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WO2000061792A1 - Genes bacteriens essentiels et leurs proteines - Google Patents

Genes bacteriens essentiels et leurs proteines Download PDF

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Publication number
WO2000061792A1
WO2000061792A1 PCT/EP2000/002713 EP0002713W WO0061792A1 WO 2000061792 A1 WO2000061792 A1 WO 2000061792A1 EP 0002713 W EP0002713 W EP 0002713W WO 0061792 A1 WO0061792 A1 WO 0061792A1
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Prior art keywords
proteins
gene
genes
coli
yjee
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German (de)
English (en)
Inventor
Heike BRÖTZ
Kerstin Ehlert
Christoph Freiberg
Frank Spaltmann
Bernd Wieland
Harald Labischinski
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Bayer AG
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Bayer AG
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Priority to AU41119/00A priority Critical patent/AU4111900A/en
Priority to JP2000611714A priority patent/JP2002541819A/ja
Priority to EP00920599A priority patent/EP1171629A1/fr
Priority to CA002366359A priority patent/CA2366359A1/fr
Publication of WO2000061792A1 publication Critical patent/WO2000061792A1/fr
Anticipated expiration legal-status Critical
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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
    • 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
    • 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
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • genes of unknown function and proteins derived from them represent new potential targets if it can be shown that they are essential for the survival of microorganisms (F. Argoni et al., Nature Biotechnology, 16: 851, 1998 ; DT Moir et al., Antimicrob. Agents Chemother. 43: 439-446, 1999; US 5821076; BJ Akerly et al,
  • Knockout technologies can be used to analyze the essentiality of such gene sequences for an organism. Knockout strategies are based on the exchange of intact (wild-type) gene sequence information in the chromosome of a
  • Knockout experiments are carried out as recombination events of homologous DNA sequences.
  • vectors are used which can controllably lose the ability to replicate the plasmid, as in the case of temperature-sensitive replicons (pKO3, pMAK 700 or pSCIOl) or vectors which cannot replicate in the desired target cell (E. coli): e.g. Gram-positive plasmids (pC194, pT181) in Gram-negative bacteria (E. coli).
  • integration vectors such as pMAK700 also carry markers such as antibiotic resistance (e.g. chloramphenicol), which can be used for selection on plasmid-carrying cells.
  • the invention relates to genes with hitherto unknown function, to show their distribution in the phylogenetically diverse bacterial realm and to select in particular those genes which have no or at most only a very distant homology in eukaryotes, and for these genes their essentiality for
  • genes to be found are to select, by suitable selection of the search strategy, only those whose derived proteins can be expected to be water-soluble due to their physicochemical properties to be expected, so that these can advantageously be used in cell-free assays.
  • gene sequences can be selected that would not have been selected in the conventional way as a target for antibiotic drug binding.
  • the gene products of said genes are expressed and purified within the scope of the invention.
  • the new proteins thus obtained are used in assays for the detection of agonists and antagonists of these proteins.
  • over- or under-expression mutants for the corresponding genes are also used in assays for the detection of agonists and antagonists.
  • the present invention relates to essential genes which encode the proteins from the group YQGF, YHBC, YGGJ, YGBP, YCHB, YGBB, YJEE, KDTB, and genes from other microorganisms which encode the corresponding orthologic gene products, and genes as indicated above for which can be shown that their controllable elimination leads to growth inhibition of the host organism, as well as genes from Escherichia coli as above, which encode gene products which are 95-100% identical and for which it can be shown that their controllable elimination to growth inhibition of the host organism, as well as genes as stated above from other microorganisms, which encode orthologic gene products which have 42-100% identical or conserved exchanged amino acids to the corresponding gene product from E. coli, and for which it can be shown that their controllable deactivation for growth inhibition of the host organism, as well as genes that are part of their nucleins acid sequence completely or partially contain at least one gene from the group of the genes specified above, and the
  • the scope of the invention further includes vectors which contain one or more of the abovementioned genes, and transformed microorganisms which contain one or more of the abovementioned genes, and the use of constructed, recombinant microorganisms in which one of the abovementioned genes can be regulatedly expressed to find substances that bind to said gene products and the use of the gene products of the above-mentioned genes in assays to find substances that bind to said gene products and to use said gene products in assays, said assays being based on the principle of affinity selection , as well as the use of said YQGF gene products in assays, the assay using a flavin mononucleotide or a flavin adenine dinucleotide as a substrate, and an indicator system which indicates the redox state of the protein, or the use of said YQGF gene products in assays, wherein in the assay the hydrolytic gap a phosphodiester bond or another ester
  • Binding to the DNA is measured, and the use of said gene products of YGGJ in assays, the key step of the assay being phosphorylation, and the use of said gene products or the use of parts of said gene products to find or produce antibodies or other proteins that bind to said gene products.
  • the scope of the invention further includes the use of substances which bind said gene products
  • the substances found by the methods listed above, which bind to the gene products mentioned above, and the medicaments produced from these substances can be used for the treatment of infections caused by pathogens in humans and animals.
  • diseases which are selected by one or more bacterial pathogens from the genera Porphyromonas gingivalis, Chlamydia trachomatis, Escherichia coli, Yersinia pestis, Staphylococcus aureus, Mycobacterium tuberculosis, Neisseria gonnorhoeaert, Neisseria meningitellaidisidellaidisidellaiditis is very particularly preferred.
  • Medicines can be used in humans and animals for the treatment of diseases and in particular for the treatment of infections affecting the blood; the cardiovascular system; the central nervous system and its appendages; the bones, the bone marrow; the muscles and fascia, the teeth; the joints and their appendages and cavities; the internal organs, their cavities, inner and outer skins and appendages; the gastrointestinal tract and its cavities, inner and outer skins and appendages; the skin, skin appendages and soft parts and their appendages; the genitourinary system and its annexes; localized and / or generalized inflammation and / or general bacteremia and sepsis, as may occur in connection with general illness, trauma, polytrauma, and / or after surgery; Opportunistic infections and sepsis also relate to other diseases such as blood disorders, viral diseases, consuming diseases and / or antineoplastic and / or immunosuppressive therapy.
  • diseases can be treated that after infection with Porphyromonas gingivalis in the area of the neck, nose, ear, head and neck, in the wound area of the oral cavity after surgical and / or dental treatment, and / or after bites by humans and animals; Escherichia coli in the area of the
  • Genitourinary system including urinary tract, bladder, kidney, pelvis, urosepsis and sepsis after surgery and other trauma; Staphylococcus aureus as a causative agent of inflammatory diseases of the respiratory tract and nasopharynx, the lungs, bones, skin, appendages and soft tissues, the heart and heart valves, as well as toxic diseases caused by the pathogens, such as haemolysis of the blood, the Epidermolysis of the skin, enterocolitis of the gastrointestinal tract, toxic shock syndrome; Mycobacterium tuberculosis as tuberculosis and / or tuberculoid diseases of the lungs, skin, and other organs and organ systems; Neisseria gonnorhoeae and the resulting clinical picture of gonorrhea; Neisseria meningitidis as a causative agent in acute purulent meningitis; Bordetella pertussis as a cause of whooping cough; Haemophil
  • Streptoccus mutans and resulting endocarditis and / or inflammation of the heart valves Streptococcus pneumoniae and resulting inflammatory diseases of the upper and lower respiratory tract such as pneumonia, otitis media, sinusitis, as well as meningitis, peritonitis, and inflammation of the cardiac membranes and the pericardium, as well as caries
  • Streptococcus pyogenes and resulting inflammatory diseases of the upper respiratory tract such as streptococcal pharyngitis, scarlet fever, otitis media, and pyoderma and erysipelas of the skin and sepsis
  • Vibrio cholerae and the resulting clinical picture of cholera Bacillus subtilis and the resulting clinical picture of oppurtunistic infections, disseminated spread and secondary infections in the context of other bacterial infections such as otitis, mastoiditis, urinary tract infection.
  • Bacteremia Bacteremia,
  • Bioinformatic analysis of various bacterial genomes The amino acid sequences of the 4289 proteins from Escherichia coli K12 MG1655 with the Genbank / EMBL entry U00096 were compared with the sequences of the complete protein sets from the Gram-positive model organism Bacillus subtilis (Genbank / EMBL accession no. AL009126) and from the Gram -negative pathogen Haemophilus influenzae (Genbank / EMBL accession no. L42023) and compared with all other amino acid sequences in the public database (Genpept, Swiss-Prot).
  • Proteins with known function or with homologies to known proteins from other organisms were excluded.
  • KDTB KDTB, SMPB, YBAB, YBAD, YBAK, YBAX, YBEA, YBEY, YCHB, YFGB,
  • YGAG, YGBB, YGBP YGGJ, YGGV, YHBC, YHBJ, YHBY, YHIN, YIBK, YIDA, YIGZ, YJEE, YJEQ, YQGF, YRAL, YRFI.
  • Basic molecular biological techniques such as plasmid isolation. Cutting with restriction endonucleases, modification of DNA (dephosphorylation, replenishment reactions, ligation), production of competent E. coli cells and transformation have been carried out using standard regulations known to the person skilled in the art, such as those described in e.g. in J. Sambrook et al., Molecular Cloning, and Ausubel et al., Current Protocols in Molecular Biology.
  • DNA polymerases were used for cloning purposes: Pwo, Taq (Boehringer, Mannheim, Germany), Akku Taq (Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany) and Pfu DNA polymerase (Stratagene, Heidelberg, Germany) .
  • Genes or their flanking regions were amplified from 100 ng chromosomal DNA from E. coli W3110 F in a reaction volume of 25 to 100 ⁇ l.
  • the primer concentrations were 1 ⁇ M and dNTP concentrations were 250 ⁇ M.
  • Twenty-five to thirty reaction cycles of 30-60s at 94 ° C, 30-60s at 48-55 ° C and 1-3 min at 72 ° C were followed by a final 5-minute
  • the vector system pBAD / His contains the sequence which codes for six histidines, including a linker for fusion with the N or C terminus of the desired recombinant protein.
  • the maximum expression of the soluble, recombinant protein is achieved by targeted activation of the araBAD promoter with arabinose.
  • the purification of the proteins was carried out with the aid of affinity chromatography on nickel-NT A-agarose.
  • the cells containing the pBAD construct must first be grown to an optical density (OD 600nm ) of 0.3 to 0.6.
  • the targeted protein production is then started with arabinose [0.002-0.2% (w / v)] and incubated for a further 2-3 hours. After completing the
  • the vector system pQE contains the sequence which codes for six histidines, including a linker for fusion with the N or C terminus of the desired recombinant protein.
  • Protein is achieved by targeted activation of the phage T5 promoter system and induction with IPTG.
  • the purification of the proteins was carried out with the aid of affinity chromatography on nickel-NT A-agarose.
  • knockout plasmids which have the temperature-sensitive origin of replication of pSCIOl. This means that these plasmids can reproduce independently in E. coli at 30 ° C., but not at 42-44 ° C. If DNA regions which are homologous to the E. coli chromosome are cloned into such plasmids, these plasmids can at 42-44 ° C using homologous recombination in the gene locus of the E. coli chromosome to be examined. The subsequent excision of the plasmid at 30 ° C can lead to the exchange of the wild type sequence for the sequence originally cloned in the plasmid.
  • flanking areas of the examined genes in the range of 400-800 bp each were cloned into pMAK705 and pKO3. Instead of the gene examined, either a tetracycline or a kanamycin resistance cassette was integrated into the respective plasmids. Two paths were taken for the cloning strategy:
  • the gene of interest including its flanking regions (400-800 bp), was amplified by means of PCR from chromosomal E. coli W3110-F DNA and converted into one
  • Cloning vector cloned (pCR blunt). With the aid of restriction digests and subsequent ligations, the major part of the coding region of the gene of interest was replaced by an antibiotic cassette or the gene was interrupted by such a cassette (tetracycline or kanamycin resistance cassette). Then the cassette with the original flanking the gene
  • the fusion PCR product could be cloned into pKO3 via restriction sites built into the 5 'ends of the external primers.
  • the fusion PCR product contained the flanking areas (400-800 bp each) as well as exactly 18 nucleotides of the 5 'end and 36 nucleotides of the 3' end of the examined
  • E. coli JM101 and E. coli MC 1061 were transformed with the plasmids according to standard protocols and incubated at 43-44 ° C. on LB agar plates with chloramphenicol.
  • 43/44 ° C clones were inoculated in 100 ml LB - chloramphenicol liquid medium and incubated at 30 ° C for 16 h. After two passages in fresh medium with renewed incubation at 30 ° for 16 h each, the plasmids were isolated from 1 ml of the last grown culture. The prepared plasmids were examined for the presence of the wild-type copy of the gene in the plasmid by means of restriction digestion. The culture in which positive plasmids were found were isolated on LB - chloramphenicol plates at 30 ° C.
  • plasmid preparation A number of colonies were picked and grown for plasmid preparation.
  • the prepared plasmids were examined by restriction digestion for the presence of the wild type copy of the gene in the plasmid. Positive clones were verified by PCR for the presence of the antibiotic resistance cassette in the corresponding chromosomal locus. In this way, a chromosomal knockout for the gene under investigation was generated in E. coli with simultaneous complementation by a functional gene copy on the plasmid pMAK705.
  • the clones form a significantly reduced number of colonies on LB chloramphenicol plates at 43/44 ° C compared to the number of colonies on LB +/- chloramphenicol at 30 ° C, since the majority of the clones at 43/44 ° C loses the pMAK705 construct and can therefore no longer develop resistance to chloramphenicol.
  • the number of colonies is reduced by at least a factor of 10. Under these conditions, the clones are not considered to be viable.
  • Criterion for chromosomal knockout in an essential gene clones which, after colony counting on LB without chloramphenicol at 43/44 ° C, formed at least 10 2 fewer colonies than at 30 ° C on LB (with or without chloramphenicol) and at most 10 times more Colonies formed as on LB plates with chloramphenicol at 43/44 ° C, according to our criteria had a chromosomal knockout in an essential gene. The clones were not viable if they lost the plasmid at 43/44 ° C on LB without chloramphenicol (measured in "number of viable colonies").
  • the clones generated with the pKO3 vector system which were chloramphenicol-sensitive and contained a kanamycin resistance cassette instead of the wild-type gene, had a chromosomal knockout in a non-essential gene. A plasmid-coded complementation no longer existed.
  • the E. coli strain MGI 655 was used, which in contrast to MC 1061 has a complete araBAD locus.
  • the araBAD genes from E. coli MGI 655 were replaced by the functional copy of the gene to be examined in each case.
  • the plasmid pAL761 was used for this.
  • the exchange of the araBAD genes for the functional copy of a gene was detected by means of PCR on the clones that were not on M9 minimal medium with 0.2% (w / v) arabinose could grow.
  • the genotype of the deleted chromosomal gene locus with kanamycin cassette was diagnosed by means of PCR among the chloramphenicol-sensitive colonies. If the clones obtained showed a dependence on the growth of arabinose (i.e. no single colony formation on LB agar plates without arabinose in contrast to the growth on LB agar plates with arabinose), the corresponding genes were considered essential.
  • YJEE is likely to be an ATP or GTP binding protein.
  • the protein may have ATPase / GTPase or ATP / GTP synthase activity.
  • KDTB is likely to be a sugar and / or nucleoside and or nucleoside derivative binding protein with phosphorylation or dehydrogenase activity.
  • YQGF may be a flavin derivative binding protein. It can bind flavomononucleotide (FMN) or flavin adenine dinucleotide (FAD) and transfer electrons to another substrate / protein. YQGF may represent hydrolysis that cleaves phosphodiester bonds or other ester bonds.
  • YHBC is a putative DNA-binding protein with a modulatory / regulatory function.
  • YGGJ is a possible phosphotransferase.
  • YGBP is probably a nucleoside diphophate derivative phosphorylase or pyrophosphorylase.
  • YGBP probably cleaves CDP sorbitol or another polyol nucleoside diphosphate with the aid of pyrophosphate or transfers the nucleotide CMP to sorbitol 1-phosphate or another polyol phosphate.
  • YCHB is a putative kinase or putative oxidoreductase.
  • YGBB is a putative hydrolase or NAD (P) H-dependent reductase.
  • the integration plasmid pMAK705 (5.5 kb) can be integrated into the chromosome of E. coli JM101 via homologous recombination.
  • the plasmid contains a chloramphenicol resistance gene for the selection of the plasmid in the host organism.
  • the coding sequence for yjeE and 420 base pairs (bp) upstream and 413bp downstream of the coding sequence for yjeE were amplified by PCR (Cycler Perkin Elmer 480). 100 chromosomal DNA from E.coli W3110 F were in 25 cycles with 45 sec. at 94 ° C, 45 sec. at 48 ° C and 3 min. at 72 ° C and a subsequent incubation at 72 ° C for 5 minutes with Pfu DNA polymerase in
  • Primer A (5'-CCT GCT GGC AAT CAA TCC CGA TA-3 ') and primer B (5'-AGG CGG TGG CGG CAC ATCGGC GTT-3') were used as amplification primers.
  • the amplification product 1482 bp was converted into the vector pCR - blunt using the "pCR - blunt cloning kit" (Invitrogen
  • a tetracycline resistance cassette was cloned into the restriction site Bpml of pCR - blunt / yjee.
  • the tetracycline cassette was isolated from plasmid pBR322 as a 1400bp EcoRI / Aval fragment, and blunt ends were made with Klenow polymerase.
  • the resulting plasmid pCR - blunt / yjeE :: tet was digested with Kpnl / Xbal restriction enzymes.
  • a 2.8 kb DNA band was isolated and cloned into the vector pMAK705 (host organism E. coli JM101) pretreated with Kpnl / Xbal.
  • the resulting construct (pMAK705yjee) was used for the integration experiments.
  • the prepared plasmids were examined by restriction digestion for the presence of the wild type copy of the gene in the plasmid.
  • Cells from the batches containing positive clones (yjeE gene from the wild-type chromosome on the plasmid) were separated on LB plates with chloramphenicol at 30 ° C.
  • the plasmid DNA of the individual clones was tested again for intact yjeE gene from the chromosome and positive colonies were identified.
  • the integration of the copy of yjeE inactivated in the chromosome by insertion of the tetracycline cassette was tested by PCR.
  • the clones checked in this way could now be used for the vitality of the target yjeE for the survival of the E.coli cell.
  • the cells were incubated on LB test plates with and without chloramphenicol at 30 ° and 43 ° C. Table 6: Cell numbers of the incubation +/- chloramphenicol at 30 ° C / 43 ° C
  • the cells can grow at 30 ° C. both with and without chloramphenicol.
  • the growth with chloramphenicol as well as without chloramphenicol is drastically reduced, ie by a factor of 10 4 .
  • DNA regions surrounding the 5 'and 3' end of the ygbP gene were amplified in two separate PCR reactions from 100 ng chromosomal E. coli W31 10-F DNA in a reaction volume of 25 ⁇ l.
  • the primer concentrations were 1 ⁇ M and dNTP concentrations were 250 ⁇ M.
  • Twenty-five reaction cycles of 30s at 94 ° C, 30s at 52 ° C and 1-2 min at 72 ° C were carried out with a final 5 minute step at 72 ° C.
  • the PCR product made with the primers YGBP1A (5'-cgcggatccCCACATGGTCACTGCCTGG-3 ') and YGBP1B (5'-cccatccactaaactgcagctCAAATGAGTGGTTGCCATGTT-3') comprises 18 bp of the 5 'bend-on end and the 6' bend-on end by ygbP.
  • the PCR product that is primed with YGBP2A (5'- agctgcagtttagtggatgggTACCTCACCCGAACCATCC-3 ') and YGBP2B (5'- cgcggatccACCTGGCAGCCTTCCAGTTG-3 '), comprises 36 bp of the 3' end of ygbP and 807 bp of the chromosomal region downstream of ygbP.
  • the inner primers YGBPIB and YGBP2A additionally contained 21mer "tags" at their 5 'ends, which were complementary to one another (small letters of the sequences mentioned above). These were used to assemble both PCR products in a second PCR step using the external primers YGBP1A and YGBP2B. To this end, 1 ⁇ l of the two previous PCR batches were mixed as template for a second PCR with YGBP1A and YGBP2B. This PCR was carried out under the conditions described above. The PCR
  • Product of the expected size was purified using an agarose gel electrophoresis using QIAEX (QIAGEN, Hilden.Germany).
  • the fusion PCR product could be cloned into the BamHI-cut, dephosphorylated pKO3.
  • the plasmid pAL759 was generated which contained the "in-frame" deletion of ygbP.
  • the kanamycin resistance cassette was cloned into pAL759 via the Pstl interface in the 21 bp "day” (5'-ctgcag-3 ') and was obtained from pUC4K by means of a Pstl digest.
  • the resulting construct was named pAL759a.
  • E. coli MC 1061 was transformed with the plasmid pAL759a and opened at 44 ° C
  • E. coli MC 1061 was transformed with the plasmid pAL759 and incubated at 44 ° C. on LB agar plates with chloramphenicol.
  • sucrose clones 100 of the resulting sucrose clones were inoculated again on LB sucrose and LB chloramphenicol plates ("replica -
  • the E. coli strain MG 1655 was used, which in contrast to MC 1061 has a complete araBAD locus.
  • the E. coli MG 1655 araBAD genes were replaced with the functional copy of ygbP.
  • the plasmid pAL763 was used for this.
  • the plasmid pAL763 was generated from the chromosomal E.
  • the primers contain the restriction sites for Xhol (YGBPT) and Nhel (YGBPR) at their 5 'termini.
  • the PCR product was cloned into the vector pAL761 via the interfaces. After transformation of E.
  • coli MG1655 with pAL763 and subsequent integration and excision steps (see above), the exchange of the araBAD genes for the functional copy of ygbP was detected by means of PCR on the clones which were not on M9 minimal medium with 0, 2% (w / v) arabinose could grow.
  • the diagnostic PCR was carried out with the primers YGBPR (see above) and ARA2B (atcgcggccgcAAAGCCGTGCTCGCGC).
  • the primer ARA2B binds in the 3 'region 865 bp downstream of the araD gene, so that together with the primer YGBPR a 1655 bp product was formed in positive clones.
  • YJEE protein was carried out using the pBAD His vector system (Invitrogen).
  • the sequence of yjeE was made from 100 chromosomal DNA from E. coli W3110F for 1 min at 94 ° C. for 1 min. at 52 ° C, 3 min at 72 ° C in 25 cycles with Pfu DNA polymerase in 100 ⁇ l reaction mixture.
  • YJEE-START (5'-TGA AGA TCT AAT CGA GTA ATT CCG CTC CCT GAT-3 ')
  • YJEE-STOP 5'-TCA GAA TTC TTA ACC GGC TAA ACG CGC CAG CAA CAA TC-3'
  • START contains the sequence for the restriction enzyme Bglll.
  • the 5 'end of YJEE-STOP contains the sequence for EcoRI.
  • the PCR mixture was separated in an agarose gel and a band was cut out at 450 bp. The band was then isolated with Qiaex (Qiagen, Hilden, Germany) and incubated with the restriction enzymes BglII and EcoRI for 3 hours at 37 ° C.
  • the DNA fragment treated in this way was cloned into the vector pBAD / HisB pretreated with BglII / EcoRI [host organism: E. coli strain TOP 10 (Invitrogen)]. Successful cloning was demonstrated by plasmid isolation of the transformants obtained and restriction with EcoRI. The clone with the plasmid pBAD / ⁇ isB-YjeE30 has been used for expression and purification.
  • the cultivation and purification of YjeE from E. coli TOP 10 with plasmid pBAD HisB-YjeE30 was carried out according to the protocol for the purification of histidine-labeled proteins on Ni-NTA agarose (The QIAexpressionist, A handbook for high-level expression and purification of 6xHis- tagged proteins, Quiagen, Hilden, Germany 1997).
  • Various concentrations of imidazole 50, 100, 150 and 200 mM were used to elute the bound protein. The highest yield was obtained at 150 raM
  • Targets of unknown function based on affinity selection and mass spectrometry
  • screening methods can be used that test substance banks with regard to their affinity for the protein.
  • a screening option is the affinity selection from substance mixtures with subsequent detection of the ligands in the mass spectrometer. Defined substance mixtures must be used for this, from which individual substances can be identified with the aid of mass detection. Mixtures of substances that have been prepared from combinatorial syntheses are therefore particularly suitable for this screening method.
  • a liquid chromatography-electrospray mass spectrometer serves as a screening apparatus, the HPLC column being replaced by an ultrafiltration chamber.
  • Ultrafiltration chamber consists of a filtration unit in which the filter disk is replaced by an ultrafiltration membrane (exclusion molecular weight 10 kDa). Mass spectra are generated using a mass spectrometer (from Hewlett-Packard; Palo Alto, CA; "5989B MS Engine Quadrupole Mass Spectrometer”). It is operated as described (van Breemen et al., Pulsed Ultrafiltration Mass Spectrometry. A New Method for Screening Combinatorial: Anal. Chem., 69 [11], 2159-2164, 1997).
  • Ammonium acetate buffer is present.
  • the protein substance mixture is at least Incubated for 15 min at room temperature. After the mixture has been injected into the ultrafiltration chamber, it is washed with water for 8 min at a flow rate of 50 l / min.
  • the mobile phase is then changed to methanol / water (50:50 v / v) in order to dissociate possible ligand-protein complexes and to filter out the ligands.
  • the ligands are detected using electrospray mass spectrometry.
  • the substances from a 96 mixture identified as possible ligands on the basis of their mass peaks are used again as individual substances in the affinity test in order to verify their affinity for the target protein (eg YJEE).
  • Substances that are noticeable in the affinity selection are subjected to further tests such as the MIC test.
  • the MIC values are determined using the microdilution method in BH medium.
  • Each test substance is dissolved in the nutrient medium.
  • Serial dilutions of the test substances are made in the microtiter plate.
  • Overculture cultures of the pathogens are used for inoculation, which are previously diluted 1: 250 in the nutrient medium. 100 ml of the inoculated solution are added to 100 ml of the diluted nutrient solutions containing the active ingredient.
  • microtiter plates are incubated at 37 ° C and read after about 20 hours or after 3 to 5 days.
  • the MIC value (mg / ml) indicates the lowest active substance concentration at which no growth can be seen.
  • Another method is to determine the minimum inhibitory concentration (MIC) in the liquid dilution test.
  • Overpower cultures of the test germs (S. aureus 133) in isosensitest broth are diluted 1: 1000 in fetal calf serum FKS) or isosensitest broth and incubated with dilutions of the test substances (dilution levels 1: 2).
  • the cultures are incubated at 37 ° C for 18 to 24 hours.
  • the lowest substance concentration at which no visible bacterial growth occurs is defined as the MIC.

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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne des acides nucléiques et leurs séquences protéiques dérivées, qui possèdent des fonctions essentielles à la vie d'Escherichia coli; des vecteurs et des cellules hôtes, transformées au moyen desdits vecteurs, qui permettent l'expression desdites protéines essentielles et les protéines produites au moyen desdits vecteurs; ainsi que l'utilisation des protéines produites au moyen desdits vecteurs dans des techniques de criblage qui permettent de rechercher des substances antibactériennes et les substances antibactériennes trouvées au moyen desdites protéines.
PCT/EP2000/002713 1999-04-10 2000-03-28 Genes bacteriens essentiels et leurs proteines Ceased WO2000061792A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU41119/00A AU4111900A (en) 1999-04-10 2000-03-28 Novel essential bacterial genes and their proteins
JP2000611714A JP2002541819A (ja) 1999-04-10 2000-03-28 新規の必須細菌遺伝子およびこれらのタンパク質
EP00920599A EP1171629A1 (fr) 1999-04-10 2000-03-28 Genes bacteriens essentiels et leurs proteines
CA002366359A CA2366359A1 (fr) 1999-04-10 2000-03-28 Genes bacteriens essentiels et leurs proteines

Applications Claiming Priority (2)

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DE19916176.3 1999-04-10
DE19916176A DE19916176A1 (de) 1999-04-10 1999-04-10 Neue essentielle bakterielle Gene und ihre Proteine

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WO2000061792A1 true WO2000061792A1 (fr) 2000-10-19

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JP (1) JP2002541819A (fr)
AU (1) AU4111900A (fr)
CA (1) CA2366359A1 (fr)
DE (1) DE19916176A1 (fr)
WO (1) WO2000061792A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002020733A3 (fr) * 2000-09-01 2003-08-14 Du Pont Genes impliques dans la production de composes isoprenoides

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20040100435A (ko) * 2003-05-23 2004-12-02 (주)대한솔루션 자동차용 적층체 및 그 제조방법과 제조장치
CN110804562B (zh) * 2018-08-02 2024-08-09 广州溯原生物科技股份有限公司 一种头孢抗性且高表达Sir2样蛋白的粪肠球菌及应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998042875A1 (fr) * 1997-03-27 1998-10-01 Case Western Reserve University PROCEDES DE DEPISTAGE D'AGENTS ANTIMICROBIENS UTILISANT (aarC) ET DE LEURS COMPOSITIONS
WO1999054474A2 (fr) * 1998-04-22 1999-10-28 Glaxo Group Limited Famille de polypeptides bacteriens

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998042875A1 (fr) * 1997-03-27 1998-10-01 Case Western Reserve University PROCEDES DE DEPISTAGE D'AGENTS ANTIMICROBIENS UTILISANT (aarC) ET DE LEURS COMPOSITIONS
WO1999054474A2 (fr) * 1998-04-22 1999-10-28 Glaxo Group Limited Famille de polypeptides bacteriens

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ARIGONI F ET AL: "A genome-based approach for teh identification of essential bacterial genes", NATURE BIOTECHNOLOGY,US,NATURE PUBLISHING, vol. 16, no. 16, September 1998 (1998-09-01), pages 851 - 856-856, XP002124132, ISSN: 1087-0156 *
BALTZ ET AL: "DNA sequence sampling of the Streptococcus pneumoniae genome to identify novel targets for antibiotic development", MICROBIAL DRUG RESISTANCE,US,LIEBERT, vol. 4, no. 1, 21 March 1998 (1998-03-21), pages 1 - 9-9, XP002112153, ISSN: 1076-6294 *
BLATTNER F R ET AL: "THE COMPLETE GENOME SEQUENCE OF ESCHERICHIA COLI K-12", SCIENCE,US,AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, vol. 277, 5 September 1997 (1997-09-05), pages 1453 - 1462, XP002069950, ISSN: 0036-8075 *
FLEISCHMANN R D ET AL: "WHOLE-GENOME RANDOM SEQUENCING AND ASSEMBLY OF HAEMOPHILUS INFLUENZAE RD", SCIENCE,US,AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, vol. 269, no. 5223, 28 July 1995 (1995-07-28), pages 496 - 498,507-51, XP000517090, ISSN: 0036-8075 *
TATUSOV R L ET AL: "METABOLISM AND EVOLUTION OF HAEMOPHILUS INFLUENZAE DEDUCED FROM A WHOLE-GENOME COMPARISON WITH ESCHERICHIA COLI", CURRENT BIOLOGY,GB,CURRENT SCIENCE,, vol. 6, no. 3, 1 March 1996 (1996-03-01), pages 279 - 291-291, XP000857871, ISSN: 0960-9822 *
TRIAS AND GORDON: "INNOVATIVE APPROACHES TO NOVEL ANTIBACTERIAL DRUG DISCOVERY", CURRENT OPINION IN BIOTECHNOLOGY,GB,LONDON, vol. 8, no. 8, 1997, pages 757 - 762-762, XP002119418, ISSN: 0958-1669 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002020733A3 (fr) * 2000-09-01 2003-08-14 Du Pont Genes impliques dans la production de composes isoprenoides
US6660507B2 (en) 2000-09-01 2003-12-09 E. I. Du Pont De Nemours And Company Genes involved in isoprenoid compound production
US7056717B2 (en) 2000-09-01 2006-06-06 E. I. Du Pont De Nemours And Company Genes involved in isoprenoid compound production

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DE19916176A1 (de) 2000-10-12
CA2366359A1 (fr) 2000-10-19
EP1171629A1 (fr) 2002-01-16
JP2002541819A (ja) 2002-12-10
AU4111900A (en) 2000-11-14

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