WO1994010204A1 - Protein fragments of the pectinatus bacterium - Google Patents

Protein fragments of the pectinatus bacterium Download PDF

Info

Publication number
WO1994010204A1
WO1994010204A1 PCT/FI1993/000431 FI9300431W WO9410204A1 WO 1994010204 A1 WO1994010204 A1 WO 1994010204A1 FI 9300431 W FI9300431 W FI 9300431W WO 9410204 A1 WO9410204 A1 WO 9410204A1
Authority
WO
WIPO (PCT)
Prior art keywords
ala
asn
val
ser
asp
Prior art date
Application number
PCT/FI1993/000431
Other languages
French (fr)
Inventor
Elias Hakalehto
Jukka Finne
Original Assignee
Elias Hakalehto
Jukka Finne
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Elias Hakalehto, Jukka Finne filed Critical Elias Hakalehto
Priority to AU51785/93A priority Critical patent/AU5178593A/en
Priority to EP93922966A priority patent/EP0666870A1/en
Priority to JP6510741A priority patent/JPH08502506A/en
Publication of WO1994010204A1 publication Critical patent/WO1994010204A1/en

Links

Classifications

    • 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
    • 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

  • the invention relates to protein fragments i.e. peptides of the Pectinatus bacteria useful in the detection and identification of the Pectinatus bac ⁇ teria.
  • the invention also relates to antibodies raised against the peptides, to a method for detecting the Pectinatus bacteria by means of the antibodies, and to the use of the peptides according to the invention as antigens.
  • Pectinatus is a Gram-negative, rod-shaped, anaerobic bacterium described for the first time in 1978 (Lee S.Y. et al . , 1978, Int . J. Syst . Bacteriol . , 28, 582-594).
  • the bacterium was isolated from turbid beer, and has thereafter been found in breweries in different parts of the world. It is a dreaded, highly harmful contaminant in breweries.
  • a characteristic feature of Pectinatus is that it has flagellae located on one side of the cell only.
  • the Pectinatus bacteria produce organic acids, such as propionic acid and sulphur compounds which spoil the flavour of beer.
  • the protein patterns of the Pectinatus bacteria have been studied.
  • the bacterial cells have been extracted by a mild hydrochloric acid solution which removes the surface layer of the cell, and the extracts have then been subjected to SDS polyacryl- amide gel electrophoresis (SDS-PAGE).
  • SDS-PAGE SDS polyacryl- amide gel electrophoresis
  • the Pectinatus bacteria have been divided into two main groups on the basis of the presence or absence of the 22 kDa protein. These groups have further been divided into at least five subgroups on the basis of other major proteins.
  • Pectinatus There are also proteins common to all groups, such as the proteins having an apparent molecular weight of about 48 to 53 kDa and about 16 kDa (Hakalehto E. et al . , 1984, Food Microbiology , 1 , 209-216).
  • the main groups of Pectinatus have more recently been divided into two species, viz. Pectinatus fri- singiensis and Pectinatus cerevisiiphilus (Schleifer K. H. et al . , 1990, Int . J. Syst. Bacteriol . 40, 19- 27).
  • the antigenic properties of the components of the surface layer of the Pectinatus bacteria have also been studied.
  • the Pectinatus bacteria can be divided into two main groups even on the basis of these results: the size of the main antigenic component of one group is about 55 to 78 kDa, depending on the strain, and that of the other group about 22 to 25 kDa (Hakalehto E. and Finne J., 1990, FEMS Microbiology Letters 67, 307-312).
  • the N- terminal fragments of the above-described proteins having the size of about 48 to 53 kDa, about 55 to 78 kDa and about 16 kDa are efficient antigens useful in the detection of all Pectinatus strains. This is particularly surprising as the 48-53 kDa protein has not been found to be antigenic as a native protein, and the 55-78 kDa protein acts as an antigen only with P . frisingiensis . Instead, the N-terminal fragments act as antigens common to both Pectinatus species.
  • N-terminal fragment of the 48-53 kDa protein was very similar to the N-terminal sequence of a 40-50 kDa protein of another beer spoilage bacterium i.e. Megasphaera resulting in cross-reactive antibodies.
  • the determined N-terminal sequence of the 48-53 kDa protein is Sequence No. 1
  • the N-terminal sequence of the 55-78 kDa protein is Sequence No. 2
  • the N-terminal sequence of the 16 kDa protein is Sequence No. 6 in the sequence list.
  • a characteristic feature of the peptide accord ⁇ ing to the invention is that its antigenic determinant is Ala Ala Asn Pro Phe Ser Asp Val Pro Ala Asp Ser Ser Ala Tyr Asp Ala Val Ser Lys Leu Ala Ala Asp Asp Ser Val (Seq. No. 1), or a subsequence or derivative thereof; or Ala Leu Val Val Lys Asn Asn Met Ser Ala Leu Asn Thr Leu Asn lie Met Ser Lys Asn Asn Lys Asn Leu Ala (Seq. No 2), or a subsequence or derivative thereof; or Met lie Lys Pro Leu Gly Asp Gin Xaa Val lie Gin Asp Ser Glu (Seq. No 6) or a subsequence or derivative thereof.
  • the three-letter abbreviations of the amino acids used herein read as follows:
  • Ala alanine
  • Asn asparagine
  • Xaa valine or isoleucine
  • the invention also relates to peptides having an antigenically active subsequence of the above sequences as an anti ⁇ genic determinant.
  • Subsequences comprising approxim ⁇ ately the first 15 N-terminal amino acids are useful. It is obvious that the deletion, addition or substitution of a few amino acids in the above amino acid sequences or their subsequences by an amino acid of the same type or a chemical derivative does not necessarily affect the antigenic properties of the peptides, and therefore the invention further relates to peptides having the antigenically active derivat- ives of the above sequences or their subsequences as antigenic determinants.
  • antigenically active refers to a compound which is able to elicit anti ⁇ bodies.
  • An antigenic determinant is a molecule portion reacting with the antibody.
  • the above-mentioned peptides can be used as antigens, thus obtaining antibodies useful in the immunological detection of Pectinatus bacteria.
  • the sample to be analyzed is thus reacted with the antibody and the amount of reactive antibodies is measured, either directly or by means of an anti- antibody or an antibody binding molecule in a conventional way.
  • the binding of the antibodies indicates the presence of the bacteria.
  • the invention thus also relates to the use of the peptides as antigens, to antibodies raised against the peptides, and to a method for detecting Pectinatus bacteria by reacting a sample suspected to contain Pectinatus with the antibodies.
  • Seq. No. 1 and subsequences and derivatives thereof it is further possible to detect Megasphaera bacteria.
  • the desired antibodies can be produced by conjugating the peptide according to the invention with a carrier molecule and then utilizing the obtained conjugate in immunization to produce the antibody.
  • the peptide Ala Leu Val Val Lys Asn Asn Met Ser Ala Leu Asn Thr Leu Asn (Seq. No. 4) or a subsequence of derivative thereof can also be used as an immunogenic carrier molecule as such.
  • the invention will be described in greater detail below.
  • Pectinatus strains were grown anaerobically, the cells were harvested, washed and extracted by HC1 to remove the surface layer. The cell debris was separ- ated and SDS-PAGE was performed on the extracts, as described by Hakalehto E. and Finne J., 1990, FEMS Microbiology Letters . 67, 307-312. Proteins of 48 to 53 kDa (about 51 kDa on the average), 55 to 78 kDa (about 63 kDa on the average) and 16 kDa were identified and their N-terminal sequences were determined by an automatic sequencer. All strains studied had identical sequences except for site 9 in the 16 kDa protein.
  • the peptides according to the N-terminal sequence were then synthesized by the solid phase method. It is, of course, also possible to synthesize the peptides by other conventional methods, such as the liquid phase synthesis or the recombinant DNA technique.
  • the peptides were used for the production of antisera by injecting them into a rabbit as such or conjugated with keyhole limpet hemocyanin. After immunization the sera were recovered.
  • the reactivity of the obtained antibodies was studied with different Pectinatus strains by the immunoblotting method (Hakalehto E. and Finne J. , 1990, FEMS Microbiology Letters 67, 307-312). It was found that the N-terminal fragments of the 48-53 kDa protein, the 55-78 kDa protein and the 16 kDa protein act as efficient antigens and that these antigens were present in both the P . frisingiensis and P. cerevisiiphilus strains.
  • the P. frisingiensis strains ATCC 33332, VT -E- 81141, VTT-E-80121, VTT-E-83170, VTT-E-87205, VTT-E- 88310 and VTT-E-87295, and the P. cerevisiiphilus strains DSM 20466 and ATCC 29359 were grown in a PYG broth (1% of peptone, 1% of yeast extract and 2% of glucose) in anaerobic Gas Pak jars at 30°C for 3 to 4 days. Oxygen was removed from the broth by boiling prior to incubation.
  • the gels were blotted on PVDF membranes (Immobilon P®, Millipore) stained by Coomassie Brilliant Blue, and the 48-53 kDa, 55-78 kDa and 16 kDa proteins were cut off and dissolved in acetonitrile for sequencing.
  • N-terminal sequences of the proteins isol ⁇ ated in Example 1 were determined by Edman degradation using a peptide sequencer of Applied Biosystems.
  • the first 27 N-terminal amino acids of the 48-53 kDa protein of the ATCC 33332 strain were determined. They were as follows: Ala Ala Asn Pro Phe Ser Asp Val Pro Ala Asp Ser Ser Ala Tyr Asp Ala Val Ser Lys Leu Ala Ala Asp Asp Ser Val (Seq. No. 1).
  • the first 20 N- terminal amino acids of the VTT-E-80121, DSM 20466, VTT-E-87295 and VTT-E-88310 strains were identical with the first 20 amino acids of the ATCC 33332 strain, and the first 10 N-terminal amino acids of the VTT-E-83170 strain were also identical with those of the above strains.
  • the first 25 N-terminal amino acids of the 55-78 kDa protein of the ATCC 33332 strain were determined. They were as follows: Ala Leu Val Val Lys Asn Asn Met Ser Ala Leu Asn Thr Leu Asn lie Met Ser Lys Asn Asn Lys Asn Leu Ala (Seq. No. 2).
  • the first 20 amino acids of the DSM 20466 and VTT-E-81141 strains, the first 15 amino acids of the VTT-E-80121 and VTT-E-83170 strains, the first 11 amino acids of the VTT-E-88310 strain, and the first 10 amino acids of the VTT-E- 87295 and ATCC 29359 strains were identical with the respective amino acids of the ATCC 33332 strain.
  • the first 15 N-terminal amino acids of the 16 kDa protein of the P. cerevisiiphilus strains ATCC 29359 and DSM 20466 were Met lie Lys Pro Leu Gly Asp Gin Val Val lie Gin Asp Ser Glu (Seq. No. 7), and the ten first N-terminal amino acids of the same protein of the P. frisingiensis strains VTT-E-80121, VTT-E- 83170, VTT-E-87205 and VTT-E-88310 were identical except for site 9, where there was a species dependent difference, i.e. the amino acid was lie instead of Val.
  • a peptide synthesizer utilizing the solid phase method (Applied Biosystems 431A Automated Peptide Synthesizer) and the Fmoc strategy were used to synthesize the protein fragments containing the first 15 N-terminal amino acids in the 48-53 kDa protein, the 55-78 kDa protein, and the 16 kDa protein, i.e. the following peptides: Ala Ala Asn Pro Phe Ser Asp Val Pro Ala Asp Ser Ser Ala Tyr (Seq. No. 3), Ala Leu Val Val Lys Asn Asn Met Ser Ala Leu Asn Thr Leu Asn (Seq. No. 4), and Met lie Lys Pro Leu Gly Asp Gin Val Val lie Gin Asp Ser Glu (Seq. No. 7) referred to below as peptide A, peptide B and peptide C.
  • the peptides (A, B and C) synthesized in Example 3 and conjugated with keyhole limpet hemocyanin were injected into rabbits under the skin and into the muscle as described earlier (Liu, F. T. et al. , 1979, Biochemistry 3J3, 690-697).
  • a cysteine had been attached to the C-terminal end of the peptides by the solid-phase peptide synthesizer.
  • Peptide B was also injected as such, without conjugation.
  • the rabbits were immunized with about 0.5 to 1 mg of peptide/rabbit, 4 to 5 times at intervals of two weeks. The sera were recovered after 1 to 4 months from the first injection.
  • peptide B pre- cipitated quite rapidly in Ca 2+ - and Mg + -free phosphate buffered saline solution (CMF-PBS). This precipitate was extremely insoluble. When observed in electron micrographs, the precipitates were found to be fibrilles, which resembled the outlook of fibrilles formed by fragments of the ⁇ A4-amyloid peptide detected in the brains of patients suffering from Altzheimer's disease (Fraser, P. et al . , 1992, J. Neurochem. , 5_9_, 1531-1540).
  • peptide B was also used as an immunogen as such without any carrier and it was found to elicit the same types of antibodies as peptide B coupled to keyhole limpet hemocyanin. Obviously peptide B could act as a carrier for other antigens.
  • Example 4 The antibodies obtained in Example 4 were used in immunoblotting in accordance with the Western blotting method.
  • SDS-PAGE was performed on the hydro- chloric acid extracts of the strains shown in Table 1, as set forth in Example 1.
  • the gel was transferred to the Trans-Blot® Cell apparatus of Bio-Rad, where the proteins contained in the gel were transferred by an electric current (90 mA) in a buffer solution to an Immobilon®-P membrane (Millipore). After a run of about 60 minutes the membranes were transferred into calcium- and magnesium-free phosphate buffered saline (CMF-PBS), pH 7.4, containing 0.5% Tween 20 and 1% milk powder for pre-incubation.
  • CMF-PBS calcium- and magnesium-free phosphate buffered saline
  • the membranes were washed in CMF-PBS containing 0.05% Tween 20 and 1% milk powder, and then incubated for 80 min together with antibodies which were obtained in Example 4 and diluted with the last-mentioned CMF-PBS, and then the membranes were rewashed, as above.
  • the membranes were transferred into a secondary antibody solution (P 217, Dako-immunoglobulins) for 60 minutes, and the washes were repeated. Staining was performed with diamino- benzidine (15 mg/30 ml) in the presence of hydrogen peroxide (15 ⁇ l of 30% hydrogen peroxide/30 ml) in the dark.
  • the results for peptides A and B are shown in Table 1.
  • the table also shows the antibody reactions of native proteins with respect to antibodies elicited by whole cells. These antibodies were prepared as described above (Hakalehto E. and Finne J., 1990, FEMS Microbiology Letters 62, 307- 312).
  • Seq. No. 5 This sequence is a subsequence of Seq. No. 1 differ- ing from it only at four sites.
  • a peptide having Seq. No. 5 was synthesized as described in Example 3, and cystein was attached to the C-terminal end of the peptide before coupling it to the keyhole limpet hemocyanin.
  • Antibodies to said peptide were prepared as described in Example 4 and immunoblotted as in Example 5 using a serum dilution of 1:100-1:1000.
  • the antibodies reacted strongly and specifically not only with the protein of 40-50 kDa of the Megasphaera strain ATCC 43254, from which the sequence was derived, but also with the 48-53 kDa protein of the Pectinatus strains ATCC 33332 and DSM 20466.
  • MOLECULE TYPE protein
  • FRAGMENT TYPE N-terminal
  • MOLECULE TYPE protein
  • FRAGMENT TYPE N-terminal
  • MOLECULE TYPE protein
  • FRAGMENT TYPE N-terminal
  • MOLECULE TYPE protein
  • FRAGMENT TYPE N-terminal
  • ORGANISM Pectinatus cerevisiiphilus

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to a peptide wherein the antigenic determinant is Ala Ala Asn Pro Phe Ser Asp Val Pro Ala Asp Ser Ser Ala Tyr Asp Ala Val Ser Lys Leu Ala Ala Asp Asp Ser Val; Ala Leu Val Val Lys Asn Asn Met Ser Ala Leu Asn Thr Leu Asn Ile Met Ser Lys Asn Asn Lys Asn Leu Ala; or Met Ile Lys Pro Leu Gly Asp Gln Xaa Val Ile Gln Asp Ser Glu; or a subsequence or derivative of said sequences. The invention further relates to the use of the peptide as an antigen, antibodies raised against the peptide, and a method for detecting Pectinatus bacteria by means of the antibodies.

Description

Protein fragments of the Pectinatus bacterium
Field of the Invention
The invention relates to protein fragments i.e. peptides of the Pectinatus bacteria useful in the detection and identification of the Pectinatus bac¬ teria. The invention also relates to antibodies raised against the peptides, to a method for detecting the Pectinatus bacteria by means of the antibodies, and to the use of the peptides according to the invention as antigens.
Technical Background
Pectinatus is a Gram-negative, rod-shaped, anaerobic bacterium described for the first time in 1978 (Lee S.Y. et al . , 1978, Int . J. Syst . Bacteriol . , 28, 582-594). The bacterium was isolated from turbid beer, and has thereafter been found in breweries in different parts of the world. It is a dreaded, highly harmful contaminant in breweries. A characteristic feature of Pectinatus is that it has flagellae located on one side of the cell only. The Pectinatus bacteria produce organic acids, such as propionic acid and sulphur compounds which spoil the flavour of beer.
To clear up contaminations and find out their origin, it is important that the contaminant can be detected and identified. For this purpose, the protein patterns of the Pectinatus bacteria, among other things, have been studied. The bacterial cells have been extracted by a mild hydrochloric acid solution which removes the surface layer of the cell, and the extracts have then been subjected to SDS polyacryl- amide gel electrophoresis (SDS-PAGE). The Pectinatus bacteria have been divided into two main groups on the basis of the presence or absence of the 22 kDa protein. These groups have further been divided into at least five subgroups on the basis of other major proteins. There are also proteins common to all groups, such as the proteins having an apparent molecular weight of about 48 to 53 kDa and about 16 kDa (Hakalehto E. et al . , 1984, Food Microbiology , 1 , 209-216). The main groups of Pectinatus have more recently been divided into two species, viz. Pectinatus fri- singiensis and Pectinatus cerevisiiphilus (Schleifer K. H. et al . , 1990, Int . J. Syst. Bacteriol . 40, 19- 27). The antigenic properties of the components of the surface layer of the Pectinatus bacteria have also been studied. The Pectinatus bacteria can be divided into two main groups even on the basis of these results: the size of the main antigenic component of one group is about 55 to 78 kDa, depending on the strain, and that of the other group about 22 to 25 kDa (Hakalehto E. and Finne J., 1990, FEMS Microbiology Letters 67, 307-312).
Summary of the Invention
It has now been unexpectedly found that the N- terminal fragments of the above-described proteins having the size of about 48 to 53 kDa, about 55 to 78 kDa and about 16 kDa are efficient antigens useful in the detection of all Pectinatus strains. This is particularly surprising as the 48-53 kDa protein has not been found to be antigenic as a native protein, and the 55-78 kDa protein acts as an antigen only with P . frisingiensis . Instead, the N-terminal fragments act as antigens common to both Pectinatus species. In addition, it has been found that the N-terminal fragment of the 48-53 kDa protein was very similar to the N-terminal sequence of a 40-50 kDa protein of another beer spoilage bacterium i.e. Megasphaera resulting in cross-reactive antibodies. The determined N-terminal sequence of the 48-53 kDa protein is Sequence No. 1, the N-terminal sequence of the 55-78 kDa protein is Sequence No. 2 and the N-terminal sequence of the 16 kDa protein is Sequence No. 6 in the sequence list.
A characteristic feature of the peptide accord¬ ing to the invention is that its antigenic determinant is Ala Ala Asn Pro Phe Ser Asp Val Pro Ala Asp Ser Ser Ala Tyr Asp Ala Val Ser Lys Leu Ala Ala Asp Asp Ser Val (Seq. No. 1), or a subsequence or derivative thereof; or Ala Leu Val Val Lys Asn Asn Met Ser Ala Leu Asn Thr Leu Asn lie Met Ser Lys Asn Asn Lys Asn Leu Ala (Seq. No 2), or a subsequence or derivative thereof; or Met lie Lys Pro Leu Gly Asp Gin Xaa Val lie Gin Asp Ser Glu (Seq. No 6) or a subsequence or derivative thereof. The three-letter abbreviations of the amino acids used herein read as follows:
Ala = alanine Asn = asparagine
Asp = aspartic acid
Cys = cysteine
Gin = glutamine
Glu = glutamic acid Gly = glycine lie = isoleucine
Leu = leucine
Lys = lysine
Met = methionine Phe = phenylalanine Pro = proline Ser = serine Thr = threonine Tyr = tyrosine Val = valine
Xaa = valine or isoleucine
In addition to peptides having the sequences set forth above as antigenic determinants, the invention also relates to peptides having an antigenically active subsequence of the above sequences as an anti¬ genic determinant. Subsequences comprising approxim¬ ately the first 15 N-terminal amino acids are useful. It is obvious that the deletion, addition or substitution of a few amino acids in the above amino acid sequences or their subsequences by an amino acid of the same type or a chemical derivative does not necessarily affect the antigenic properties of the peptides, and therefore the invention further relates to peptides having the antigenically active derivat- ives of the above sequences or their subsequences as antigenic determinants. The term antigenically active refers to a compound which is able to elicit anti¬ bodies. An antigenic determinant is a molecule portion reacting with the antibody. The above-mentioned peptides can be used as antigens, thus obtaining antibodies useful in the immunological detection of Pectinatus bacteria. The sample to be analyzed is thus reacted with the antibody and the amount of reactive antibodies is measured, either directly or by means of an anti- antibody or an antibody binding molecule in a conventional way. The binding of the antibodies indicates the presence of the bacteria. The invention thus also relates to the use of the peptides as antigens, to antibodies raised against the peptides, and to a method for detecting Pectinatus bacteria by reacting a sample suspected to contain Pectinatus with the antibodies. Regarding Seq. No. 1 and subsequences and derivatives thereof, it is further possible to detect Megasphaera bacteria. The desired antibodies can be produced by conjugating the peptide according to the invention with a carrier molecule and then utilizing the obtained conjugate in immunization to produce the antibody. The peptide Ala Leu Val Val Lys Asn Asn Met Ser Ala Leu Asn Thr Leu Asn (Seq. No. 4) or a subsequence of derivative thereof can also be used as an immunogenic carrier molecule as such. The invention will be described in greater detail below.
Detailed Description of the Invention
Pectinatus strains were grown anaerobically, the cells were harvested, washed and extracted by HC1 to remove the surface layer. The cell debris was separ- ated and SDS-PAGE was performed on the extracts, as described by Hakalehto E. and Finne J., 1990, FEMS Microbiology Letters .67, 307-312. Proteins of 48 to 53 kDa (about 51 kDa on the average), 55 to 78 kDa (about 63 kDa on the average) and 16 kDa were identified and their N-terminal sequences were determined by an automatic sequencer. All strains studied had identical sequences except for site 9 in the 16 kDa protein. The peptides according to the N-terminal sequence were then synthesized by the solid phase method. It is, of course, also possible to synthesize the peptides by other conventional methods, such as the liquid phase synthesis or the recombinant DNA technique. The peptides were used for the production of antisera by injecting them into a rabbit as such or conjugated with keyhole limpet hemocyanin. After immunization the sera were recovered.
The reactivity of the obtained antibodies was studied with different Pectinatus strains by the immunoblotting method (Hakalehto E. and Finne J. , 1990, FEMS Microbiology Letters 67, 307-312). It was found that the N-terminal fragments of the 48-53 kDa protein, the 55-78 kDa protein and the 16 kDa protein act as efficient antigens and that these antigens were present in both the P . frisingiensis and P. cerevisiiphilus strains.
The invention will be illustrated by the follow¬ ing non-limiting examples.
All publications cited in this patent application are incorporated herein by reference.
Example 1
The P. frisingiensis strains ATCC 33332, VT -E- 81141, VTT-E-80121, VTT-E-83170, VTT-E-87205, VTT-E- 88310 and VTT-E-87295, and the P. cerevisiiphilus strains DSM 20466 and ATCC 29359 were grown in a PYG broth (1% of peptone, 1% of yeast extract and 2% of glucose) in anaerobic Gas Pak jars at 30°C for 3 to 4 days. Oxygen was removed from the broth by boiling prior to incubation. Cells were harvested by centrifugation and treated with 0.05 M hydrochloric acid to detach and remove the surface layer, as described by Hakalehto E. et al . , 1984, Food Microbiology , JL, 209-216. After neutralization with 0.05 M NaOH the samples were boiled in an electrophoresis sample buffer for 10 minutes, and then SDS-PAGE was performed as described by Laemmli (Laemmli, U.K., 1970, Nature (London) 227, 680-685) by using 8-12% polyacrylamide gels. The gels were blotted on PVDF membranes (Immobilon P®, Millipore) stained by Coomassie Brilliant Blue, and the 48-53 kDa, 55-78 kDa and 16 kDa proteins were cut off and dissolved in acetonitrile for sequencing.
Example 2
The N-terminal sequences of the proteins isol¬ ated in Example 1 were determined by Edman degradation using a peptide sequencer of Applied Biosystems. The first 27 N-terminal amino acids of the 48-53 kDa protein of the ATCC 33332 strain were determined. They were as follows: Ala Ala Asn Pro Phe Ser Asp Val Pro Ala Asp Ser Ser Ala Tyr Asp Ala Val Ser Lys Leu Ala Ala Asp Asp Ser Val (Seq. No. 1). The first 20 N- terminal amino acids of the VTT-E-80121, DSM 20466, VTT-E-87295 and VTT-E-88310 strains were identical with the first 20 amino acids of the ATCC 33332 strain, and the first 10 N-terminal amino acids of the VTT-E-83170 strain were also identical with those of the above strains.
The first 25 N-terminal amino acids of the 55-78 kDa protein of the ATCC 33332 strain were determined. They were as follows: Ala Leu Val Val Lys Asn Asn Met Ser Ala Leu Asn Thr Leu Asn lie Met Ser Lys Asn Asn Lys Asn Leu Ala (Seq. No. 2). The first 20 amino acids of the DSM 20466 and VTT-E-81141 strains, the first 15 amino acids of the VTT-E-80121 and VTT-E-83170 strains, the first 11 amino acids of the VTT-E-88310 strain, and the first 10 amino acids of the VTT-E- 87295 and ATCC 29359 strains were identical with the respective amino acids of the ATCC 33332 strain.
The first 15 N-terminal amino acids of the 16 kDa protein of the P. cerevisiiphilus strains ATCC 29359 and DSM 20466 were Met lie Lys Pro Leu Gly Asp Gin Val Val lie Gin Asp Ser Glu (Seq. No. 7), and the ten first N-terminal amino acids of the same protein of the P. frisingiensis strains VTT-E-80121, VTT-E- 83170, VTT-E-87205 and VTT-E-88310 were identical except for site 9, where there was a species dependent difference, i.e. the amino acid was lie instead of Val.
Example 3
A peptide synthesizer utilizing the solid phase method (Applied Biosystems 431A Automated Peptide Synthesizer) and the Fmoc strategy were used to synthesize the protein fragments containing the first 15 N-terminal amino acids in the 48-53 kDa protein, the 55-78 kDa protein, and the 16 kDa protein, i.e. the following peptides: Ala Ala Asn Pro Phe Ser Asp Val Pro Ala Asp Ser Ser Ala Tyr (Seq. No. 3), Ala Leu Val Val Lys Asn Asn Met Ser Ala Leu Asn Thr Leu Asn (Seq. No. 4), and Met lie Lys Pro Leu Gly Asp Gin Val Val lie Gin Asp Ser Glu (Seq. No. 7) referred to below as peptide A, peptide B and peptide C.
Example 4
The peptides (A, B and C) synthesized in Example 3 and conjugated with keyhole limpet hemocyanin were injected into rabbits under the skin and into the muscle as described earlier (Liu, F. T. et al. , 1979, Biochemistry 3J3, 690-697). For the conjugation a cysteine had been attached to the C-terminal end of the peptides by the solid-phase peptide synthesizer. Peptide B was also injected as such, without conjugation. The rabbits were immunized with about 0.5 to 1 mg of peptide/rabbit, 4 to 5 times at intervals of two weeks. The sera were recovered after 1 to 4 months from the first injection.
Surprisingly, it was found that peptide B pre- cipitated quite rapidly in Ca2+- and Mg+-free phosphate buffered saline solution (CMF-PBS). This precipitate was extremely insoluble. When observed in electron micrographs, the precipitates were found to be fibrilles, which resembled the outlook of fibrilles formed by fragments of the βA4-amyloid peptide detected in the brains of patients suffering from Altzheimer's disease (Fraser, P. et al . , 1992, J. Neurochem. , 5_9_, 1531-1540). This preparation of peptide B was also used as an immunogen as such without any carrier and it was found to elicit the same types of antibodies as peptide B coupled to keyhole limpet hemocyanin. Obviously peptide B could act as a carrier for other antigens.
Example 5
The antibodies obtained in Example 4 were used in immunoblotting in accordance with the Western blotting method. SDS-PAGE was performed on the hydro- chloric acid extracts of the strains shown in Table 1, as set forth in Example 1. The gel was transferred to the Trans-Blot® Cell apparatus of Bio-Rad, where the proteins contained in the gel were transferred by an electric current (90 mA) in a buffer solution to an Immobilon®-P membrane (Millipore). After a run of about 60 minutes the membranes were transferred into calcium- and magnesium-free phosphate buffered saline (CMF-PBS), pH 7.4, containing 0.5% Tween 20 and 1% milk powder for pre-incubation. The membranes were washed in CMF-PBS containing 0.05% Tween 20 and 1% milk powder, and then incubated for 80 min together with antibodies which were obtained in Example 4 and diluted with the last-mentioned CMF-PBS, and then the membranes were rewashed, as above. The membranes were transferred into a secondary antibody solution (P 217, Dako-immunoglobulins) for 60 minutes, and the washes were repeated. Staining was performed with diamino- benzidine (15 mg/30 ml) in the presence of hydrogen peroxide (15 μl of 30% hydrogen peroxide/30 ml) in the dark. The results for peptides A and B are shown in Table 1. For comparison, the table also shows the antibody reactions of native proteins with respect to antibodies elicited by whole cells. These antibodies were prepared as described above (Hakalehto E. and Finne J., 1990, FEMS Microbiology Letters 62, 307- 312).
Table 1 Antibody reactions of native proteins when using the immunoblotting method
Antigen used in the production of the antibody
Tested Peptide A Peptide B Whole cells of the strain
Pectinatus strains Native protein reactivity in the antibody reaction
48-53 kDa 55-78 kDa 48-53 kDa 55-78 kDa 48-53 kDa 55-78 kDa
ATCC 333321 + + + VTT-E-821651 + + + VTT-E-872941 + + + VTT-E-873071 + + + DSM 204651 + + + DSM 204662 + + ATCC 293592 + +
x) P. frisingiensis 2) P. cerevisiiphilus
The results in Table 1 show that both of the peptides A and B are antigenic with all strains test¬ ed, and the antibodies of the peptides react specif¬ ically with the protein from which the peptides are fragments. This is true for the native protein of both the P. frisingiensis and P. cerevisiiphilus str¬ ains, even though the native protein itself acts as an antigen only when analysing the antibodies of the P. frisingiensis strains (55-78 kDa) or does not at all act as an antigen (48-53 kDa).
The antisera obtained from rabbits immunized with peptide C gave a strong specific reaction with the 16 kDa band of P. cerevisiiphilus DSM 20466 and that of P. frisingiensis ATCC 33332 even when diluted 1:5,000 and 1:500, respectively. These results show that peptide C is useful in the detection of both Pectinatus strains.
Example 6
Megasphaera cerevisiae ATCC 43254 was grown in a PYG broth and the surface layer of the harvested cells was removed and subjected to gel electro- phoresis as described in Example 1. A strong protein band was obtained at about 40-50 kDa (about 46.5 kDa on the average). In the Western blotting according to Example 5, it was surprisingly found that said band cross-reacted strongly with the antibodies prepared against peptide A (Seq. No. 3). The protein was isolated and purified and the N-terminal sequence was determined according to Example 2 as:
Ala Asn Pro Phe Val Asp Val Pro Ala Asp Ser Ser Ala Tyr Lys Ser Asp (Seq. No. 5) . This sequence is a subsequence of Seq. No. 1 differ- ing from it only at four sites. A peptide having Seq. No. 5 was synthesized as described in Example 3, and cystein was attached to the C-terminal end of the peptide before coupling it to the keyhole limpet hemocyanin. Antibodies to said peptide were prepared as described in Example 4 and immunoblotted as in Example 5 using a serum dilution of 1:100-1:1000. The antibodies reacted strongly and specifically not only with the protein of 40-50 kDa of the Megasphaera strain ATCC 43254, from which the sequence was derived, but also with the 48-53 kDa protein of the Pectinatus strains ATCC 33332 and DSM 20466.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: Elias Hakalehto
(B) STREET: Kasarmikatu 12 C 1
(C) CITY: KUOPIO
(E) COUNTRY: FINLAND
(F) POSTAL CODE (ZIP): 70110 KUOPIO
(A) NAME: Jukka Finne
(B) STREET: Katajanokanranta 3 A 5
(C) CITY: HELSINKI
(E) COUNTRY: FINLAND
(F) POSTAL CODE (ZIP): 00160 HELSINKI
(ii) TITLE OF INVENTION: Protein fragments of the Pectinatus bacterium
(iii) NUMBER OF SEQUENCES: 7
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0,
Version #1.25 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: FI 924835
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Pectinatus frisingiensis
(B) STRAIN: ATCC 33332 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
Ala Ala Asn Pro Phe Ser Asp Val Pro Ala Asp Ser Ser Ala Tyr Asp 1 5 10 15
Ala Val Ser Lys Leu Ala Ala Asp Asp Ser Val 20 25
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Pectinatus frisingiensis
(B) STRAIN: ATCC 33332
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Ala Leu Val Val Lys Asn Asn Met Ser Ala Leu Asn Thr Leu Asn He 1 5 10 15
Met Ser Lys Asn Asn Lys Asn Leu Ala 20 25
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Ala Ala Asn Pro Phe Ser Asp Val Pro Ala Asp Ser Ser Ala Tyr 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Ala Leu Val Val Lys Asn Asn Met Ser Ala Leu Asn Thr Leu Asn 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Megashpaera cerevisiae
(B) STRAIN: ATCC 43254
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Ala Asn Pro Phe Val Asp Val Pro Ala Asp Ser Ser Ala Tyr Lys Ser 1 5 10 15
Asp (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Met He Lys Pro Leu Gly Asp Gin Xaa Val He Gin Asp Ser Glu 1 5 10 15
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Pectinatus cerevisiiphilus
(B) STRAIN: ATCC 29359 and DSM 20466
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Met He Lys Pro Leu Gly Asp Gin Val Val He Gin Asp Ser Glu 1 5 10 15

Claims

Claims :
1. Peptide wherein the antigenic determinant is Ala Ala Asn Pro Phe Ser Asp Val Pro Ala Asp Ser Ser Ala Tyr Asp Ala Val Ser Lys Leu Ala Ala Asp Asp Ser Val (Seq. No. 1); Ala Leu Val Val Lys Asn Asn Met Ser Ala Leu Asn Thr Leu Asn lie Met Ser Lys Asn Asn Lys Asn Leu Ala (Seq. No 2); or Met lie Lys Pro Leu Gly Asp Gin Xaa Val lie Gin Asp Ser Glu (Seq. No. 6); or a subsequence or derivative of said sequences.
2. Peptide according to claim 1 wherein the antigenic determinant is Ala Ala Asn Pro Phe Ser Asp Val Pro Ala Asp Ser Ser Ala Tyr (Seq. No. 3); or Ala Leu Val Val Lys Asn Asn Met Ser Ala Leu Asn Thr Leu Asn (Seq. No. 4); or a subsequence or derivative of said sequences.
3. Peptide according to claim 1 having the amino acid sequence Ala Ala Asn Pro Phe Ser Asp Val Pro Ala Asp Ser Ser Ala Tyr (Seq. No 3) , or an anti- genically active derivative thereof.
4. Peptide according to claim 1 having the amino acid sequence Ala Leu Val Val Lys Asn Asn Met Ser Ala Leu Asn Thr Leu Asn (Seq. No 4), or an anti¬ genically active derivative thereof. 5. Peptide according to claim 1 having the amino acid sequence Ala Asn Pro Phe Val Asp Val Pro Ala Asp Ser Ser Ala Tyr Lys Ser Asp (Seq. No.
5), or an antigenically active derivative thereof.
6. Peptide according to claim 1 having the amino acid sequence Met lie Lys Pro Leu Gly Asp Gin
Val Val lie Gin Asp Ser Glu (Seq. No. 7), or an anti¬ genically active derivative thereof.
7. Antibody directed against a peptide accord¬ ing to any of claims 1 to 6.
8. Antibody according to claim 7 prepared by conjugating a peptide according to any of claims 1 to 6 with a carrier molecule and using the obtained conjugate in immunization to produce the antibody.
9. Method for detecting Pectinatus bacteria wherein the sample suspected to contain Pectinatus bacteria is reacted with the antibody according to claim 7.
10. Method for detecting Pectinatus and/or Megasphaera bacteria wherein a sample suspected to contain said bacteria is reacted with antibodies raised against the peptide of claim 3 or 5.
11. Use of a peptide according to any of claims 1 to 6 as an antigen.
12. Use of the peptide Ala Leu Val Val Lys Asn Asn Met Ser Ala Leu Asn Thr Leu Asn (Seq. No 4) or a subsequence or derivative thereof as an immunogenic carrier molecule.
PCT/FI1993/000431 1992-10-23 1993-10-21 Protein fragments of the pectinatus bacterium WO1994010204A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU51785/93A AU5178593A (en) 1992-10-23 1993-10-21 Protein fragments of the (pectinatus) bacterium
EP93922966A EP0666870A1 (en) 1992-10-23 1993-10-21 PROTEIN FRAGMENTS OF THE $i(PECTINATUS) BACTERIUM
JP6510741A JPH08502506A (en) 1992-10-23 1993-10-21 Protein fragment of Pectinatus bacteria

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FI924835A FI94051C (en) 1992-10-23 1992-10-23 Pectinatus bacterial protein fragments
FI924835 1992-10-23

Publications (1)

Publication Number Publication Date
WO1994010204A1 true WO1994010204A1 (en) 1994-05-11

Family

ID=8536105

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/FI1993/000431 WO1994010204A1 (en) 1992-10-23 1993-10-21 Protein fragments of the pectinatus bacterium

Country Status (5)

Country Link
EP (1) EP0666870A1 (en)
JP (1) JPH08502506A (en)
AU (1) AU5178593A (en)
FI (1) FI94051C (en)
WO (1) WO1994010204A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997020071A1 (en) * 1995-11-28 1997-06-05 Asahi Breweries, Ltd. Detection of bacterium belonging to the genus pectinatus

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Dialog Information Services, File 5, BIOSIS, Dialog Accession No. 10000338, BIOSIS Number: 95000338, KLUGBAUER N. et al.: "Subunit Beta of Adenosine Triphosphate Synthase of Pectinatus-Frisingensis and Lactobacillus-Casei", Syst Appl Microbiol 15 (3), 1992, 323-330. *
FEMS Microbiology Letters, Volume 67, 1990, ELIAS HAKALEHTO et al., "Identification by Immunoblot Analysis of Major Antigenic Determinants of the Anaerobic Beer Spoilage Bacterium Genus Pectinatus", page 307 - page 312. *
International Journal of Systematic Bacteriology, Volume 33, No. 3, July 1983, C.-H. LAI et al., "Centipeda Periodontii Gen. Nov., Sp. Nov. from Human Periodontal Lesions", page 628 - page 635. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997020071A1 (en) * 1995-11-28 1997-06-05 Asahi Breweries, Ltd. Detection of bacterium belonging to the genus pectinatus
EP0806483A1 (en) * 1995-11-28 1997-11-12 Asahi Breweries, Ltd. Detection of bacterium belonging to the genus pectinatus
EP0806483A4 (en) * 1995-11-28 2000-06-07 Asahi Breweries Ltd Detection of bacterium belonging to the genus pectinatus

Also Published As

Publication number Publication date
FI94051B (en) 1995-03-31
FI94051C (en) 1995-07-10
FI924835A0 (en) 1992-10-23
EP0666870A1 (en) 1995-08-16
JPH08502506A (en) 1996-03-19
AU5178593A (en) 1994-05-24
FI924835A (en) 1994-04-24

Similar Documents

Publication Publication Date Title
EP0474313B1 (en) Nucleotide sequence coding for an outer membrane protein from Neisseria meningitidis and use of said protein in vaccine preparations
Nachamkin et al. Campylobacter jejuni
Pancholi et al. Isolation and characterization of the cell-associated region of group A streptococcal M6 protein
EP0378881B1 (en) Synthetic peptides and their use as universal carriers for the preparation of immunogenic conjugates suitable for the development of synthetic vaccines
NO179164B (en) Process for preparing an immunogen conjugate
CA2549188A1 (en) Use of proteins and peptides coded by the genome of a novel strain of sars-associated coronavirus
Ambler The amino acid sequence of cytochrome c′ from Alcaligenes sp. NCIB 11015
EP0500736B1 (en) Recombinant vaccine for porcine pleuropneumonia
Singh et al. Antigenic determinants of the OmpC porin from Salmonella typhimurium
Ullmann et al. Immunochemistry of the cell walls of Listeria monocytogenes
Kraus et al. Vimentin-cross-reactive epitope of type 12 streptococcal M protein
EP2262828B1 (en) Compositions, methods and kits
JPH07502896A (en) Multifunctional surface proteins of streptococci
Wietzerbin et al. Structural study of the poly-l-Glutamic acid of the cell wall of Mycobacterium tuberculosis var hominis, strain Brevannes
Pugsley et al. Comparison of colicins B-K260 and D-CA23: purification and characterization of the colicins and examination of colicin immunity in the producing strains
WO1994010204A1 (en) Protein fragments of the pectinatus bacterium
WO2000039310A9 (en) Rubredoxin fusion proteins, protein expression system and methods
EP1414860A2 (en) Pan-specific monoclonal antibody
Finlay et al. Major antigenic determinants of F and ColB2 pili
Olson et al. Identification of functional epitopes of Pseudomonas aeruginosa exotoxin A using synthetic peptides and subclone products
Baleux et al. Novel version of multiple antigenic peptide allowing incorporation on a cysteine functionalized lysine tree
Seidl et al. The Immunochemistry of Peptidoglycan: Antibodies against a Synthetic Immunogen Cross‐Reacting with an Interpeptide Bridge of Peptidoglycan
US5840513A (en) Antibodies directed to pagC peptides for the detection of salmonellae
WO1988006594A1 (en) Synthetic anti-caries vaccine
US5919640A (en) Streptococcus suis adhesin protein and method for producing it

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR BY CA CH CZ DE DK ES FI GB HU JP KP KR KZ LK LU LV MG MN MW NL NO NZ PL PT RO RU SD SE SK UA US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref country code: US

Ref document number: 1995 416818

Date of ref document: 19950414

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1993922966

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1993922966

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: CA

WWW Wipo information: withdrawn in national office

Ref document number: 1993922966

Country of ref document: EP