EP0959903A1 - NOVEL FabD - Google Patents
NOVEL FabDInfo
- Publication number
- EP0959903A1 EP0959903A1 EP97953035A EP97953035A EP0959903A1 EP 0959903 A1 EP0959903 A1 EP 0959903A1 EP 97953035 A EP97953035 A EP 97953035A EP 97953035 A EP97953035 A EP 97953035A EP 0959903 A1 EP0959903 A1 EP 0959903A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- polypeptide
- polynucleotide
- fabd
- seq
- amino acid
- Prior art date
- Legal status (The legal status 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 status listed.)
- Withdrawn
Links
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Classifications
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/14—Decongestants or antiallergics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/16—Otologicals
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- 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
- This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses.
- the invention relates to novel polynucleotides and polypeptides of the malonyl- CoA:ACP family, hereinafter referred to as "FabD”. BACKGROUND OF THE INVENTION
- Streptococci make up a medically important genera of microbes known to cause several types of disease in humans, including, for example, otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection of cerebrospinal fluid.
- Streptococcus pneumoniae Since its isolation more than 100 years ago, Streptococcus pneumoniae has been one of the more intensively studied microbes. For example, much of our early understanding that DNA is, in fact, the genetic material was predicated on the work of Griffith and of Avery, Macleod and McCarty using this microbe.
- Streptococcal genes and gene products as targets for the development of antibiotics.
- Streptococcus pneumoniae infections has risen dramatically in the past few decades. This has been attributed to the emergence of multiply antibiotic resistant strains and an increasing population of people with weakened immune systems. It is no longer uncommon to isolate Streptococcus pneumoniae strains which are resistant to some or all of the standard antibiotics. This phenomenon has created a demand for both new antimicrobial agents, vaccines, and diagnostic tests for this organism.
- the pathway for the biosynthesis of saturated fatty acids is very similar in prokaryotes and eukaryotes. However, whilst the chemical reactions may not vary, the organisation of the biosynthetic apparatus is very different.
- the acyl carrier protein (ACP) is an integral part of the complex.
- ACP acyl carrier protein
- Mycobacteria are unique in that they possess both type I and II FASs; the former is involved in basic fatty acid biosynthesis whereas the latter is involved in synthesis of complex cell envelope lipids such as mycolic acids. There therefore appears to be considerable potential for selective inhibition of the bacterial systems by broad spectrum antibacterial agents (Rock, C.
- the first step in the biosynthetic cycle is the condensation of malonyl-ACP with acetyl-CoA by FabH.
- malonyl-ACP Prior to this, malonyl-ACP is synthesised from ACP and malonyl- CoA by FabD, malonyl CoA:ACP transacylase.
- malonyl-ACP is condensed with the growing-chain acyl-ACP (FabB and FabF, synthases I and II respectively).
- the second step in the elongation cycle is ketoester reduction by NADPH- dependent ⁇ -ketoacyl-ACP reductase (FabG).
- the polypeptide of the present invention has the conserved residues, and have amino acid sequence homology to known transacylase protein.
- polynucleotides that encode FabD polypeptides particularly polynucleotides that encode the polypeptide herein designated FabD.
- the polynucleotide comprises a region encoding FabD polypeptides comprising a sequence set out in Table 1 [SEQ ID NO 1 or 3 or 5] which includes a full length gene, or a variant thereof
- FabD protein from Streptococcus pneumoniae comprising the amino acid sequence of Table I [SEQ ID NO 2 or 4 or 6], or a va ⁇ ant thereof
- an isolated nucleic acid molecule encoding a mature polypeptide expressible by the Streptococcus pneumoniae 0100993 strain contained in the deposited strain
- a further aspect of the invention there are provided isolated nucleic acid molecules encoding FabD, particularly Streptococcus pneumoniae FabD, including mRNAs, cDNAs, genomic DNAs Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful va ⁇ ants thereof, and compositions composing the same.
- a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization
- the particularly preferred embodiments of the invention are naturally occurring allelic va ⁇ ants of FabD and polypeptides encoded thereby
- novel polypeptides of Streptococcus pneumoniae referred to herein as FabD as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
- variants of FabD polypeptide encoded by naturally occurring alleles of the FabD gene are variants of FabD polypeptide encoded by naturally occurring alleles of the FabD gene.
- inhibitors to such polypeptides useful as antibacterial agents, including, for example, antibodies.
- FabD polypeptide or polynucleotide for assessing FabD expression, treating disease, assaying genetic variation, and administering a FabD polypeptide or polynucleotide to an organism to raise an immunological response against a bacteria, especially a Streptococcus pneumoniae bacteria.
- polynucleotides that hybridize to FabD polynucleotide sequences, particularly under stringent conditions.
- antibodies against FabD polypeptides there are provided antibodies against FabD polypeptides.
- FabD agonists and antagonists preferably bacteriostatic
- compositions comprising a FabD polynucleotide or a FabD polypeptide for administration to a cell or to a multicellular organism.
- the invention relates to novel FabD polypeptides and polynucleotides as described in greater detail below.
- the invention relates to polypeptides and polynucleotides of a novel FabD of Streptococcus pneumoniae. which is related by amino acid sequence homology to FabD polypeptide of a different species.
- the invention relates especially to FabD having the nucleotide and amino acid sequences set out in Table 1 as SEQ ID NO: 1 and SEQ ID NO: 2 respectively, and to the FabD nucleotide sequences of the DNA in the deposited strain and amino acid sequences encoded thereby.
- E Streptococcus pneumoniae FabD polypeptide sequence deduced from the polynucleotide ORF sequence in this table [SEQ ID NO:4] N ' -terminus of a fabD polypeptide embodiment.
- H 2 -1 MTKTAFLFAG QGAQYLGMGR DFYDQYPIVK ETIDRASQVL GYDLRYLIDT 51 EEDKLNQTRY TQPAILATSV AIYRL QEKG YQPDMVAGLS LGEYSA VAS 101 GALDFEDAVA VAKRGAYME EA-COOH
- a fabD polypeptide sequence embodiment comprising a putative C-terminus.
- NCIMB National Collections of Indust ⁇ al and Ma ⁇ ne Bacte ⁇ a Ltd.
- Streptococcus peumnoiae 0100993 DNA library in E. coll was similarly depositedwith the NCIMB and assigned deposit number 40800
- the Streptococcus pneumoniae strain deposit is referred to herein as "the deposited strain” or as "the DNA of the deposited strain.”
- the deposited strain contains the full length FabD gene
- the sequence of the polynucleotides contained in the deposited strain, as well as the ammo acid sequence of the polypeptide encoded thereby, are controlling in the event of any conflict with any desc ⁇ ption of sequences herein.
- the deposit of the deposited strain has been made under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for Purposes of Patent Procedure.
- the strain will be irrevocably and without restriction or condition released to the public upon the issuance of a patent.
- the deposited strain is provided merely as convenience to those of skill in the art and is not an admission that a deposit is required for enablement, such as that required under 35 U.S.C. ⁇ 1 12.
- a license may be required to make, use or sell the deposited strain, and compounds derived therefrom, and no such license is hereby granted.
- polypeptides of the invention include a polypeptide of Table 1 [SEQ ID NO:2 or
- polypeptide 4 or 6 (in particular the mature polypeptide) as well as polypeptides and fragments, particularly those which have the biological activity of FabD, and also those which have at least 70% identity to a polypeptide of Table 1 [SEQ ID NO:l or 3 or 5]or the relevant portion, preferably at least 80% identity to a polypeptide of Table 1 [SEQ ID NO:2 or 4and more preferably at least 90% similarity (more preferably at least 90% identity) to a polypeptide of Table 1 [SEQ ID NO:2 or 4 or 6] and still more preferably at least 95% similarity (still more preferably at least 95% identity) to a polypeptide of Table 1 [SEQ ID NO:2 or 4 or 6] and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
- the invention also includes polypeptides of the formula:
- R j and R3 are any amino acid residue, m is an integer between 1 and 1000 or zero, n is an integer between 1 and 1000 or zero, and R 2 is an amino acid sequence of the invention, particularly an amino acid sequence selected from Table 1. In the formula above R 2 is oriented so that its amino terminal residue is at the left, bound to R ⁇ and its carboxy terminal residue is at the right, bound to R3.
- a fragment is a variant polypeptide having an amino acid sequence that entirely is the same as part but not all of the amino acid sequence of the aforementioned polypeptides.
- fragments may be "free-standing,” or comprised within a larger polypeptide of which they form a part or region, most preferably as a single continuous region, a single larger polypeptide.
- Preferred fragments include, for example, truncation polypeptides having a portion of an amino acid sequence of Table 1 [SEQ ID NO:2 or 4 or 6], or of variants thereof, such as a continuous series of residues that includes the amino terminus, or a continuous series of residues that includes the carboxyl terminus.
- Degradation forms of the polypeptides of the invention in a host cell, particularly a Streptococcus pneumoniae are also preferred.
- fragments characterized by structural or functional attributes such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet-forming regions, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
- biologically active fragments which are those fragments that mediate activities of FabD, including those with a similar activity or an improved activity, or with a decreased undesirable activity. Also included are those fragments that are antigenic or immunogenic in an animal, especially in a human. Particularly preferred are fragments comprising receptors or domains of enzymes that confer a function essential for viability of Streptococcus pneumoniae or the ability to initiate, or maintain cause disease in an individual, particularly a human.
- Variants that are fragments of the polypeptides of the invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, these variants may be employed as intermediates for producing the full-length polypeptides of the invention.
- X or "Xaa” may also be used in describing certain polypeptides of the invention.
- "X” and “Xaa” mean any of the twenty naturally occuring amino acids may appear at such a designated position in the polypeptide sequence.
- Polynucleotides Another aspect of the invention relates to isolated polynucleotides, including the full length gene, that encode the FabD polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2 or 4 or 6] and polynucleotides closely related thereto and variants thereof.
- a polynucleotide of the invention encoding FabD polypeptide may be obtained using standard cloning and screening methods, such as those for cloning and sequencing chromosomal DNA fragments from bacte ⁇ a using Streptococcus pneumoniae 0100993 cells as starting material, followed by obtaining a full length clone
- a polynucleotide sequence of the invention such as a sequence given in Table 1 [SEQ ID NO 1 or 3 or 5]
- typically a library of clones of chromosomal DNA of Streptococcus pneumoniae 0100993 in E coli or some other suitable host is probed with a radiolabeled ohgonucleotide, preferably a 17-mer or longer, derived from a partial sequence Clones carrying DNA identical to that of the probe can then be distinguished using stringent
- the DNA sequence set out in Table 1 contains an open reading frame encoding a protein having about the number of amino acid residues set forth in Table 1 [SEQ ID NO 2 or 4 or 6] with a deduced molecular weight that can be calculated using amino acid residue molecular weight values well known in the art
- the polynucleotide of SEQ ID NO 1, between nucleotide number 1 and the stop codon underlined in Table 1(A) [SEQ ID NO 1] encodes the polypeptide of SEQ ID NO.2.
- FabD of the invention is structurally related to other proteins of the malonyl- CoA ACP family, as shown by the results of sequencing the DNA encoding FabD of the deposited strain
- the invention provides a polynucleotide sequence identical over its entire length to a coding sequence in Table 1 [SEQ ID NO 1 or 3 or 5]
- the coding sequence for the mature polypeptide or a fragment thereof by itself as well as the coding sequence for the mature polypeptide or a fragment in reading frame with other coding sequence, such as those encoding a leader or secretory sequence, a pre-, or pro- or prepro- protein sequence.
- the polynucleotide may also contain non-coding sequences, including for example, but not limited to non-coding 5' and 3' sequences, such as the transcribed, non- translated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence which encode additional amino acids.
- non-coding sequences including for example, but not limited to non-coding 5' and 3' sequences, such as the transcribed, non- translated sequences, termination signals, ribosome binding sites, sequences that stabilize mRNA, introns, polyadenylation signals, and additional coding sequence which encode additional amino acids.
- a marker sequence that facilitates purification of the fused polypeptide can be encoded.
- the marker sequence is a hexa-histidine peptide, as provided in the pQE vector (Qiagen, Inc.) and described in Gentz et al., Proc. Natl. Acad.
- Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene and its naturally associated sequences that control gene expression.
- a preferred embodiment of the invention is a polynucleotide of comprising nucleotide 1 to the nucleotide immediately upstream of the stop codon set forth in SEQ ID NO: 1 of Table 1, both of which encode the FabD polypeptide.
- the invention also includes polynucleotides of the formula:
- R 1 X-(R 1 ) m -(R 2 )-(R 3 )n-Y
- X is hydrogen
- Y is hydrogen or a metal
- R j and R3 is any nucleic acid residue
- m is an integer between 1 and 3000 or zero
- n is an integer between 1 and 3000 or zero
- R is a nucleic acid sequence of the invention, particularly a nucleic acid sequence selected from Table 1.
- R is oriented so that its 5' end residue is at the left, bound to R l and its 3' end residue is at the right, bound to R3.
- Any stretch of nucleic acid residues denoted by either R group, where m and or n is greater than 1, may be either a heteropolymer or a homopolymer, preferably a heteropolymer.
- m and/or n is an integer between 1 and 1000.
- polynucleotides of the inventions are derived from Streptococcus pneumoniae. however, they may preferably be obtained from organisms of the same taxonomic genus. They may also be obtained, for example, from organisims of the same taxonomic family or order.
- polynucleotide encoding a polypeptide encompasses polynucleotides that include a sequence encoding a polypeptide of the invention, particularly a bacterial polypeptide and more particularly a polypeptide of the Streptococcus pneumoniae FabD having an amino acid sequence set out in Table 1 [SEQ ID NO:2 or 4 or 6].
- the term also encompasses polynucleotides that include a single continuous region or discontinuous regions encoding the polypeptide (for example, interrupted by integrated phage or an insertion sequence or editing) together with additional regions, that also may contain coding and/or non-coding sequences.
- the invention further relates to variants of the polynucleotides described herein that encode for variants of the polypeptide having a deduced amino acid sequence of Table 1 [SEQ ID NO:2 or 4 or 6].
- Variants that are fragments of the polynucleotides of the invention may be used to synthesize full-length polynucleotides of the invention.
- Further particularly preferred embodiments are polynucleotides encoding FabD variants, that have the amino acid sequence of FabD polypeptide of Table 1 [SEQ ID NO:2 or 4 or 6] in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination. Especially preferred among these are silent substitutions, additions and deletions, that do not alter the properties and activities of FabD.
- Further preferred embodiments of the invention are polynucleotides that are at least
- polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly prefe ⁇ ed polynucleotides.
- those with at least 95% are especially preferred.
- those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly prefe ⁇ ed, with at least 99% being the more prefe ⁇ ed.
- Prefe ⁇ ed embodiments are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature polypeptide encoded by a DNA of Table 1 [SEQ ID NO: 1 or 3 or 5].
- the invention further relates to polynucleotides that hybridize to the herein above- described sequences.
- the invention especially relates to polynucleotides that hybridize under stringent conditions to the herein above-described polynucleotides.
- stringent conditions and “stringent hybridization conditions” mean hyb ⁇ dization will occur only if there is at least 95% and preferably at least 97% identity between the sequences
- An example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% formamide, 5x SSC ( 150mM NaCl, 15mM t ⁇ sodium citrate), 50 mM sodium phosphate (pH7 6), 5x Denhardt s solution, 10% dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the hybridization support in 0 lx SSC at about 65°C Hybridization and wash conditions are well known and exemplified in Sambrook, et al , Molecular Cloning A Laboratory Manual Second Edition, Cold Spring Harbor, N Y , ( 1989), particularly Chapter 1 1 therein
- the invention also provides a polynucleotide consisting essentially of a polynucle
- polynucleotide assays of the invention may be used as a hyb ⁇ dization probe for RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding FabD and to isolate cDNA and genomic clones of other genes that have a high sequence simila ⁇ ty to the FabD gene
- probes generally will compose at least 15 bases
- probes will have at least 30 bases and may have at least 50 bases
- Particularly prefe ⁇ ed probes will have at least 30 bases and will have 50 bases or less
- the coding region of the FabD gene may be isolated by screening using a DNA sequence provided in Table 1 [SEQ ID NO 1 or 3] to synthesize an oligonucleotide probe A labeled oligonucleotide having a sequence complementary to that of a gene of the invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine which members of the library the probe hyb ⁇ dizes to
- polynucleotides and polypeptides of the invention may be employed, for example, as research reagents and mate ⁇ als for discovery of treatments of and diagnostics for disease particularly human disease, as further discussed herein relating to polynucleotide assays
- Polynucleotides of the invention that are ohgonucleotides derived from the sequences of Table 1 [SEQ ID NOS 1 or 2 or 3 or 4] may be used in the processes herein as described, but preferably for PCR, to determine whether or not the polynucleotides identified herein in whole or in part are transcribed in bacteria in infected tissue It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
- the invention also provides polynucleotides that may encode a polypeptide that is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids inte ⁇ or to the mature polypeptide (when the mature form has more than one polypeptide chain, for instance) Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half-life or may facilitate manipulation of a protein for assay or production, among other things As generally is the case in vivo, the additional amino acids may be processed away from the mature protein by cellular enzymes.
- a precursor protein, having the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide When prosequences are removed such inactive precursors generally are activated Some or all of the prosequences may be removed before activation Generally, such precursors are called proproteins.
- a polynucleotide of the invention may encode a mature protein, a mature protein plus a leader sequence (which may be refe ⁇ ed to as a preprotein), a precursor of a mature protein having one or more prosequences that are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed du ⁇ ng processing steps that produce active and
- the invention also relates to vectors that comprise a polynucleotide or polynucleotides of the invention, host cells that are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
- Cell-free translation systems can also be employed to produce such proteins using RNAs de ⁇ ved from the DNA constructs of the invention
- host cells can be genetically engineered to incorporate expression systems or portions thereof or polynucleotides of the invention
- Introduction of a polynucleotide into the host cell can be effected by methods desc ⁇ bed in many standard laboratory manuals, such as Davis et al , BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al , MOLECULAR CLONING A LABORATORY MANUAL, 2nd Ed , Cold Sp ⁇ ng Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, N Y ( 1989), such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection.
- bacte ⁇ al cells such as streptococci, staphylococci, enterococci E coli, streptomyces and Bacillus subtihs cells
- fungal cells such as yeast cells and Aspergillus cells
- insect cells such as Drosophila S2 and
- Spodoptera Sf9 cells animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and
- vectors include, among others, chromosomal, episomal and virus-de ⁇ ved vectors, e g , vectors de ⁇ ved from bacte ⁇ al plasmids, from bacte ⁇ ophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors de ⁇ ved from combinations thereof, such as those derived from plasmid and bacte ⁇ ophage genetic elements, such as cosmids and phagemids
- the expression system constructs may contain control regions that regulate as well as engender expression Generall) , any system or vector suitable to maintain, propagate or express polynucleotides and or to express a poly
- approp ⁇ ate secretion signals may be incorporated into the expressed polvpeptide These signals may be endogenous to the polypeptide or they may be heterologous signals
- Polypeptides of the invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography Most preferably, high performance liquid chromatography is employed for pu ⁇ fication Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or pu ⁇ fication
- This invention is also related to the use of the FabD polynucleotides of the invention for use as diagnostic reagents Detection of FabD in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease Eukaryotes (herein also " ⁇ nd ⁇ v ⁇ dual(s)”), particularly mammals, and especially humans, particularly those infected or suspected to be infected with an organism comp ⁇ sing the FabD gene may be detected at the nucleic acid level by a va ⁇ ety of techniques.
- Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as bone, blood, muscle, cartilage, and skin Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification technique p ⁇ or to analysis RNA, cDNA and genomic DNA may also be used in the same ways Using amplification, characterization of the species and strain of prokaryote present in an individual, may be made by an analysis of the genotype of the prokaryote gene Deletions and insertions can be detected by a change in size of the amplified product in compa ⁇ son to the genotype of a reference sequence Point mutations can be identified by hyb ⁇ dizing amplified DNA to labeled FabD polynucleotide sequences Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures DNA sequence differences may also be detected by alterations in the electrophoretic mobility of the DNA fragments in gels, with or
- the invention further provides a process for diagnosing, disease, preferably bacte ⁇ al infections, more preferably infections by Streptococcus pneumoniae, comprising determining from a sample derived from an individual a increased level of expression of polynucleotide having a sequence of Table 1 [SEQ ID NO 1 or 3] Increased or decreased expression of FabD polynucleotide can be measured using any on of the methods well known in the art for the quantation of polynucleotides, such as, for example, amplification, PCR, RT-PCR, RNase protection, Northern blotting and other hyb ⁇ dization methods.
- a diagnostic assay in accordance with the invention for detecting over- expression of FabD protein compared to normal control tissue samples may be used to detect the presence of an infection, for example Assay techniques that can be used to determine levels of a FabD protein, in a sample de ⁇ ved from a host are well-known to those of skill in the art Such assay methods include radioimmun
- polypeptides of the invention or va ⁇ ants thereof, or cells expressing them can be used as an immunogen to produce antibodies immunospecific for such polypeptides
- Antibodies as used herein includes monoclonal and polyclonal antibodies, chime ⁇ c, single chain, simianized antibodies and humanized antibodies, as well as Fab fragments, including the products of an Fab immunolglobulin expression library
- Antibodies generated against the polypeptides of the invention can be obtained by administe ⁇ ng the polypeptides or epitope-bea ⁇ ng fragments, analogues or cells to an animal, preferably a nonhuman. using routine protocols.
- any technique known in the art that provides antibodies produced by continuous cell line cultures can be used. Examples include various techniques, such as those in ohler, G. and Milstein. C, Nature 256: 495-497 (1975); Kozbor et al.. Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).
- phage display technology may be utilized to select antibody genes with binding activities towards the polypeptide either from repertoires of PCR amplified v- genes of lymphocytes from humans screened for possessing anti-FabD or from naive libraries (McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al., (1992) Biotechnology 10, 779-783).
- the affinity of these antibodies can also be improved by chain shuffling (Clackson, T. et al.. (1991) Nature 352, 624-628).
- each domain may be directed against a different epitope - termed 'bispecific' antibodies.
- the above-described antibodies may be employed to isolate or to identify clones expressing the polypeptides to purify the polypeptides by affinity chromatography.
- antibodies against FabD- polypeptide may be employed to treat infections, particularly bacterial infections.
- Polypeptide variants include antigenically, epitopically or immunologically equivalent variants that form a particular aspect of this invention.
- the term "antigenically equivalent derivative” as used herein encompasses a polypeptide or its equivalent which will be specifically recognized by certain antibodies which, when raised to the protein or polypeptide according to the invention, interfere with the immediate physical interaction between pathogen and mammalian host.
- the term “immunologically equivalent derivative” as used herein encompasses a peptide or its equivalent which when used in a suitable formulation to raise antibodies in a vertebrate, the antibodies act to interfere with the immediate physical interaction between pathogen and mammalian host.
- the polypeptide such as an antigenically or immunologically equivalent derivative or a fusion protein thereof is used as an antigen to immunize a mouse or other animal such as a rat or chicken.
- the fusion protein may provide stability to the polypeptide.
- the antigen may be associated, for example by conjugation, with an immunogenic carrier protein for example bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).
- BSA bovine serum albumin
- KLH keyhole limpet haemocyanin
- a multiple antigenic peptide comprising multiple copies of the protein or polypeptide, or an antigenically or immunologically equivalent polypeptide thereof may be sufficiently antigenic to improve immunogenicity so as to obviate the use of a ca ⁇ ier.
- a prefe ⁇ ed fusion protein comprises a heterologous region from immunolglobulin that is useful to solubilize or purify polypeptides.
- EP-A-0 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another protein or part thereof.
- proteins have been fused with antibody Fc portions for the purpose of high-throughput screening assays to identify antagonists. See, D. Bennett et al, Journal of Molecular Recognition, Vol. 8 52-58 (1995) and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471 (1995).
- the antibody or variant thereof is modified to make it less immunogenic in the individual.
- the antibody may most preferably be "humanized”; where the complimentarity determining region(s) of the hybridoma-derived antibody has been transplanted into a human monoclonal antibody , for example as described in Jones, P. et al. ( 1986), Nature 321 , 522-525 or Tempest et al., ( 1991) Biotechnology 9, 266-273.
- a polynucleotide of the invention in genetic immunization will preferably employ a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992, 1 :363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNA complexed with specific protein ca ⁇ iers (Wu et al., J Biol Chem. 1989: 264, 16985), coprecipitation of DNA with calcium phosphate (Benvenisty & Reshef, PNAS USA, 1986:83,9551).
- a suitable delivery method such as direct injection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992, 1 :363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNA complexed with specific protein ca ⁇ iers (Wu et al., J Biol Chem. 1989: 264, 16985),
- Polypeptides of the invention may also be used to assess the binding of small molecule substrates and ligands in. for example, cells, cell-free preparations, chemical libraries, and natural product mixtures.
- substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics See, e g , Cohgan et al , Current Protocols in Immunology 1(2) Chapter 5 (1991)
- T e invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of FabD polypeptides or polynucleotides, particularly those compounds that are bacte ⁇ ostatic and/or bacte ⁇ ocidal.
- the method of screening may involve high-throughput techniques
- a synthetic reaction mix for example, to screen for agonists or antagoists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, comp ⁇ sing FabD polypeptide and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be a FabD agonist or antagonist
- the ability of the candidate molecule to agonize or antagonize the FabD polypeptide is reflected in decreased binding of the labeled ligand or decreased production of product from such substrate
- Molecules that bind gratuitously, / e without inducing the effects of FabD polypeptide are most likely to be good antagonists
- Molecules that bind well and increase the rate of product production from substrate are agonists
- Detection of the rate or level of production of product from substrate may be enhanced by using a reporter
- an assay for FabD antagonists is a competitive assay that combines FabD and a potential antagonist with FabD-bmding molecules, recombinant FabD binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under approp ⁇ ate conditions for a competitive inhibition assay FabD can be labeled, such as by radioactivity or a colo ⁇ met ⁇ c compound, such that the number of FabD molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.
- Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide or polypeptide of the invention and thereby inhibit or extinguish its activity
- Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, without inducing FabD-induced activities, thereby preventing the action of FabD by excluding FabD from binding
- Potential antagonists include a small molecule that binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented.
- small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules.
- Other potential antagonists include antisense molecules (see Okano, J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, FL (1988), for a description of these molecules).
- Preferred potential antagonists include compounds related to and variants of FabD.
- Each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds.
- the encoded protein upon expression, can be used as a target for the screening of antibacterial drugs.
- the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct antisense sequences to control the expression of the coding sequence of interest.
- the invention also provides the use of the polypeptide, polynucleotide or inhibitor of the invention to interfere with the initial physical interaction between a pathogen and mammalian host responsible for sequelae of infection.
- the molecules of the invention may be used: in the prevention of adhesion of bacteria, in particular gram positive bacteria, to mammalian extracellular matrix proteins on in-dwelling devices or to extracellular matrix proteins in wounds; to block FabD protein-mediated mammalian cell invasion by, for example, initiating phosphorylation of mammalian tyrosine kinases
- the antagonists and agonists of the invention may be employed, for instance, to inhibit and treat diseases.
- H. pylori Helicobacter pylori bacteria infect the stomachs of over one-third of the world's population causing stomach cancer, ulcers, and gastritis (International Agency for Research on Cancer (1994) Schistosomes, Liver Flukes and Helicobacter Pylori (International Agency for Research on Cancer, Lyon, France; http://www.uicc.ch/ecp/ecp2904.htm). Moreover, the international Agency for Research on Cancer recently recognized a cause-and-effect relationship between H.
- Preferred antimicrobial compounds of the invention agonists and antagonists of FabD found using screens provided by the invention, particularly broad-spectrum antibiotics, should be useful in the treatment of H pylori infection
- Such treatment should decrease the advent of H py/ ⁇ -induced cancers, such as gastrointestinal carcinoma
- Such treatment should also cure gastric ulcers and gast ⁇ tis Vaccines
- Another aspect of the invention relates to a method for inducing an immunological response in an individual, particularly a mammal which comprises inoculating the individual with FabD, or a fragment or variant thereof, adequate to produce antibody and/ or T cell immune response to protect said individual from infection, particularly bacterial infection and most particularly Streptococcus pneumoniae infection Also provided are methods whereby such immunological response slows bacte ⁇ al replication Yet another aspect of the invention relates to a method of inducing immunological response in an individual which comprises delivering to such individual a nucleic acid vector to direct expression of FabD, or a fragment or a variant thereof, for expressing FabD, or a fragment or a va ⁇ ant thereof m vivo in order to induce an immunological response, such as, to produce antibody and/ or T cell immune response, including, for example, cytokine- producing T cells or cytotoxic T cells, to protect said individual from disease, whether that disease is already established within the individual or not
- One way of administering the gene is by accelerating
- a further aspect of the invention relates to an immunological composition which, when introduced into an individual capable or having induced within it an immunological response, induces an immunological response in such individual to a FabD or protein coded therefrom, wherein the composition comprises a recombinant FabD or protein coded therefrom comprising DNA which codes for and expresses an antigen of said FabD or protein coded therefrom
- the immunological response may be used therapeutically or prophylactically and may take the form of antibody immunity or cellular immunity such as that arising from CTL or CD4+ T cells
- a FabD polypeptide or a fragment thereof may be fused with co-protein which may not by itself produce antibodies, but is capable of stabilizing the first protein and producing a fused protein which will have immunogenic and protective properties
- fused recombinant protein preferably further comprises an antigenic co-protein, such as hpoprotein D from Hemophdus influenzae, Glutathione-S-transferase (GST) or beta- galactosidase, relatively large co-proteins which solubihze the protein and facilitate production and purification thereof
- the co-protein may act as an adjuvant in the sense of providing a generalized stimulation of the immune system
- the co-protein may be attached to either the amino or carboxy terminus of the first protein
- compositions particularly vaccine compositions, and methods comprising the polypeptides or polynucleotides of the invention and immunostimulatory DNA sequences, such as those described in Sato. Y et al Science 273. 352 (1996).
- the polypeptide may be used as an antigen for vaccination of a host to produce specific antibodies which protect against invasion of bacteria, for example by blocking adherence of bacteria to damaged tissue
- tissue damage include wounds in skin or connective tissue caused, e g , by mechanical, chemical or thermal damage or by implantation of indwelling devices, or wounds in the mucous membranes, such as the mouth, mammary glands, urethra or vagina.
- the invention also includes a vaccine formulation which comprises an immunogenic recombinant protein of the invention together with a suitable earner Since the protein may be broken down in the stomach, it is preferably administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, or intradermal
- Formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacte ⁇ ostats and solutes which render the formulation insotonic with the bodily fluid, preferably the blood, of the individual, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents
- the formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials and may be stored in a freeze-d ⁇ ed condition requiring only the addition of the sterile liquid ca ⁇ ier immediately prior to use
- the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity
- Polypeptides and other compounds of the invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
- compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intrape ⁇ toneal, intramuscular, subcutaneous, mtranasal or intradermal routes among others.
- the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably lsotonic.
- the composition may be formulated for topical application for example in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwash, impregnated dressings and sutures and aerosols, and may contain appropriate conventional additives, including, for example, preservatives, solvents to assist drug penetration, and emollients in ointments and creams.
- Such topical formulations may also contain compatible conventional ca ⁇ iers, for example cream or ointment bases, and ethanol or oleyl alcohol for lotions.
- Such ca ⁇ iers may constitute from about 1 % to about 98% by weight of the formulation; more usually they will constitute up to about 80% by weight of the formulation.
- the daily dosage level of the active agent will be from 0.01 mg/kg to 10 mg/kg, typically around 1 mg kg.
- the physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual.
- the above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
- In-dwelling devices include surgical implants, prosthetic devices and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time.
- Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.
- CAPD continuous ambulatory peritoneal dialysis
- composition of the invention may be administered by injection to achieve a systemic effect against relevant bacteria shortly before insertion of an in-dwelling device. Treatment may be continued after surgery during the in-body time of the device.
- composition could also be used to broaden perioperative cover for any surgical technique to prevent bacterial wound infections, especially Streptococcus pneumoniae wound infections.
- compositions of this invention may be used generally as a wound treatment agent to prevent adhesion of bacteria to matrix proteins exposed in wound tissue and for prophylactic use in dental treatment as an alternative to, or in conjunction with, antibiotic prophylaxis
- the composition of the invention may be used to bathe an indwelling device immediately before insertion
- the active agent will preferably be present at a concentration of l ⁇ g/ml to lOmg/ml for bathing of wounds or indwelling devices
- a vaccine composition is conveniently in injectable form
- Conventional adjuvants may be employed to enhance the immune response
- a suitable unit dose for vaccination is 0 5-5 microgram/kg of antigen, and such dose is preferably administered 1-3 times and with an interval of 1-3 weeks With the indicated dose range, no adverse toxicological effects will be observed with the compounds of the invention which would preclude their administration to suitable individuals
- D ⁇ sease(s) means and disease caused by or related to infection by a bacte ⁇ a, including otitis media, conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, and most particularly meningitis, such as for example infection ot cerebrospinal fluid.
- “Host cell” is a cell which has been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence
- Identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by compa ⁇ ng the sequences
- identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences "Identity” and "simila ⁇ ty ' can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Biocomputing Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993, Computer Analysis of Sequence Data.
- Prefe ⁇ ed computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 ( 1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. et al.. J. Molec. Biol. 215: 403-410 (1990).
- the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al. J. Mol. Biol. 215: 403-410 ( 1990).
- a polynucleotide having a nucleotide sequence having at least, for example, 95% "identity" to a reference nucleotide sequence of SEQ ID NO: 1 it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence of SEQ ID NO: 1.
- a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
- These mutations of the reference sequence may occur at the 5 or 3 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
- a polypeptide having an amino acid sequence having at least, for example, 95% identity to a reference amino acid sequence of SEQ ID NO:2 is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of SEQ ID NO: 2.
- up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
- These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
- Isolated means altered “by the hand of man” from its natural state, i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
- a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated”, as the term is employed herein.
- Polynucleotide(s) generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
- Polynucleotide(s) include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double- stranded regions.
- polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
- the strands in such regions may be from the same molecule or from different molecules.
- the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
- One of the molecules of a triple-helical region often is an oligonucleotide.
- the term "polynucleotide(s)” also includes DNAs or RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotide(s)" as that term is intended herein.
- DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
- polynucleotide(s) as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides.
- Polynucleot ⁇ de(s) also embraces short polynucleotides often refe ⁇ ed to as ohgonucleot ⁇ de(s).
- Polypept ⁇ de(s) refers to any peptide or protein comp ⁇ sing two or more amino acids joined to each other by peptide bonds or modified peptide bonds
- Polypept ⁇ de(s) refers to both short chains, commonly refe ⁇ ed to as peptides, ohgopeptides and oligomers and to longer chains generally refe ⁇ ed to as proteins
- Polypeptides may contain amino acids other than the 20 gene encoded amino acids
- Polypept ⁇ de(s)” include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques Such modifications are well desc ⁇ bed in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide Also, a given polypeptide may contain many types of modifications Modifications can occur anywhere in a polypeptide, including
- Polypeptides may be branched or cyclic, with or without branching Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
- Variant(s) is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains essential properties.
- a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
- a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
- a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
- a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
- a variant of a polynucleotide or polypeptide may be a naturally occu ⁇ ing such as an allelic variant, or it may be a variant that is not known to occur naturally.
- Non-naturally occu ⁇ ing variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
- Example 1 Strain selection, Library Production and Sequencing The polynucleotide having a DNA sequence given in Table 1 [SEQ ID NO: 1 or 3 or 5] was obtained from a library of clones of chromosomal DNA of Streptococcus pneumoniae in E. coli. The sequencing data from two or more clones containing overlapping Streptococcus pneumoniae DNAs was used to construct the contiguous DNA sequence in SEQ ID NO: 1. Libraries may be prepared by routine methods, for example: Methods 1 and 2 below.
- Total cellular DNA is mechanically sheared by passage through a needle in order to size-fractionate according to standard procedures.
- DNA fragments of up to 1 l bp in size are rendered blunt by treatment with exonuclease and DNA polymerase, and EcoRI linkers added. Fragments are ligated into the vector Lambda ZapII that has been cut with EcoRI, the library packaged by standard procedures and E.coli infected with the packaged library.
- the library is amplified by standard procedures.
- Total cellular DNA is partially hydrolyzed with a one or a combination of restriction enzymes appropriate to generate a series of fragments for cloning into library vectors (e.g., Rsal, Pall, Alul, Bshl235I), and such fragments are size-fractionated according to standard procedures.
- EcoRI linkers are ligated to the DNA and the fragments then ligated into the vector Lambda ZapII that have been cut with EcoRI, the library packaged by standard procedures, and E.coli infected with the packaged library.
- the library is amplified by standard procedures.
- FabD malonyl-CoA.ACP transacylase activity can be readily assayed via the incorporation of ⁇ C -malonate from trichloroacetic acid soluble ' ⁇ C-malonyl-CoA into TCA insoluble ⁇ C-malonyl-ACP, using ACP as acceptor. Test compounds may be added to this assay to determine whether they agonise or antagonise enzyme activity.
- ACACCTGCAC AAATCGTCAT TGCTGGAGAA GTGGTTGCAG TTGATCGAGC GGTTGAACTT 540
- GACAAACTCA ATCAGACCCG CTATACGCAA CCAGCCATTC TAGCGACTTC GGTTGCTATC 300
- AGGCATAAGC AACTTATTCG AGATTGGACC GGGGAAAGTC TTGTCAGGTT TTGTTAAAAA 480
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Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US3116096P | 1996-11-18 | 1996-11-18 | |
US31160P | 1996-11-18 | ||
PCT/US1997/020992 WO1998022133A1 (en) | 1996-11-18 | 1997-11-14 | NOVEL FabD |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0959903A1 true EP0959903A1 (en) | 1999-12-01 |
EP0959903A4 EP0959903A4 (en) | 2002-04-24 |
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ID=21857935
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP97953035A Withdrawn EP0959903A4 (en) | 1996-11-18 | 1997-11-14 | NOVEL FabD |
Country Status (3)
Country | Link |
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EP (1) | EP0959903A4 (en) |
JP (1) | JP2001505421A (en) |
WO (1) | WO1998022133A1 (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0843016A2 (en) * | 1996-11-13 | 1998-05-20 | Smithkline Beecham Corporation | Staphylococcus FabD (malonylCoA:ACP transacylase) protein |
-
1997
- 1997-11-14 JP JP52379398A patent/JP2001505421A/en active Pending
- 1997-11-14 EP EP97953035A patent/EP0959903A4/en not_active Withdrawn
- 1997-11-14 WO PCT/US1997/020992 patent/WO1998022133A1/en not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0843016A2 (en) * | 1996-11-13 | 1998-05-20 | Smithkline Beecham Corporation | Staphylococcus FabD (malonylCoA:ACP transacylase) protein |
Non-Patent Citations (8)
Title |
---|
BOUQUIN N ET AL: "Resistance to trifluoroperazine, a calmodulin inhibitor, maps to the fabD locus of Escherichia coli" MOL. GEN. GENET, XX, XX, vol. 246, no. 5, 10 March 1995 (1995-03-10), pages 628-637, XP002119516 * |
DATABASE SWISS-PROT [Online] 1992 MAGNUSON ET AL.: "MALONYL COA-ACYL CARRIER PROTEIN TRANSACYLASE (EC 2.3.1.39) (MCT)" retrieved from EBI Database accession no. P25715 XP002191152 * |
DATABASE SWISS-PROT [Online] 1995 FLEISCHMANN ET AL.: "Malonyl CoA-Acyl carrier protein transacylase (EC 2.3.1.39) MCT" retrieved from EBI Database accession no. P43712 XP002191153 * |
DATABASE SWISS-PROT [Online] 25 August 1996 (1996-08-25) CRONAN ET AL.: "Bacillus subtiulis PlsX, Malonyl-CoA: Acyl carrier protein transacylase (fabD) and 3-ketoacyl-acyl carrier protein reductase (fabG) genes, complete cds, and acyl carrier protein (acpP) gene, partial cds." retrieved from EBI Database accession no. U59433 XP002191154 * |
GARWIN J L ET AL: "STRUCTURAL, ENZYMATIC, AND GENETIC STUDIES OF BETA-KETOACYL-ACYL CARRIER PROTEIN SYNTHASES I AN DII OF ESCHERICHIA COLI" JOURNAL OF BIOLOGICAL CHEMISTRY, THE AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, INC.,, US, vol. 255, no. 24, 1980, pages 11949-11956, XP002911268 ISSN: 0021-9258 * |
MAGNUSON K ET AL: "Cloning and nucleotide sequence of the fabD gene encoding malonyl coenzyme A-acyl carrier protein transacylase from Escherichia coli" FEBS LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 299, no. 3, March 1992 (1992-03), pages 262-266, XP002016568 ISSN: 0014-5793 * |
MORBIDONI H R ET AL: "Bacillus subtilis acyl carrier protein is encoded in a cluster of lipid biosynthesis genes" JOURNAL OF BACTERIOLOGY, WASHINGTON, DC, US, vol. 178, no. 16, August 1996 (1996-08), pages 4794-4800, XP002119514 ISSN: 0021-9193 * |
See also references of WO9822133A1 * |
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JP2001505421A (en) | 2001-04-24 |
WO1998022133A1 (en) | 1998-05-28 |
EP0959903A4 (en) | 2002-04-24 |
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