WO1992001001A1 - Production of outer membrane (om) proteins in gram-positive bacteria and recovery of protective epitopes - Google Patents

Production of outer membrane (om) proteins in gram-positive bacteria and recovery of protective epitopes Download PDF

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Publication number
WO1992001001A1
WO1992001001A1 PCT/FI1991/000212 FI9100212W WO9201001A1 WO 1992001001 A1 WO1992001001 A1 WO 1992001001A1 FI 9100212 W FI9100212 W FI 9100212W WO 9201001 A1 WO9201001 A1 WO 9201001A1
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outer membrane
membrane protein
protein
cloned
gram
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PCT/FI1991/000212
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English (en)
French (fr)
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Matti Sarvas
Sarah Butcher
Marjatta Nurminen-Kalliokoski
Kate Runeberg-Nyman
Susanna Muttilainen
Eva WAHLSTRÖM
Ilona IDÄNPÄÄN-HEIKKILÄ
Ritvaleena Puohiniemi
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The Finnish National Public Health Institute
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Priority to AU81873/91A priority Critical patent/AU668075B2/en
Priority to JP3511658A priority patent/JPH06511140A/ja
Publication of WO1992001001A1 publication Critical patent/WO1992001001A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • OMP outer membrane proteins
  • Tne conformation of the OMP's seems to depend on their association with LPS's and other specific factors in the environment. What these other factors are, and how they affect conformation, are not well understood.
  • epitopes on native proteins comprise about 15-25 amino acid residues, which are made up of two or three discontinuous surface loops. See for example. Layer et al, Cell 61:553-556 (1990). Tne protective epitopes contained in the outer membrane proteins are exposed on the cell surface of the bacterium, where they are capable of inducing antibodies that can protect an animal against infection by that strain of bacteria. Energetic calculations suggest that a smaller subset of 5-6 amino acid residues of an epitope contributes most of the binding energy to the antibodies, with the surrounding residues merely aiding in
  • the epitope regions are loops connecting so-called beta-regions which protrude out of the membrane. These loops are hydrophiiic and may be locally water soluble, even though this is not true of the whole OMP.
  • Purified outer membrane proteins could be used in medicine as vaccines to prevent diseases caused by pathogenic gram-negative bacteria or as reagents to diagnose such diseases by immunological methods.
  • Neisseria meningitidis bacteria meningococci
  • purulent meningitis Pieris-associated meningitis
  • capsular polysaccharide vaccines were shown to be efficacious against two meningococcal serogroups, A and C (World Health Organization Study Group. Technical Report Series No. 588. World Health Organization. Geneva, 1976).
  • MenB The capsule of the third major serogroup, B (hereinafter MenB) proved, however, to be structurally closely similar to the saccharide part of glycoprot ⁇ ins in some human tissues (Finne, J., Leinonen, M., and Gurkelä, P.H. Lancet ii:355-357 (1983)). Tne resulting strons immunolo ⁇ ical cross-reactivitv mav exoiain whv attemots to produce a capsular polysaccharide vaccine for MenB have failed.
  • Alteanative candidates for a MenB vaccine have been sought.
  • the outer membrane of Neisseria meningitidis bacteria, as the surface structure of the bacteria in immediate contact with the environment, is one such candidate. Protection afforded by monoclonal antibodies directed to different OM components has been studied in an infant rat model
  • OM complex vaccines consisting of semi-purified OM components, have been prepared and tested in several field trials (Frasch, C.E.
  • Neisseria meningitidis and other pathogens such as Neisseria gonorrhoeae, Haemophilus infiuenzae, Yersinia sp. , and Brucella sp., their preparation and purification from gram-negative bacteria is difficult with conventional biochemical methods.
  • a special problem is the tight association of the OMP's with lipopolysaccharide (Hitchcock, P.J., and Morrison, D.C. in E.T. Rietschel (ed.) Handbook of Endotoxin, vol. I. Chemistry of Endotoxin, Elsevier Science Publishing. Inc. New York, 1984.), which is toxic.
  • the problem of removing toxic LPS has so far not been solved in a satisfactory manner, even using harsh methods.
  • proteins When proteins are produced using the methods of genetic engineering, it is often a goal to produce the recombinant proteins in large amounts. Sometimes when large amounts of recombinant proteins are produced in bacteria, the proteins form insoluble aggregates. These insoluble aggregates are referred to as inclusion bodies. In inclusion bodies the proteins are in an unnatural state devoid of their authentic configuration and epitopes. Before restoration of the biological activity (e.g., enzymatic or receptor activities, or protective epitopes) the recombinant proteins must be returned to their native conformations, i.e., need to be correctly folded.
  • biological activity e.g., enzymatic or receptor activities, or protective epitopes
  • Chaotropic agents and detergents can be used to solubilize some proteins and return them to their native conformations. (Chaotropic agents prevent the random coils among hydrophilic regions , which are characteristic of denatured lipophilic proteins in aqueous solutions, from occurring.) Unfortunately, the proteins produced in bacteria as inclusion bodies are very resistant to many chaotropic agents and detergents (see generally, Methods of Enzymology, 1990) and are soluble, if at all, only in high concentrations of urea or guanidine hydrochloride, or SDS. This is true even though the native proteins were water soluble.
  • an enzyme protein can be regained after removal of the chaotropic agent, e.g., active prourokinase is formed from inactive prourokinase inclusion bodies by solubilization with 6 M guanidine hydrochloride and 2-mercaptoethanol and subsequent refolding of the protein for 24 h at 15 o C in a buffer containing 2 M urea (Orsini, G. et al, Eur. J. Biochem. 195:691-697 (1991)).
  • Some recombinant proteins are soluble in sarkosyl (Puohiniemi, R., M. Karvonen, J. Vuopio-Varkila, A. Muotiala, I.M. Helander and M. Sarvas. Inf. Imm. 58:1691-1696 (1990);
  • the outer membrane proteins of gram-negative bacteria are intimately bound to lipopolysaccharide (LPS) and perhaps other membrane components; their proper conformation is dependent on this association with such components of the membrane environment.
  • LPS lipopolysaccharide
  • these stabilizing components must be replaced by other components that mimic their function, so as to ensure the stability of the OMP in its native beta-barrel conformation.
  • the beta structure is the well-ordered part of the OMP that is complexed with LPS.
  • regulation and expression sequence it is meant to include within the scope of the instant invention the DNA sequence of a gene preceding the DNA sequence encoding a polypeptide; the DNA sequence is needed for the transcription and translation of the DNA sequence encoding that polypeptide. Such sequence typically includes the promoter and ribosomal binding site and possibly binding sites for regulatory proteins.
  • the “regulation and expression sequence” may be any biologically active fragment thereof.
  • the regulation and expression sequence may include also a DNA sequence encoding an N-terminal fragment of the polypeptide, if that fragment of the polypeptide is not a functional signal sequence for export.
  • outer membrane protein and by “mature outer membrane protein” it is meant an outer membrane protein, which is devoid of a signal peptide for export, or devoid of a functional signal peptide.
  • vector any autonomous element capable of replicating in a host independently of the host's chromosome, into which additional sequences of DNA may be incorporated.
  • vectors include, but are not limited to, bacterial plasmids and phages.
  • the regulation and expression sequence including the promoter, controls the initiation of expression of the polypeptide encoded by the structural gene; there may be a DNA sequence derived from the same gene as the promoter or any other DNA sequence between the promoter and the initiation of the polypeptide to enhance the expression of the polypeptide.
  • This DNA sequence may encode a peptide that remains fused to the polypeptide, but the said peptide must not be a functional signal for export.
  • Recombinamly produced when referring to the production of OMP, means that the OMP's of one species of bacteria, or DNA encoding for such protein, are expressed in the cells of a bacterium from another species. Recombinamly produced OMP will have been produced using the techniques of gene expression and genetic engineering. Recombinamly produced OMP's are cloned OMP's, and are to be
  • the invention provides a method for producing cloned outer membrane (OM) protein from pathogenic gram-negative bacteria.
  • the invention also provides a method for renaturing the cloned outer membrane protein thus produced so the cloned OM protein regains immunologically active epitopes which are capable of eliciting production of antibodies, in mammals and other animals, that are bactericidal and can provide protection against infection by the pathogenic gram -negative bacteria.
  • DNA encoding outer membrane protein from gram-negative bacteria known to be pathogenic in humans and animals, is expressed in a gram-positive bacterial host.
  • the recombinant or cloned OM protein thus produced is then renatured so as to regain biologically or immunologically active epitopes which are capable of eliciting production of antibodies in animals and humans; the antibodies are bactericidal and protect the animals and humans from infection by the pathogenic gram-negative bacteria from which the gene encoding the cloned OMP's was derived.
  • a Bacillus or other suitable gram-positive bacterial host containing a recombinant DNA molecule comprising the regulation and expression sequence of a gene expressed well in the host is operationally linked to a DNA sequence encoding an outer membrane protein from a gram-negative bacteria known to be pathogenic to animals and humans.
  • the regulation and expression signal is typically an effective promoter and ribosomal binding site.
  • the DNA sequence encoding the outer membrane protein is devoid of functional signal sequence, as is the regulation and expression sequence.
  • the presence of signal sequences decreases the amount of recombinant outer membrane protein expressed in the gram-positive host.
  • the recombinant DNA molecule may be introduced into the Bacillus or other suitable gram-positive host by transforming the host with a vector that is capable of replicating in several copies in the host strain.
  • the recombinant DNA molecule may be integrated into the chromosome of the Bacillus or other host strain. Using the method, more than one hundred milligrams of product per liter of culture are obtained when ordinary laboratory media are used. The amount can be higher in high density cultures.
  • the product may be aggregated
  • the conditions for refolding the OM proteins are such that allow the OM proteins to take the same or partially the same conformation as they have in their natural environment, in the outer membrane. While such conditions can be achieved by using the same amphiphilic compounds present in OM, e.g., LPS, use of LPS is not preferred since LPS is toxic. Non-toxic derivatives of LPS can used. Other non-toxic lipids or their derivatives or analogs, may also be used, alone or in conjunction with detergents, variations in pH and/or temperature, the addition of chemicals, e.g., sugars and amino acids, as well as other conditions which may favor refolding of protective epitopes.
  • LPS amphiphilic compounds present in OM
  • Other non-toxic lipids or their derivatives or analogs may also be used, alone or in conjunction with detergents, variations in pH and/or temperature, the addition of chemicals, e.g., sugars and amino acids, as well as other conditions which may favor refolding of protective epitopes.
  • any of these methods and agents, or combinations of agents may be used as long as they permit (a) proper refolding of the epitopic loops and (b) their accessibility to the immune system, i.e., the epitopic loops must protrude into the primarily aqueous serum of an animal or human after injection and subsequent dilution.
  • the method of the invention is exemplified by the production of cloned and renatured class 1 outer membrane protein from Neisseria meningitidis, class 3 OM protein of Neisseria meningitidis and the OM protein OmpA of Escherichia coli, all in Bacillus subtilis.
  • the method can be used to produce other outer membrane proteins, including, but not limited to, other OM proteins of Neisseria meningitidis, and the OM proteins of Neisseria gonorrhoeae,
  • One objective of the invention is to use the method for the production of safe and effective vaccines. Another objective is to provide diagnostic antigens for the identification of infections caused by
  • FIG. 1 The sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE, Coomassie Blue staining) of 12 transformant colonies tested (see Example 3). Lane 1, the molecular weight standard;
  • Lane 3 Mock preparation made similarly to the OmpA preparation in lane 2 from IH6418, a strain containing the secretion vector without any insert. Lanes 4-15, samples of separate transformant colonies.
  • the figures to the left show the position and size (kDa) of molecular weight markers. Symbol on the right indicate position of OmpA.
  • Oligonucleotides used to amplify the DNA coding for the class 1 proteins in a PCR reaction.
  • the oligonucleotide of Sec. ID 1 consists of 4 nucleotides (AACC), a HindIII site, and nucleotides 125-155 of Barlow, et al, Mol. Microbiol. 3:131-139 (1989) coding for the first 10 amino acids of the mature protein.
  • the oligonucleotide of Seq. ID 2 consists of 4 nucleotides (AACC), the reverse sequence of the following: a HindIII site (including part of the stop codon), stop codon and nucleotides 1246-1217 of Barlow, et al, Mol. Microbiol. 3:131-139 (1989). The sequences are included in the specification, just preceding the claims.
  • Sequence ID Number 3 (Seq. ID 3). DNA sequence of the pKTH250 insert; P1.7,16. Nucleotide 1 of the sequence shown, corresponds to nucleotide 125 of the sequence from Barlow et al, Mol. Microbiol. 3:131-139 (1989).
  • Suitable expression vectors may be used in the present invention, and are known to those of skill in the art of recombinant genetics.
  • a preferred vector is disclosed in PCT WO 90FI41, filed 2 Feb 1990 and published 23 Aug 1990 as WO 9009448: New Recombinant DNA Molecules for Producing Proteins and Peptides in Bacillus Strains.
  • the transfer vector may be any plasmid or phage capable of replicating in several copies in a Bacillus strain or other gram-positive bacterium.
  • a multitude of such vectors are available, the most representative of them being the plasmids isolated from Staphy lococcus, Bacillus or
  • Streptococcus or their derivatives.
  • the regulation and expression 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-linkers so that the genes to be expressed may be joined downstream from the regulation and expression sequence of the vector.
  • a DNA sequence encoding the OM protein (or epitopically functional portion(s) thereof) is ligated to a suitable vector.
  • the DNA sequences need not be identical to the DNA sequences encoding a particular OM protein as found in a particular natural gene. They may be derived from the sequences of natural genes of OM proteins, but modified in ways that may alter the properties of the resulting protein. Suitable sequences may also be made synthetically or semisynthetically.
  • the selected host may be transformed and cultivated by conventional methods.
  • the choice of suitable transformation systems and cultivation conditions depends on the selected host. Purification of OM Proteins Produced Intracellularly in Bacillus Host or in Other Gram-Positive Bacteria
  • the OM proteins produced with the method of the invention often form intracellular inclusion bodies.
  • the main advantage of producing inclusion bodies (or intracellular aggregates of overproduced protein) is that they are easy to purify (Marston and Hartley, Methods of Enzymology
  • the bacterial cells are disrupted by sonication, passage through French pressure cells, lysozyme-treated or by other suitable means.
  • the inclusion bodies can be pelleted with low speed centrifugation and usually further washed with a mild detergent.
  • the inclusion bodies are soluble only in chaotropic agents like urea or guanidine hydrochloride or strong detergents like SDS.
  • the proteins can be further purified with conventional purification methods.
  • OM protein produced in Bacillus is not present in inclusion bodies, modifications of the methods above may be applied.
  • the purification may also involve solubilization and differential extraction with various types of detergents, and chromatography and electrophoresis in the presence and absence of detergents.
  • compositions of the recombinant polypeptides produced by the method of the invention may be prepared.
  • the purified OM proteins or their fragments may be used alone to prepare a pharmaceutically-acceptable dosage form and they may be mixed together in any combination.
  • the recovery of the native epitopes may involve addition of solubihzation and/or denaturing agents such as urea, guanidine hydrochloride and SDS, which may be later removed. It may also involve addition of compounds like
  • the preparation may be in a form of liposomes or in another form.
  • Immunoadjuvants such as aluminium hydroxide and
  • pharmacologically-acceptable preservatives such as thiomersal may be added to the composition.
  • the D ⁇ A fragment coding for the mature protein was acquired as follows: using the published nucleotide sequence of meningococcal class 1, P1.7,16 protein (Barlow et al, Mol Microbiol 3:131-139 (1989)) two
  • oligonucleotides (primer 1 and primer 2; primer 1 is shown herein as Seq. ID 1; primer 2 is Seq. ID 2) were synthesized and used to amplify the D ⁇ A coding for the mature class 1 protein in a polymerase chain reaction (PCR) with chromosomal meningococcal D ⁇ A .
  • the meningococcal D ⁇ A was isolated from strain IH5341 (P1.7,16) grown on protease-peptone plates containing 15% agarose in lieu of agar. After treatment with a zwitterionic detergent in citrate buffer (Domenico et al, J Microbial Methods 9:211-19 (1989)) to remove e.g., capsule, the D ⁇ A was isolated.
  • the PCR reaction was performed using a GeneAmpTM kit using the methods described by the manufacturer (Perkin Elmer Cetus).
  • the amplified D ⁇ A fragments were of two sizes when separated by agarose gel electrophoresis. The bigger fragment seemed to be the expected size for class 1, i.e., about 1100 bp whereas the other fragment was smaller, about 900 bp.
  • the amplified D ⁇ A mixture was purified by phenol extraction, ethanol precipitated, resuspended in TE (10 mM Tris-HCl pH8, 1 mM EDTA) and digested with the restriction enzyme HindIII.
  • the plasmid vector pUC18 was digested with the restriction enzyme HindIII.
  • the linearized vector D ⁇ A was ligated with the
  • HindIII-digested amplified D ⁇ A The ligation mixture was used to transform competent Escherichia coli K12 TGI cells which were grown, after
  • Plasmid pKTH250 (pUC18 containing DNA coding for the cloned class 1 protein P1.7,16) was digested with HindIII to release the cloned class 1 gene. Also plasmid pKTH2S9 was digested with HindIII to release the extra adapter copy and to linearize the vector (see Fig. 1). The two HindIII digested plasmids were ligated and the ligation mixture was used to transform IH6140. Cells that had received at least pKTH2S9 were selected on the basis of kanamycin resistance.
  • the size of plasmids present in the colonies which grew on Luria plates containing kanamycin was checked by cell lysis and running samples in an agarose gel by standard methods. Colonies which contained plasmids of the expected size were analyzed for class 1 protein expression by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAG ⁇ ) and SDS-PAG ⁇ followed by immunoblotting (Western blot). One strain IH6627 containing plasmid pKTH290 which expressed the class 1 protein was further analyzed. The cloning site was checked by Sanger dideoxy sequencing of pKTH290 to ensure that only one copy of the adapter was present. Screening for the Expression of Class 1 Proteins
  • class 1 P1.7,16 protein in Bacillus transformants was screened using SDS-PAGE in the following way: a loopful of bacteria grown on Luria agar plates was suspended in Laemmli sample buffer and after heating at 100 o C, a sample was applied to SDS-PAGE. The class 1 protein was visualized with Coomassie Blue staining (Fig. 3) and with immunostaining of the SDS-PAGE after blotting the proteins onto millipore filter (Western blot). Antisera KH 1110 prepared by immunization of a rabbit with an extract of MenB:15:P1.7,16 bacteria, was used in
  • the bacteria were grown either in liquid or on solid Luria medium containing 10 - 30 ⁇ g kanamycin per liter. When growing one liter of Luria broth about 10 g bacterial cells (wet weight) were obtained.
  • the bacteria were disrupted with lysozyme in the following way: one gram of bacteria grown on Luria agar plates containing kanamycin was suspended in 5 ml of 20 sucrose in buffer (25 mM TrisHCl, pH 8.0; 15 mMMgCl 2 containing 1 mg lysozyme/ml). After an incubation of 30 min at 37 ° C the protoplasts were collected by cemrifugation (10,000xg) and lysed by suspending them in 5 ml of 50 mM TrisHCl, pH 8,0. DNa se (1 mg/ml) was added and after five minutes inclusion bodies were collected by centrifugation 10,000xg for 10 min.
  • BacP1.7,16 protein isolated as inclusion bodies was called BacP1.7,16-IB.
  • the inclusion body fraction derived from 10 g of bacteria contained 300 mg protein, as measured by the Lowry method (Lowry, O.H. et al, J. Biol Chem. 193:265-275 (1951)).
  • the amount of BacP1.7,16 protein in this fraction was roughly estimated by visual inspection of the SDS-PAGE (Fig. 6) to be at least 120 mg. That means that more than 1/3 of the protein present in the inclusion bodies from
  • IH6627 is BacP1.7,16 protein. It can be calculated that BacP1.7,16 protein comprised at least 25 % of the total cellular protein of IH6627. It can also be calculated that there was more than one hundred mg of BacP1.7,16 protein per liter of culture.
  • the size of the BacP1.7,16 protein is roughly the same as that of the authentic protein of N. meningitidis in SDS-Page.
  • the plasmid pKTH 217 (described in Puohiniemi, R., M.
  • Karvonen, J. Vuopio-Varkila, A. Muotiala. I.M. Helander and M. Sarvas. Inf. Imm. 58:1691-1696 (1990)) contains a 2.5 Kb HindIII-BamHI fragment which encodes, starting at the HindIII terminus, the 8 to 325 amino acid residues of the OmpA protein of E. coli. The fragment is flanked at the HindIII terminus by a unique Clal-HindIII fragment. To construct the plasmid pKTH3125, this ClaI-HindIII fragment was replaced by the ClaI-HindIII fragment of the plasmid pKTH288 shown in Figure 1.
  • the latter fragment contains the promoter and truncated, nonfunctional signal sequence of ⁇ -amylase.
  • pKTH3125 they were fused to the DNA fragment encoding the OmpA protein.
  • the plasmids pKTH217 and 288 were digested with endonucleases Clal and HindIII, phenol extracted, ethanol precipitated and resuspended in water. Then 1.2 ⁇ g of digested pKTH217 was mixed with 2.5 ⁇ g of digested pKT ⁇ 288, treated with polynucleotide kinase and then ligated. All DNA manipulations were performed as described in Maniatis et al, (Maniatis, T., E. F. Fritsch, and J. Sambrook, Molecular Cloning. A
  • Competent cells of B. subtilis strain IH6140 were then transformed with the ligation mixture as described in Maniatis et al, and plated on Luria-plates containing 10 ⁇ g of kanamycin.
  • the expression of OmpA was tested as follows from 24 of more than 10 4 transformant colonies obtained. About half of the colony was mixed with 10 ⁇ l of SDS-PAGE sample buffer, heated to 100o C for 5 minutes and the sample was electrophoresed in SDS-PAGE (Laemmli, U. K. Nature, (London) 227:680-685 (1970)). As judged by the presence of a major band of the size of the mature OmpA protein (Fig. 4, lanes 4, 6-9, 13, 14), several colonies contained OmpA protein. This band also reacted with OmpA serum in immunoblotting. One of them was designated IH6649, and the plasmid in this strain pKTH3125.
  • IH6649 was grown overnight at 37 oC with shaking (250 rpm) in liquid culture in twofold-concentrated L-broth containing 10 mg of NaCl per liter, 10 ⁇ g of kanamycin per ml, and 30 ⁇ l of potato extract per ml (Kallio, P., M. Simonen, I. Palva, and M. Sarvas, J. Gen. Microbiol. 132:677-678 (1986)).
  • particulate material was then pelleted by centrifugation at 16,000xg, for 10 min. OmpA was not a major band in the supernatant, as analyzed with
  • subtilis IH6649 it forms aggregates or inclusion bodies that can be collected by centrifuging at 2,000-5,000xg.
  • the amount of OmpA in the 2,000xg pellet after washing and the 2.000xg supernatant after pelleting at 5,000xg and washing was estimated visually by comparing the intensity of the OmpA bands in SDS-PAGE-(Fig. 5, lanes 13 and 14) with the intensity of the molecular weight standard bands
  • the 43 kDa band contains 1.47 ⁇ g protein according to the manufacturer. Pharmacia).
  • the band in lane 13 was estimated to contain 1.5 ⁇ g of OmpA which makes the total amount 20 mg in the 2.000xg pellet.
  • the band in lane 14 was estimated to contain 0.8 ⁇ g of OmpA which makes the total amount 13 mg of OmpA in the 5,000xg pellet. The total yield of purified OmpA was thus about 60 mg/l of culture.
  • the recovery of protective epitopes was tested by immunizing groups of ten mice with 20 ⁇ g of BacP1.7,16 protein treated in various ways, given in two injections.
  • the immunizing injection was either subcutaneous or intraperitoneal (i.p.) with 0.1 ml of antigen diluted in PBS. The interval between the two doses was six weeks.
  • the first injection contained an adjuvant as indicated in Tables 1-4.
  • Ten days after the second injection the mice were bled, and the pooled sera analyzed.
  • EIA Enzyme Immunoassay
  • Anti-meningococcal antibodies were measured by EIA (Jalonen et al, J. Infect. 19:127-134 (1989)) using P1.7,16 meningococcal OM
  • the pups were randomized in groups of 6 pups each and injected i.p. with 100 ⁇ l of the immune sera in several dilutions (1:10, 1:100, 1:1000). One hour later a bacterial challenge 10 6 bacteria/ml) was injected i.p. in a volume of 100 ⁇ l. Tne development of bacteremia and meningitis was assessed by taking the appropriate samples 6 hours after the challenge to enumerate the viable bacteria by culture.
  • BacP1.7,16-IB protein is wholly soluble in SDS, guanidine hydrochloride and urea, but only partially in sarkosyl or cetylammonium bromide. The, protein precipitates if the solubilizing agent is removed. If the agent was guanidine hydrochloride, the precipitated protein is referred to as
  • BacP1.7,16-Gu is prepared from BacP1.7,16-IB in the following way: 5 mg of BacP1.7,16-IB protein was solubilized with 1 ml of
  • BacP1.7,16-Gu differs from BacP1.7,16-IB in being fairly soluble both in sarkosyl and cetylammonium bromide (CTB).
  • mice were immunized with BacP1.7,16-IB and -Gu preparations, and with preparations dissolved in the above mentioned detergents (1 mg of protein/1 ml of 2% detergent in 10 mM TrisHCl, pH 8.0 containing 5 mM EDTA) and diluted 1:5 in PBS.
  • Trisbuffer pH 8.0. Before immunization the sample was diluted 1:5 in PBS.
  • the immune sera were analyzed using the methods disclosed above. The result of the tests are shown in Table 3. All the sera contained antibodies against BacP1.7,16 protein and MenB:P1.7,16-membranes, were bactericidal and protective, which indicates that protective epitopes were obtained by this refolding method.
  • BacP1.7,16-IB or BacP1.7,16-Gu was suspended in 1 ml of 2% sarkosyl, and 0.1 ml of soybean lecithin (25 mg/ml of 2% sarkosyl) was added. Before immunization the samples were diluted 1:5 in 0.9% NaCl. In each case the mice received 0.2 ml of the antigen preparation.
  • the immune sera were analyzed using the methods disclosed above. The result of the tests are shown in Table 4.
  • BacOmpA-SS ⁇ BacOmpA 228 OmpA 228 -SS protein as a positive control.
  • the BacOmpA-SS ⁇ as shown in Table 5, was able to
  • BacOmpA 228 OmpA 228 -SS( + LPS) inhibited 83% of the phage binding, whereas 75 ⁇ g of BacOmpA-ss ⁇ ( +LPS) was needed to inhibit 70% of the phage binding. Hence binding by BacOmpA-ss ⁇ +LPS is less efficient. It can be said that the phage binding capacity of BacOmpA-SS ⁇ can be restored with LPS to a limited extent.
  • Plasmid pKTH290 in Bacillus subtilis in the parent strain IH6140 (the recombinant strain denoted as strain IH6627) was deposited on July 5, 1990 with the Deutsche Sammlung von Mikroorganismen (DSM),
  • the invention provides a method for producing cloned and renatured outer membrane (OM) protein from pathogenic gram-negative bacteria.
  • the renatured (OM) proteins produced by the method of the invention have immunologically active epitopes which are capable of eliciting production of antibodies, in mammals and other animals, that are bactericidal and can provide protection against infection by pathogenic gram-negative bacteria.
  • These cloned renatured OM proteins are useful as diagnostic antigens for the identification of infections caused by gram-negative bacteria. They are also useful as vaccines or as components of vaccines.
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Neisseria meningitidis
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Neisseria meningitidis
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Neisseria meningitidis

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PCT/FI1991/000212 1990-07-06 1991-07-05 Production of outer membrane (om) proteins in gram-positive bacteria and recovery of protective epitopes WO1992001001A1 (en)

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AU81873/91A AU668075B2 (en) 1990-07-06 1991-07-05 Production of outer membrane (OM) proteins in gram-positive bacteria and recovery of protective epitopes
JP3511658A JPH06511140A (ja) 1990-07-06 1991-07-05 グラム陽性細菌中での外層膜タンパク質の生産及び保護的エピトープの回復

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WO1995020048A1 (de) * 1994-01-19 1995-07-27 Forschungszentrum Jülich GmbH Sekretion von proteinen der äusseren membran gram-negativer bakterien mittels gram-positiver wirtsorganismen
US5439808A (en) * 1993-07-23 1995-08-08 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane group B porin proteins from Neisseria meningitidis
US5747287A (en) * 1995-04-28 1998-05-05 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane group B porin proteins from Neisseria meningitidis
WO1998056817A1 (en) * 1997-06-09 1998-12-17 Karolinska Innovations Ab Composition and methods for the treatment of inflammatory diseases
FR2785293A1 (fr) * 1998-10-30 2000-05-05 Pasteur Merieux Serums Vacc Acides nucleiques et polypeptides specifiques des souches pathogenes du genre neisseria
US6153406A (en) * 1993-07-23 2000-11-28 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane protein P2 from Haemophilus influenzae type B
US6350449B1 (en) 1995-06-07 2002-02-26 Baxter International Inc. Antibodies to meningococcal polysaccharide conjugate vaccines

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WO1988007374A1 (en) * 1987-03-30 1988-10-06 The Texas A & M University System Vaccine against brucella abortus

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Dialog Information Services, File 154, Medline 85-91, Dialog Accession No. 06452432, CARBONETTI N.H. et al.: "Molecular cloning and characterization of the structural gene for protein I, the major outer membrane protein of Neisseria gonorrhoeae", & Proc Natl Acad Sci USA Dec 1987, 84 (24) p9084-8. *
Dialog Information Services, File 154, Medline 85-91, Dialog Accession No. 06768281, TARKKA E. et al.: "Cloning of an outer membrane protein of Neisseria meningitidis in Escherichia coli", & Microb Pathog Dec 1987, 3 (6) p445-53. *
Dialog Information Services, File 154, Medline 85-91, Dialog Accession No. 06777316, MUNSON R., Jr. et al.: "Molecular cloning, expression, and primary sequence of outer membrane protein P2 of Haemophilus influenzae type b", & Infect Immun Jan 1989, 57 (1) p88-94. *
Dialog Information Services, File 154, Medline 85-91, Dialog Accession No. 07349245, PUOHINIEMI R. et al.: "A strong antibody response to the periplasmic C-terminal domain of the OmpA protein of Escherichia coli is produced by immunization with purified OmpA or with whole E. coli or Salmonella typhimurium *
Dialog Information Services, File 154, Medline 85-91, Dialog Accession No. 07371344, McGUINNESS B. et al.: "Deduced amino acid sequences of class i protein (PorA) from three strains of Neisseria meningitidis. Synthetic peptides define the epitopes responsible for serosubtype specificity", & J Exp Med Jun 1 1990 171 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5439808A (en) * 1993-07-23 1995-08-08 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane group B porin proteins from Neisseria meningitidis
US5879686A (en) * 1993-07-23 1999-03-09 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane group B porin proteins from Neisseria meningitidis
US6013267A (en) * 1993-07-23 2000-01-11 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane group B porin proteins from Neisseria meningitidis
US6153406A (en) * 1993-07-23 2000-11-28 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane protein P2 from Haemophilus influenzae type B
WO1995020048A1 (de) * 1994-01-19 1995-07-27 Forschungszentrum Jülich GmbH Sekretion von proteinen der äusseren membran gram-negativer bakterien mittels gram-positiver wirtsorganismen
US5747287A (en) * 1995-04-28 1998-05-05 North American Vaccine, Inc. Method for the high level expression, purification and refolding of the outer membrane group B porin proteins from Neisseria meningitidis
US6350449B1 (en) 1995-06-07 2002-02-26 Baxter International Inc. Antibodies to meningococcal polysaccharide conjugate vaccines
WO1998056817A1 (en) * 1997-06-09 1998-12-17 Karolinska Innovations Ab Composition and methods for the treatment of inflammatory diseases
FR2785293A1 (fr) * 1998-10-30 2000-05-05 Pasteur Merieux Serums Vacc Acides nucleiques et polypeptides specifiques des souches pathogenes du genre neisseria
WO2000026375A2 (fr) * 1998-10-30 2000-05-11 Aventis Pasteur ACIDES NUCLEIQUES ET POLYPEPTIDES SPECIFIQUES DES SOUCHES PATHOGENES DU GENRE $i(NEISSERIA)
WO2000026375A3 (fr) * 1998-10-30 2000-08-17 Pasteur Merieux Serums Vacc ACIDES NUCLEIQUES ET POLYPEPTIDES SPECIFIQUES DES SOUCHES PATHOGENES DU GENRE $i(NEISSERIA)

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IL98727A0 (en) 1992-07-15
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ZA915234B (en) 1993-02-24
JPH06511140A (ja) 1994-12-15
AU8187391A (en) 1992-02-04
EP0538318A1 (en) 1993-04-28
AU668075B2 (en) 1996-04-26

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