amino acids, having the same or substantially the same activity as the pertussis toxin subunits or any combinations of subunits. By "vector" it is meant any autonomous element capable of replicating in a host independently of the host's chromosome, into which additional sequences of DNA may be incorporated into the autonomous element's genome. Such vectors include, but are not limited to, bacterial plasmids and phages.

By "operatively linked" it is meant that the prσtnoter controls the initiation of expression of the polypeptide encoded by the structural gene.

By "polypeptide" it is meant to include within the scope of the invention the entire sequence or biologically active fragment thereof.

The production of pertussis toxin subunits by transform ng a Bacillus host

The construction of the expression vector a) The construction of the regulation and the secretion signal sequence of a gene coding for a secretory protein of a

Bacillus strain.

Bacillus expression or secretion vectors may be constructed in several different ways among which may be mentioned: 1) isolating first a gene encoding a certain secretory protein and thereafter the regulation and secretion signal sequences of said protein;

2) using the regulation and secretion signal sequences of genes known to be effectively expressed (it will be appreciated that parts of the sequences or any combinations thereof can be used);

3) using the promoter probe vectors or secretion probe vectors to facilitate the random isolation of the regulation and the secretion signals of bacterial or phage genomes; and

4) synthesizing the parts of any regulation or secretion system and using them either as such or by joining them to previously isolated signal sequences or parts thereof. Secretion vectors constructed according to the invention may or may not have a deleted nonfunctional signal sequence of a gene encoding a secretory protein as defined herein. It is to be understood that such secretion vectors are within the scope of the present invention.

A wide variety of suitable secretion vectors may be used in the present invention, and are known to those of skill in the art of recombinant genetics. Some of these, for example, are listed in Tables 1 and 2 of Kallio, "Expression and Regulation of the Bacillus amyloliquefaciens α-Amylase Gene in Bacillus subtilis." Thesis Dissertation (1987), available at the library of the University of Helsinki, Helsinki, Finland. b) Isolation and purification of the transfer vector. The transfer vector may be any plasmid or phage capable of replicating in several copies on a Bacillus strain. A multitude of such vectors are available, the most represen¬ tative of them being the plasmids isolated from Staphylococ- cus. Bacillus or Streptococcus or their derivatives. c) The ligation of the regulation and the secretion signal sequence or the regulation and deleted nonfunctional secretion signal sequence to the transfer vector.

The regulation and/or secretion sequence is in most cases first ligated to the transfer vector to be used and is thereafter modified for example by the aid of DNA-1inkers so that the gene to be expressed may be joined downstream of the regulation/secretion sequence of the vector. d) The cloning of the pertussis toxin gene and the subcloning of the subunits.

Pertussis toxin is a so-called A-B toxin in which part A has enzymatic activity and part B binds to a receptor on the host cell. PT consists of 5 different subunits called

Si, S₂, S3, S4, and S5. S2-S5 make up part B, the receptor binding site, whereas S_j contains the enzymatically active part.

The isolated and purified chromosomal DNA from J _ pertussis was digested with the restriction endoncleases Ba HI and EcoRI under standard conditions. The resulting- DNA fragments were separated according to their size by gel electrophoresis. By using the fragments of lambda phage DNA as size markers, chromosomal DNA fragments of 3-8 kb were cut from the gel as several separate size fractions. THe DNA fragments were then isolated from the agarose gel', ethanol precipitated and dissolved in TE (lOmM Tris-HCl pH 7.5, ImM EDTA). A DNA sample of 1 μ\ from each fraction was denatured in 0.4 M NaOH and immobilized onto a nylon filter (Zetaprobe; BioRad, Cambridge, MA 02139). The filter, containing the immobilized DNA fractions, was washed, prehybridized and hybridized with a labeled oligonu- cleotide probe.

The oligonucleotide probe used in hybridization was obtained by synthesizing the DNA sequence containing the first 30 nucleotides from the 5' end of the structural gene of the subunit Si. The oligonucleotide was labeled by radioactive gamma-³^P-ATP using T4 polynucleotide kinase and purif ed by Sephadex G50 (super fine) chromatography using triethano'lamine buffer (pH 3.5) for elution. After the hybridization reactions the oligonucleotide probe unspecifically bound was removed by washing the filter and then exposed to X-ray film at -70*C.

The DNA fraction found to be positive in the hybridiza¬ tion test, i.e., the DNA fragment containing the gene' coding for the whole B. pertussis toxin was about 4.5 kb correspond¬ ing to the size of the B. pertussis toxin genes found in other B. pertussis strains and to a previous Southern blot result with the chromosomal B. pertussis DNA and oligonucleotide probe mentioned above.

The plas id vector pGEM4blue (Promega Biotech., Madison, WI 53711)) was digested with the restriction enzymes BamHI and EcoRI and dephosphorylated with calf intestinal phospha- tase. The linearized vector DNA was ligated with the hybridi¬ zation positive DNA fraction. The ligation mixture was used to transform competent JL. coli K12 TGI cells which were grown, after transformation, on Luria plates containing 100 μg/ml ampicillin, 40 μg/ml Xgal and 0.5 mM IPTG. Ten percent of the colonies grown overnight were blue, thus representing the background caused by the vector. Nine hundred white colonies, containing chromosomal inserts, were tested by colony hybridi¬ zation (Grunstein et al.. PNAS 72:3961-3965 (1975)) using the oligonucleotide probe mentioned above. One of the colonies became positive in the hybridization test and after plasmid isolation a 4.5 kb insert, containing the gene encoding the whole pertussis toxin, was found in this clone.

To subclone the DNA fragments coding for different pertussis toxin subunits separately into the Bacillus secre¬ tion vectors or vectors constructed for cytoplasm!c produc¬ tion, the following further modifications were performed. The 4.5 kb EcoRI/BamHI fragment (S_j to S5) was first inserted into a M13mpl9 or Bluescript vector. Oligonucleotides were synthesized to create Hindlll sites at the 5' ends of each gene encoding for the mature toxin subunit. The Amersham jn vitro mutagenesis kit (Amersham Corp. Arlington Heights, IL 60005) was utilized to perform the oligonucleotide mutagen¬ esis. Alternatively, direct synthesis of modified 5' frag- ments was used. After the putative mutants were characterized by r?striction enzyme analysis and DNA sequencing, the correct

HindiII fragments were isolated from the RF-for (replicative form) of M13 derivatives to be joined to the Bacillus vectors. e) The ligation of the DNA sequence coding for pertus- sis toxin subunit to the vector.

Before the transformation of the host, a DNA sequence encoding the pertussis toxin subunit is ligated to the vector constructed as described in (c). A part of the DNA sequence may be used as well. The only condition is that a pertussis toxin subunit related polypeptide is synthesized.

Also a combination of DNA sequences encoding different subunits or their parts may be constructed. Here again the only condition is that the combination must be functional so that a pertussis toxin subunit related polypep- tide is synthesized, i.e., there are no inappropriate stop codons and the reading frames are correct.

The DNA sequences need not be identical to the DNA sequences encoding pertussis toxin subunits Sj to S5 previous¬ ly described. They may be derived from these sequences. Suitable sequences capable of hybridizing with said sequences may also be made synthetically or semisynthetically. DNA sequences that are capable of being expressed in a Bacillus host so that a polypeptide of pertussis toxin subunit type is produced may also be employed. As a useful alternative to produce pertussis toxins without secretion of protein into the medium, a recσtob-inant molecule, vector and host can be produced from which the signal sequence of a gene encoding a secretory protein has been deleted or omitted. Ordinary methods for producing such vectors and hosts can be used. The yields are good and easily recovered after breakage and centrifugation of the Bacillus cells. Example 2 presents a preferred non-limiting embodiment of this aspect of the invention.

The choice of the Bacillus host, its transformation and cultivation.

The suitability of the Bacillus bacteria as hosts depends upon the extent to which the product is degraded by proteases of the Bacillus host. It is also dependent upon the suit¬ ability of the protein to the expression and secretion system of the host. Any Bacillus hosts fitting these criteria may be used in the method of the invention.

One suitable candidate for the production of pertussis toxin subunit type polypeptides is Bacillus subtilis strain BRB41 (formerly designated strain IH6140) (Palva, Gene 22:229- 235 (1983)). It was found that the pertussis toxin subunit S_j secreted by this strain is remarkably stable in the presence of the proteases secreted by said host, although the whole pertussis toxin is not. The same strain has also successfully been used for intracellular production of all subunits (Sl- S5), as described in Example 2.

The selected host may be transformed and cultivated by conventional methods. The choice of the suitable transforma¬ tion system and cultivation conditions depends on the selected host.

Chromosomal integration of the recombinant DNA molecule

Methods of integrating the gene to be expressed into the genome of Bacillus and to multiply the integrated gene are known to those of skill in the art of recombinant genetics. These are described, for example, by Davis et al .. "Advanced Bacterial Genetics," Cold Spring Harbor Laboratory, New York (1980), and by Kallio, "Expression and Regulation of the Bacillus amyloliαuefaciens α-Amylase Gene in Bacillus sub¬ tilis," supra.

Purification of pertussis toxin subunits secreted by a Bacillus host

There are many ways to purify the product secreted by the Bacillus host. Some chro atographic methods are mentioned in the following as an example.

The method for producing subunits of pertussis toxin prepared from holotoxin made by Bordetella pertussis and chromatographed using different ion exchange resins has jeen previously described (Tamura, et al ., Biochemistry 21.15516- 5522 (1982)). These methods can be applied to purify, the subunits of pertussis toxin secreted by Bacillus bacteria. Ion exchangers like phosphocellulose, DEAE-cellulose, DEAE- Sephadex^R and CM-Sephadex^R among others can be used.

The solubility of the subunits may be improved by performing chromatography with buffers containing, for example, high concentrations of urea. Ion exchangers may also be applied to harvest and concentrate the product.

Other chromatographic methods available are, for example, hydrophobic chromatography using Phenylsepharose^R, chroj to- graphy in hydroxyapatite and affinity chromatography using as the solid phase, e.g., anti-pertussis toxin antibodies Cibacrom Blue or haptoglobin.

The preparation of the composition

There are several different ways in which a composition of the polypeptides produced by the method of the invention may be prepared; known to those of skill. The purified pertussis toxin subunit(s) or their biologically active frag¬ ment^) and analogs may be used as such to prepare a phar¬ maceutically acceptable dosage form or they be mixed together in any combination. The preparation of the combination may involve addition of solubilization and/or denaturation agents such as urea, which may be later removed. Immunoadjuvants such as aluminum hydroxide and pharmacologically acceptable preservatives such as thiomersal may be added to the composi¬ tion. These methods are described, for example, in Reming¬ ton's Pharmaceutical Science. 16th Ed., Mac. Eds. (1980).

Example 1

The secretion of pertussis toxin subunit S_} in Bacillus subtilis IH 6140 by using the secretion vector pKTH114

Construction of the secretion vector PKTH114 The secretion vector pKTH114 was constructed as previous¬ ly described (Pettersson et al.. Gene 24.15-27 (1983)). A chemically synthesized oligonucleotide that encodes the translation termination codon TGA in all three reading frames was joined to the plasmid pBR322 at different sites. The insert situated between the Hindlll site and the Ba HI site was excised and transferred to a Bacillus secretion plasmid pKTH97 to yield pKTH114. The Bacillus secretion plasmid p TH114 thus contains the regulation part and the secretion signal of the α-amylase gene from Bacillus amyloliouefaciens joined to the STOP oligonucleotide. The gene coding for any protein can be cloned at the end of the secretion signal.

Cloning of the DNA sequence coding for the subunit S_j

A DNA fragment consisting of the nucleotides between the Sau3A site at nucleotide 612 (Locht et al.. Science 232:1258-

1264 (1986)) and the Pstl site at nucleotide 1558 was isolated from Bordetella pertussis and ligated to pUC18 digested with

BamHI and Pstl. The ensuing plasmid was digested with BamHI, Klenow treated (fill in reaction), and Hindlll linkers were added by blunt end ligation. Hindlll digestion of_' the construct gave rise to a 960 bp long fragment (there is a Hindlll site in pUC18 next to the Pstl site). This fragment was purified and used for the construction of the expression plasmid pKTH212 as described below.

Transforming and selecting the Bacillus subtilis host

The recombinant plasmid pKTH212 was transferred into the competent Bacillus subtilis strain IH 6140. Cells that had received the plasmid were selected on the basis of kanamycin resistance and the cells were cultivated on nutrient medium plates containing kanamycin.

The size of plasmids present in the colonies was checked by cell lysis and running samples in an agarose gel by standard methods. Liquid cultures were made of colonies which contained plasmids of the expected size. The strains were grown in double strength L-broth to which had been added 3 % potato-extract and 10 μg/ml kanamycin. The cultures were grown overnight on a shaker at 37°C. The cultures ^"were centrifuged and the supernatants were recovered. Samples of the supernatant were run in an SDS-PAGE gel transferred electrophoretically to nitrocellulose filter (Western έlot) and probed with antipertussis toxin sera. One strain (IH 6540) which secreted the Sj protein was further analyzed.

Cultivation of Bacillus subtilis IH 6540, isolation and partial purification of the S\ Subunit. ⁴ 't

The B. subtilis strain IH 6540 was cultivated in L-broth of double strength (20 g of Bacto Tryptone, Difco; W - of

Yeast Extract, Difco; 10 g of NaCl per liter, pH 7.0), supplemented with kanamycin (30 /xg/ml) and with 30 rol l of potato extract (Kallio et al .. J. Gen. Microbiol. 132:677-687 (1982)). The growth took place in 2000 ml vials containing 250 ml of medium incubated at 37^βC in a rotary shaker incubator (250 rmp) . Bacteria were removed by centrifugation at 4'C, 8 hours after the optical density of the culture had reached 100 units as measured with Klett-Summerson colorimeter (red filter). The supernatant was stored at -60°C until used. 50 ml of above supernatant was dialyzed against 10 mM K- phosphate buffer (pH 5.0) and applied then to a phospho- cellulose (Pll, Whatman) column (about 12 ml of packed resin) equilibrated previously with the buffer above. The column was eluted with a linear gradient from 10 mN -phosphate (pH 5.0, 40 ml) to 500 mM K-phosphate (pH 5.0, 40 ml). Fractions of the eluate were assayed for the presence of S_j with i - munoblotting (dot blot) using polyclonal rabbit antipertussis toxin serum. The Sj was found in fractions between 45 ml and 60 ml of the eluate. Analysis of these fractions with SDS- PAGE and immunoblotting (Western blot) showed that they all contained both 28 kDa and 20 kDa protein. The 28 kDa protein is of the same size as native S_} and the lower one is assumed to be a truncated product of S_j.

S_j containing fractions were pooled, pH was adjusted to 6.0 and about 0.3 g (dry weight) of Blue Sepharose (Pharmacia, Piscataway, NJ 08864; washed as described by the manufacturer) was added to the solution. After overnight gentle stirring at 4^βC, the solution containing Blue Sepharose was packed on to a small column (about 1 ml) and eluted sequentially with 1 ml of following buffers: 100 mM K-phosphate (pH 6.0), 100 mM Tris- HC1 (pH 7.4) and 100 mM Tris-HCl, 750 mM MgCl₂ (pH 7.4).

The eluate was collected in approximately 0.2 ml frac- tions. Dot blot of fractions showed that there was Si protein in fractions 5 through 9. These were pooled and analyzed by SDS-PAGE stained with Coomassie Blue, and by Western blotting. These analyses showed that again both 28 kDa and 20 kDa S_j proteins were present. The total amount of S_j protein was approximately 20 μq. The total amount of protein in the preparation was about 400 jug (estimated on SDS-PAGE analysis described above).

Immunization with semipurified S_j subunit

Sj-protein made in B. subtilis and purified as djescribed above was used for immunization of a rabbit with the lymph node immunization method as described by Leinonen M., in, "Enterobacterial Surface Antigens: Methods for Molecular

Characterization," Korhenen et al., eds., Elsevier Science

Publishers, Amsterdam (1985). 150 μl of the se ipurified S_j- preparation was injected with Freunds complete adjuvant into the popliteal lymph node of the rabbit at days 0, 21 and 78, and serum collected at day 90.

Figure 1 shows the results when the serum obtained (Serum. KH974) was analyzed with Western blotting using purified pertussis toxin and Sj protein made in B. subtilis as antigens. When purified authentic pertussis toxin of J _. pertussis was used as antigen only the S_j subunit, which has the size of 28 kDa, was labeled by the serum KH974 (Lane D). When this same pertussis toxin preparation was analyzed with a serum raised against purified pertussis holotoxin (serum KH863) all five subunits were labeled (Lane A) showing their presence in the preparation (subunits S4 „and S5 were not separated in this gel system). These results show that the S|-protein made in B. subtilis is clearly immunogenic in rabbits and the im unological response is specific in the expected way (only anti Sj-antibodies were made and there were no antibodies against other subunits of the pertussis toxin). When the antigen was S_j protein made in B. subtilis, both 28 kDa and 20 kDa Sj-proteins were recognized by the serum KH974. Furthermore, when the antigen in the Western blot was derived from cell free culture supernatant of the strain IH 6540, again the main proteins labeled were the 28 kDA and 20 kDa Si-proteins (Lanes E_j and E_j). There was only one more labeled protein, having apparent molecular weight of about 35 kDa, visible in the blot. This protein was seen also in the control (Lane F), where the antigen was derived from culture supernatant of the strain IH6396, which does not produce any S_j-protein. This indicates that the 35 kDa protein is a bacillary protein, which has been present as a contaminant in the semipurified S_j-preparation used as immunogen for the serum KH974. The lack of antibodies against other bacillary proteins indicates that the S_j-proteins have been the main immunogenic proteins of the preparation used for immunization, although the preparation was only semipurified and contained also bacillary proteins.

The stability of the product

The amount of the product in the supernatant as a function of the incubation time was monitored by Western blot analysis. It was found that the amount of S_j in the super¬ natant increased during the exponential growth phase and peaked at the stationary phase.

The overnight culture supernatant of IH 6540 contained several mg/1 of polypeptides reacting with anti-Si. The amount of native size S_j produced was estimated on the basis of Western blot to be about 10 mg/1 whereas the truncated products of Sj seen in Western blot were present in even larger amounts, e.g., around 100 mg/1 of a 20 kDa size protein. The pertussis toxin subunit Si was found to be remarkably resistant to proteases present in the culture supernatant. S_j was not appreciably decreased during a 4 hour incubation at 37^βC, whereas over 90% of the subunit S_j was degraded after a 3 hour incubation when purified whole pertussis toxin* was incubated with bacillary supernatant.

In addition to the SI construction described above, a similar modified SI gene has also been constructed as follows: According to the published nucleotide sequence of Pertussis toxin (Locht et al .. Science 232:1258-1264 (198&)) a modified DNA fragment coding for the mature part of the Si subunit was constructed as follows: The PT operon was cloned into a M13mpl9 vector to generate single stranded DNA. Using oligonucleotide-directed in vitro mutagenesis (Amersham kit), Hindlll restriction sites were synthesized at the 5' end of the Si gene coding for the mature part of the S\ subunit and downstream from the translation stop-codon. After isolation of the RF (replicative form) DNA of the M13 clone, the Hindlll restriction fragment (745 bp) containing the modified S_j was purified and joined to the secretion vector pKTH 114. .The expression of this construction in B. subtilis IH 6140*was indistinguishable from that of pKTH 212. * -,

_ '^*"<*t Example 2

Expression of pertussis toxin subunits inside Bacilllis subtilis

From Bordetella pertussis strain Tohama a 4.5 kb EcoRI- BamHI chromosomal DNA restriction enzyme fragment, containing the pertussis toxin operon, was cloned, utilizing the previ¬ ously published DNA sequence (Locht, C. et al . , Science 232:1258-1264 (1986)). Using Amersha 's in vitro oligo- directed mutagenesis system, a Hindlll restriction enzyme site was created at the 5' end of the DNA sequence coding for the mature part of pertussis toxin subunits Si, S , S4 and S5. The Hindlll site in the gene coding for S3 was created with an oligolinker ligated into the Fokl site positioned immediately downstream of the 5' end of the DNA sequence coding for the mature part of S3.

To express the pertussis toxin subunits, Hindlll frag¬ ments containing the coding regions for the subunits were ligated with Hindlll cut expression vectors (Figure 2). In this way, the transcription of the subunit's genes could be controlled by the a ylase promoter (Palva, I., et al ., Gene 15:43-51 (1981)) in the vector. The procedure for expressing S4 was different. The Hindlll fragments coding for S4 were subjected to a fill-in reaction with Klenow fragment, and the expression vector, to.be ligated with the S4 coding fragment, was digested with EcoRI, followed by a partial S_j exonuclease digestion and a fill-in reaction with Klenow fragment. Bacillus subtilis strain BRB41 (Palva, I., et al .. Gene 22:229-235 (1983)) was transformed with the ligation mixes. The ability of the clones to express the pertussis toxin subunits was analyzed by SDS-polyacrylamide gel electro- phoresis, followed by Western blotting. A rabbit antiserum recognizing all subunits and the Protoblot immunoscreening system was used to visualize the i munoreactive proteins. All subunits could be expressed inside Bacillus subtilis as seen in Figure 4. The predicted N-terminal amino acids of the subunits produced in this way are illustrated in Figure 3.

Claims

WHAT IS CLAIMED IS:

1. A method for the production of pertussis toxin subunits comprising: (a) introducing into a Bacillus host a recombinant

DNA molecule selected from the group consisting of: ^j»

(i) the regulation and secretion signal sequence of a gene encoding a secretory protein of a Bacillus strain, operatively linked to at least one DNA sequence encoding a pertussis toxin subunit selected from the group consisting of Si, S₂, S3, S4 and S5;

(ii) the regulation and secretion signal sequence of a gene encoding a secretory protein of a Bacillus strain, operatively linked to at least one DNA sequence which hybridizes to any of the DNA sequences of (i) and which codes for a pertussis toxin subunit;

(iii) the regulation and secretion signal sequence of a gene encoding a secretory protein of a Bacillus strain, operatively linked to at least one DNA sequence derived from any of the DNA sequences of (i) and which codes for a pertussis toxin subunit;

(iv) the regulation sequence and deleted nonfunctional secretion signal sequence of a gene encoding a secretory protein of a Bacillus strain, operatively linked to at least one DNA sequence encoding a pertussis toxin subunit selected from the group consisting of S_j, S₂, S3, S4 and S5;.

(v) the regulation sequence and ^"deleted nonfunctional secretion signal sequence of a gene encoding a secretory protein of a Bacillus strain, operatively linked to at least one DNA sequence which hybridizes to any of the DNA sequences of (i) and which codes for a pertussis toxin subunit; and (vi) the regulation sequence and deleted nonfunctional secretion signal sequence of a gene encoding a secretory protein of a Bacillus strain, operatively linked to at least one DNA sequence derived from any of the DNA sequences of (i) and which codes for a pertussis toxin subunit;

(b) cultivating said Bacillus host containing said recombinant DNA molecule in a suitable culture medium and under conditions allowing expression of said DNA sequences; and

(c) recovering said pertussis toxin subunits.

2. The method of claim 1 wherein said regulation and secretion signal sequence of (a)(i) or a part thereof is linked to at least part of the signal sequence of a pertussis toxin subunit selected from the group consisting of Si, S , S3, S4 and S5.

3. The method of claims 1 or 2 wherein said regulation and secretion signal sequence or said regulation and deleted nonfunctional secretion signal sequence is or is derived from the regulation and secretion signal sequence of the α-amylase gene of Bacillus amyloliquefaciens.

4. The method of claims 1 or 2 wherein said DNA sequence encoding the pertussis toxin subunit is the DNA sequence encoding the pertussis toxin subunit S .

5. The method of claims 1 or 2 wherein said Bacillus host is transformed with a vector comprising said recombinant DNA molecule, said vector being capable of replicating in several copies in a Bacillus strain.

6. The method of claims 1 or 2 wherein said recombinant DNA molecule is integrated into the chromosome of the Bacillus host.

7. A Bacillus host comprising a reco binanl.- DNA molecule for the production of a pertussis toxin subunit, said molecule selected from the group consisting of:

(a) the regulation and secretion signal sequence of a gene encoding a secretory protein of a Bacillus strain, operatively linked to at least one DNA sequence encoding a pertussis toxin subunit selected from the group consisting of

(b) the regulation and secretion signal sequence of a gene encoding a secretory protein of a Bacillus strain, operatively linked to at least one DNA sequence which hybri¬ dizes to any of the DNA sequences of (a) and which codes for a pertussis toxin subunit;

(c) the regulation and secretion signal sequence of a gene encoding a secretory protein of a Bacillus strain, operatively linked to at least one DNA -sequence derived frp any of the DNA sequences of (a) and which codes for a pertussis toxin subunit;

(d) the regulation sequence and deleted nonfunc¬ tional secretion signal sequence of a gene encoding a secre- tory protein of a Bacillus strain, operatively linked to at least one DNA sequence encoding a pertussis toxin subunit selected from the group consisting of Si, S₂, S3, S4 and S5;

(e) the regulation sequence and deleted nonfunc¬ tional secretion signal sequence of a gene encoding a se re- tory protein of a Bacillus strain, operatively linked to at least one DNA sequence which hybridizes to any of the DNA sequences of (a) and which codes for a pertussis toxin subunit; and *^s (f) the regulation sequence and deleted nonfunc¬ tional secretion signal sequence of a gene encoding a secre¬ tory protein of a Bacillus strain, operatively linked to at least one DNA sequence derived from any of the DNA sequences of (a) and which codes for a pertussis toxin subunit.

8. The host of claim 7 wherein said regulation and secretion signal sequence of (a) or a part thereof is linked to at least part of the signal sequence of a pertussis toxin subunit selected from the group consisting of Si, S_2. S3, S4 and S5.

9. The host of claims 7 or 8 wherein said regulation and secretion signal sequence or said regulation and deleted nonfunctional secretion signal sequence is or is derived from the regulation and secretion signal sequence of the α-amylase gene of Bacillus amyloliαuefaciens.

10. The host of claims 7 or 8 wherein the Bacillus host is transformed with a vector comprising said recombinant DNA molecule, said vector being capable of replicating in several copies in a Bacillus strain.

11. The host of claims 7 or 8 wherein said recombinant DNA molecule is integrated into the chromosome of the Bacillus host.

12. A recombinant DNA molecule for the production of a pertussis toxin subunit in a Bacillus host, selected from the group consisting of:

(a) the regulation and secretion signal sequence of a gene encoding a secretory protein of a Bacillus strain, operatively linked to at least one DNA sequence encoding a pertussis toxin subunit selected from the group consisting of i, S2, S3, S4 and S5;

(b) the regulation and secretion signal sequence of a gene encoding a secretory protein of a Bacillus strain, operatively linked to at least one DNA sequence which hybri¬ dizes to any of the DNA sequences of (a) and which codes for a pertussis toxin subunit; '

(c) the regulation and secretion signal sequence of a gene encoding for a secretory protein of a Bacillus strain, operatively linked to at least one DNA sequence derived from any of the DNA sequences of (a) and which codes for a pertussis toxin subunit;

(d) the regulation sequence and deleted nonfunc¬ tional secretion signal sequence of a gene encoding a secretory protein of a Bacillus strain, operatively linked to at least one DNA sequence encoding a pertussis toxin subunit selected from the group consisting of S , S2, S3, S4 and S5;

(e) the regulation sequence and deleted nonfunc¬ tional secretion signal sequence of a gene encoding, a secretory protein of a Bacillus strain, operatively linked to at least one DNA sequence which hybridizes to any of the DNA sequences of (a) and which codes for a pertussis toxin subunit; and

(f) the regulation sequence and deleted nonfunc- tional secretion signal sequence of a gene encoding a secretory protein of a Bacillus strain, operatively linked to at least one DNA sequence derived from any of the DNA sequences of (a) and which codes for a pertussis toxin subunit.

13. The recombinant DNA molecule of claim 12 wherein said regulation and secretion signal sequence of (a) or a part thereof is linked to at least part of the signal sequence of a pertussis toxin subunit selected from the group consisting of ^sl_. S2, S3, S4 and S5.

14. The recombinant DNA molecule of claims 12 or 13 wherein said regulation and secretion signal sequence or said regulation and deleted nonfunctional signal sequence is or is derived from the regulation and secretion signal sequence of the α-amylase gene of Bacillus amyloliouefaciens.

15. A vector comprising the recombinant DNA molecule of claims 12 or 13 wherein said recombinant DNA molecule is inserted into a vector, said vector being capable of replicat¬ ing in several copies in a Bacillus strain.

16. The recombinant DNA molecule of claims 12 or 13 wherein said recombinant DNA molecule is integrated into the chromosome of the Bacillus host.

17. A method for producing a recombinant DNA molecule for the production of a pertussis toxin subunit in a Bacillus host comprising:

(a) preparing a recombinant DNA molecule selected from the group consisting of:

(i) at least one DNA sequence encoding a pertussis toxin subunit selected from the group consisting of ^sl> ^s2» S3, S4 and S5;

(ii) at least one DNA sequence which hybridizes to any of the DNA sequences of (i) and which codes for a pertussis toxin subunit; and (iii) at least one DNA sequence derived from any of the DNA sequences of (i) and which codes for a pertussis toxin subunit; and ^■27-

(b) ligating said DNA sequence to a regulation and secretion signal sequence or to a regulation and deleted nonfunctional signal sequence of a gene encoding a secretory protein of a Bacillus strain.

18. The method of claim 17 wherein said regulation and secretion signal sequence or said regulation and deleted nonfunctional signal sequence is linked to at least part of the signal sequence of a pertussis toxin subunit selected from the group consisting of S_j, S2, S3, S4 and S5.

19. The method of claims 17 or 18, wherein said regulation and secretion signal sequence or said regulation and deleted nonfunctional signal sequence is or is derived from the regulation and secretion signal sequence of the α- amylase gene of Bacillus amyloliquefaciens.

20. A polypeptide prepared by recombinant DNA techniques in a Bacillus host comprising a pertussis toxin subunit selected from the group consisting of Sj, S2, S3, S4 and S5.

21. A composition comprising the polypeptide of claim 20 wherein said composition is in a pharmaceutically acceptable dosage form.