WO2002008393A2 - Serine protease from yersinia enterocolitica - Google Patents
Serine protease from yersinia enterocolitica Download PDFInfo
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- WO2002008393A2 WO2002008393A2 PCT/GB2001/003357 GB0103357W WO0208393A2 WO 2002008393 A2 WO2002008393 A2 WO 2002008393A2 GB 0103357 W GB0103357 W GB 0103357W WO 0208393 A2 WO0208393 A2 WO 0208393A2
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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
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
Definitions
- This invention relates to a novel protein, termed CAB46586.1, herein identified as a serine protease and to the use of this protein, fragments of this protein and nucleic acid sequence from the encoding gene in the diagnosis, prevention and treatment of disease. All publications, patents and patent applications cited herein are incorporated in full by reference.
- bioinformatics tools increase in potency and in accuracy, these tools are rapidly replacing the conventional techniques of biochemical characterisation. Indeed, the advanced bioinformatics tools used in identifying the present invention are now capable of outputting results in which a high degree of confidence can be placed.
- This tool is a database system, termed the BiopendiumTM search database, that is the subject of co-pending International Patent Application No. PCT/GBOl/01105.
- This database system consists of an integrated data resource created using proprietary technology and containing information generated from an all-by-all comparison of all available protein or nucleic acid sequences.
- sequence data from separate data resources is to combine as much data as possible, relating both to the sequences themselves and to information relevant to each sequence, into one integrated resource. All the available data relating to each sequence, including data on the three-dimensional structure of the encoded protein, if this is available, are integrated together to make best use of the information that is known about each sequence and thus to allow the most educated predictions to be made from comparisons of these sequences.
- the annotation that is generated in the database and which accompanies each sequence entry imparts a biologically-relevant context to the sequence information.
- Proteases are enzymes that irreversibly hydrolyse amide bonds in peptides and proteins. Proteases are widely distributed and are involved in many different biological processes, from activation of proteins and peptides to degradation of proteins. Despite the fact that proteases have been shown to be involved in many different diseases, drugs targeted to proteases are still rare in pharmacy, although inhibitors of angiotensin converting enzyme have been among the most successful antihypertensive drugs for several years. Proteases have recently received substantial publicity as valuable therapeutic targets following the approval of HIV protease inhibitors. Proteases can be divided in large Families.
- proteases is used to describe a group of proteases in which each member shows an evolutionary relationship to at least one other member, either throughout the whole sequence or at least in the part of the sequence responsible for catalytic activity.
- the name of each Family reflects the catalytic activity type of the proteases in the Family.
- serine proteases belong to the S family
- threonine proteases belong to the T family
- aspartyl proteases belong to the A family
- cysteine proteases belong to the C family
- metalloproteases belong to the M family.
- Certain proteases have an unknown mechanism of action and belong to the "U" family.
- the serine proteases are grouped into 7 clans, containing 39 families and over 750 different members from various species. Of these 39 families, only 9 contain members that are expressed in animals. Families with particularly interesting members include the SI
- the S26 Family includes bacterial leader peptidase I, which has been implicated as an important agent in bacterial infection (Paetzel, M., et al, Proteins (1995), (l):122-5; Tschantz, W.R., Methods Enzymol. (1994), 244:285-301).
- the SI Family is the largest of these 9 families and the family with by far the greatest number of members of significance to health and disease.
- This family includes clotting factors such as factors VII, IX, X, XI and XII, proteases of the complement system, plasmin, trypsin, UP A, elastases such as leukocyte elastase, chymase, thrombin, cathepsin G, plasminogen, tryptase, urokinase and many others.
- the prototypic protease of this family is chymotrypsin.
- the catalytic activity of the SI family of proteases is provided by a charge relay system involving an aspartic acid residue that is hydrogen-bonded to a histidine, which itself is hydrogen-bonded to a serine.
- the sequences in the vicinity of the active site serine and histidine residues are well conserved in this family of proteases.
- Serine proteases have been shown to play a role in diverse physiological functions, many of which can play a role in disease processes (see Table 1) such as cardiovascular disease (Kohler, H.T., et al, (2000) N. Engl. J. Med. 342(24): 1792-801; Hamsten, A., et al, (2000) Thromb. Haemost. 83(3):397-403; Califf, R.M., et al, (2000) Circulation, 101(19):2231-8; Krendel, S., et al, (2000) Ann. Emerg. Med. 35(5):502-5), cancer (Schmidt, M., et al, (1999) Acta Otolaryngol.
- a further need in the art is to treat and prevent the incidence of diseases that are caused by bacterial pathogens.
- the pathogen Yersinia enterocolitica is ubiquitous, being isolated frequently from soil, water, animals, and a variety of foods. This pathogen make up a biochemically heterogeneous group that can grow at refrigeration temperatures. Based on their biochemical heterogeneity and DNA relatedness, members of the Yersinia genus are separated into a number of species, of which three are potentially pathogenic to humans: 7. pestis, Y. pseudotuberculosis, and 7 enter ocolitica. Of these, 7. enterocolitica is most important as a cause of foodborne illness.
- a therapeutic agent specific for this organism would be of great value in the diagnosis and treatment of disease caused by this organism.
- the invention is based on the discovery that a protein whose sequence is recorded in a publicly-available database as CAB46586.1 (NCBI Genbank nucleotide accession number AJ132945 and a Genbank protein accession number CAB46586.1) functions as a serine protease.
- CAB46586.1 NCBI Genbank nucleotide accession number AJ132945 and a Genbank protein accession number CAB46586.1
- Proteases are enzymes that irreversibly hydrolyse amide bonds in peptides and proteins.
- Serine proteases have an active site catalytic serine residue, although this could conceivably be substituted conservatively by a threonine residue.
- a molecule is described herein as possessing activity as a serine protease, this is intended to mean that the described molecules are active to hydrolyse amide bonds irreversibly in peptides and proteins using an active site catalytic serine or threonine residue.
- the invention provides a polypeptide, which polypeptide:
- (i) has the amino acid sequence as recited in SEQ ID NO:2; (ii) is a fragment thereof having serine protease activity or having an antigenic determinant in common with the polypeptide of (i); or
- the polypeptide having the sequence recited in SEQ ID NO:2 is referred to hereafter as "the SP2 polypeptide”.
- a preferred polypeptide fragment according to part ii) above includes the region of the protein predicted as that responsible for serine protease activity (hereafter, the "serine protease region"), and variants thereof that possess the catalytic residues His64, AsplOl and Serl78, or equivalent residues, and which possesses activity as a serine protease.
- the serine protease region of the SP2 polypeptide is considered to extend between, at the most, residue 44 and residue 212, and at the least, residue 54 and residue 197 of the SP2 polypeptide sequence.
- This aspect of the invention also includes fusion proteins that incorporate polypeptide fragments and variants of these polypeptide fragments as defined above, provided that said fusion proteins possess activity as a serine protease.
- the invention provides a purified nucleic acid molecule which encodes a polypeptide according to the first aspect of the invention.
- the purified nucleic acid molecule has the nucleic acid sequence as recited in SEQ ID NO:l (encoding the SP2 polypeptide) or is a redundant equivalent or fragment of this sequence.
- a preferred nucleic acid fragment is one that encodes a polypeptide fragment according to part ii) above, preferably a polypeptide fragment that includes the serine protease region of the SP2 polypeptide, or that encodes a variant of this fragment as this term is defined above.
- the invention provides a purified nucleic acid molecule which hydridizes under high stringency conditions with a nucleic acid molecule of the second aspect of the invention.
- the invention provides a vector, such as an expression vector, that contains a nucleic acid molecule of the second or third aspect of the invention.
- the invention provides a host cell transformed with a vector of the fourth aspect of the invention.
- the invention provides a ligand which binds specifically to, and which preferably inhibits the serine protease activity of, a polypeptide of the first aspect of the invention.
- the invention provides a compound that is effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
- a compound of the seventh aspect of the invention may either increase (agonise) or decrease (antagonise) the level of expression of the gene or the activity of the polypeptide.
- the identification of the function of the region defined herein as the serine protease region of the SP2 polypeptide, as possessing serine protease activity allows for the design of screening methods that are capable of identifying compounds that are effective in the treatment and/or diagnosis of diseases in which serine proteases, and in particular, the organism Yersinia enter ocolitica, are implicated.
- the invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a ligand of the fifth aspect of the invention, or a compound of the sixth aspect of the invention, for use in therapy or diagnosis.
- These molecules may also be used in the manufacture of a medicament for the treatment of a bacterial infection, particularly a bacterial infection caused by the organism Yersinia enterocolitica.
- the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide of the first aspect of the invention or the activity of a polypeptide of the first aspect of the invention in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of disease.
- a method will preferably be carried out in vitro.
- Similar methods may be used for monitoring the therapeutic treatment of disease in a patient, wherein altering the level of expression or activity of a polypeptide or nucleic acid molecule over the period of time towards a control level is indicative of regression of disease.
- serine protease of the first aspect of the invention is implicated are bacterial infections.
- the serine protease whose sequence is presented in SEQ ID NO:2 is implicated herein in the pathogenicity of the organism Yersinia enterocolitica.
- ligands to this polypeptide, and in particular, to the serine protease region of the SP2 polypeptide as this is defined herein are likely to be effective in controlling diseases that are caused by this organism.
- this polypeptide, and in particular, a polypeptide fragment including the serine protease region of the SP2 polypeptide sequence provides a potential component for a vaccine against this organism and the diseases that it causes.
- a preferred method for detecting polypeptides of the first aspect of the invention comprises the steps of: (a) contacting a ligand, such as an antibody, of the sixth aspect of the invention with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.
- a number of different such methods according to the ninth aspect of the invention exist, as the skilled reader will be aware, such as methods of nucleic acid hybridization with short probes, point mutation analysis, polymerase chain reaction (PCR) amplification and methods using antibodies to detect aberrant protein levels. Similar methods may be used on a short or long term basis to allow therapeutic treatment of a disease to be monitored in a patient.
- the invention also provides kits that are useful in these methods for diagnosing disease.
- the invention provides for the use of a polypeptide of the first aspect of the invention as a serine protease.
- the invention also provides for the use of a nucleic acid molecule according to the second or third aspect of the invention to express a protein that possesses serine protease activity.
- the invention also provides a method for cleaving a polypeptide comprising contacting said polypeptide with a polypeptide of the first aspect of the invention.
- the invention provides a pharmaceutical composition comprising a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, in conjunction with a pharmaceutically-acceptable carrier.
- the present invention provides a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention, for use in the manufacture of a medicament for the diagnosis or treatment of a disease, such as a bacterial infection.
- the invention provides a method of treating a disease in a patient comprising administering to the patient a polypeptide of the first aspect of the invention, or a nucleic acid molecule of the second or third aspect of the invention, or a vector of the fourth aspect of the invention, or a ligand of the sixth aspect of the invention, or a compound of the seventh aspect of the invention.
- the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an agonist.
- the polypeptide, nucleic acid molecule, ligand or compound administered to the patient should be an antagonist.
- antagonists include antisense nucleic acid molecules, ribozymes and ligands, such as antibodies.
- the invention provides transgenic or knockout non-human animals that have been transformed to express higher, lower or absent levels of a polypeptide of the first aspect of the invention.
- Such transgenic animals are very useful models for the study of disease and may also be using in screening regimes for the identification of compounds that are effective in the treatment or diagnosis of such diseases.
- polypeptide includes any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e. peptide isosteres. This term refers both to short chains (peptides and oligopeptides) and to longer chains (proteins).
- the polypeptide of the present invention may be in the form of a mature protein or may be a pre-, pro- or prepro- protein that can be activated by cleavage of the pre-, pro- or prepro- portion to produce an active mature polypeptide.
- the pre-, pro- or prepro- sequence may be a leader or secretory sequence or may be a sequence that is employed for purification of the mature polypeptide sequence.
- polypeptide of the first aspect of the invention may form part of a fusion protein.
- additional amino acid sequences which may contain secretory or leader sequences, pro-sequences, sequences which aid in purification, or sequences that confer higher protein stability, for example during recombinant production.
- the mature polypeptide may be fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol).
- Polypeptides may contain amino acids other than the 20 gene-encoded amino acids, modified either by natural processes, such as by post-translational processing or by chemical modification techniques which are well known in the art.
- glycosylation lipid attachment, sulphation, gamma-carboxylation, for instance of glutamic acid residues, hydroxylation and ADP-ribosylation.
- Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
- blockage of the amino or carboxyl terminus in a polypeptide, or both, by a covalent modification is common in naturally-occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention.
- the modifications that occur in a polypeptide often will be a function of how the polypeptide is made.
- the nature and extent of the modifications in large part will be determined by the post-translational modification capacity of the particular host cell and the modification signals that are present in the amino acid sequence of the polypeptide in question. For instance, glycosylation patterns vary between different types of host cell.
- polypeptides of the present invention can be prepared in any suitable manner.
- Such polypeptides include isolated naturally-occurring polypeptides (for example purified from cell culture), recombinantly-produced polypeptides (including fusion proteins), synthetically-produced polypeptides or polypeptides that are produced by a combination of these methods.
- the functionally-equivalent polypeptides of the first aspect of the invention may be polypeptides that are homologous to the SP2 polypeptide.
- Two polypeptides are said to be "homologous", as the term is used herein, if the sequence of one of the polypeptides has a high enough degree of identity or similarity to the sequence of the other polypeptide. "Identity” indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. "Similarity” indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences.
- Homologous polypeptides therefore include natural biological variants (for example, allelic variants or geographical variations within the species from which the polypeptides are derived) and mutants (such as mutants containing amino acid substitutions, insertions or deletions) of the SP2 polypeptide or of a fragment of the SP2 polypeptide.
- mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code.
- Such substitutions are among Ala, Val, Leu and lie; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr.
- Particularly preferred are variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or added in any combination.
- silent substitutions, additions and deletions which do not alter the properties and activities of the protein. Also especially preferred in this regard are conservative substitutions.
- Such mutants also include polypeptides in which one or more of the amino acid residues includes a substituent group.
- polypeptides of the first aspect of the invention have a degree of sequence identity with the SP2 polypeptide, or with active fragments thereof, of greater than 30%. More preferred polypeptides have degrees of identity of greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectively with the SP2 polypeptide, or with active fragments thereof.
- preferred active fragments of the SP2 polypeptide are those that include the serine protease region of the SP2 polypeptide and which possess the residues His64, AsplOl and Serl78, or equivalent residues.
- residues that are equivalent to the residues that form the catalytic triad of the serine protease region may replace one or more of the three catalytic residues, provided that the serine protease region retains activity as a serine protease.
- the Histidine residue may be replaced by Lysine or Arginine.
- the Aspartate residue may be replaced by Glutamate.
- the Serine residue may be replaced by Threonine.
- this aspect of the invention includes polypeptides that have degrees of identity of greater than 30%, preferably, greater than 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectively, with the serine protease region of the SP2 polypeptide and which possess His64, AsplOl and Serl78, or equivalent residues.
- the serine protease region of the SP2 polypeptide is considered to extend between, at the most, residue 44 and residue 212, and at the least, residue 54 and residue 197 of the SP2 polypeptide sequence.
- the functionally-equivalent polypeptides of the first aspect of the invention may also be polypeptides which have been identified using one or more techniques of structural alignment.
- the Inpharmatica Genome ThreaderTM technology that forms one aspect of the search tools used to generate the BiopendiumTM search database may be used (see co-pending International patent application PCT/GBOl/01105) to identify polypeptides of presently-unknown function which, while having low sequence identity as compared to the SP2 polypeptide, or more particularly, with the serine protease region of the SP2 polypeptide, possess His64, AsplOl and Ser 178, or equivalent residues and are predicted to have serine protease activity, by virtue of sharing significant structural homology with these polypeptide sequences and possessing the catalytic triad.
- the Inpharmatica Genome ThreaderTM predicts two proteins to share structural homology with a certainty of at least 10% more preferably, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and above.
- the certainty value of the Inpharmatica Genome ThreaderTM is calculated as follows. A set of comparisons was initially performed using the Inpharmatica Genome ThreaderTM exclusively using sequences of known structure. Some of the comparisons were between proteins that were known to be related (on the basis of structure). A neural network was then trained on the basis that it needed to best distinguish between the known relationships and known not-relationships taken from the CATH structure classification (www.biochem.ucl.ac.uk/bsm/cath).
- polypeptides of the first aspect of the invention include fragments of the SP2 polypeptide, functional equivalents of the fragments of the SP2 polypeptide, and fragments of the functional equivalents of the SP2 polypeptide, provided that those functional equivalents and fragments retain serine protease activity or have an antigenic determinant in common with the SP2 polypeptide.
- fragment refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of the amino acid sequence of the SP2 polypeptide or one of its functional equivalents.
- the fragments should comprise at least n consecutive amino acids from the sequence and, depending on the particular sequence, n preferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more). Small fragments may form an antigenic determinant.
- Preferred polypeptide fragments according to this aspect of the invention are fragments that include the region defined herein as the serine protease region of the SP2 polypeptide.
- This region of the SP2 polypeptide defines the region of the protein that has been annotated as a serine protease.
- This region of the SP2 polypeptide is considered to extend between, at the most, residue 44 and residue 212, and at the least, residue 54 and residui 197.
- Variants of this fragment are included as embodiments of this aspect of the invention provided that these variants possess activity as a serine protease.
- the term "variant” is meant to include extended or truncated versions of this polypeptide fragment.
- the serine protease region of the SP2 polypeptide will fold correctly and show serine protease activity if additional residues C terminal and/or N terminal of these boundaries in the SP2 polypeptide sequence are included in the polypeptide fragment.
- additional residues C terminal and/or N terminal of these boundaries in the SP2 polypeptide sequence are included in the polypeptide fragment.
- an additional 5, 10, 20, 30, 40 or ever 50 or more amino acid residues from the SP2 polypeptide sequence, or a homologous sequence may be included at either or both the C terminal and/or N terminal of the boundaries of the serine protease region of the SP2 polypeptide without prejudicing the ability of the polypeptide fragment to fold correctly and to exhibit serine protease activity.
- one or more amino acid residues may be deleted at either or both the C terminus or the N terminus of the serine protease region of the SP2 polypeptide, although the catalytic triad (His64, AsplOl and Serl78 or equivalent residues) that forms the active site of the polypeptide molecule should be maintained intact; deletions should not extend so far into the polypeptide sequence that any of these residues are deleted.
- the term "variant" includes homologues of the polypeptide fragments described above, that possess significant sequence homology with the serine protease region of the SP2 polypeptide and which possess the catalytic residues His64, AsplOl and Serl78, or equivalent residues, provided that said variants retain activity as a serine protease.
- Homologues include those polypeptide molecules that possess greater than 30% identity with the serine protease region of the SP2 polypeptide.
- variant homologues of polypeptide fragments of this aspect of the invention have a degree of sequence identity with the serine protease region of the SP2 polypeptide of greater than 40%. More preferred variant polypeptides have degrees of identity of greater than 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectively with the serine protease region of the SP2 polypeptide, provided that said variants retain activity as a serine protease.
- Variant polypeptides also include homologues of the truncated forms of the polypeptide fragments discussed above, provided that said variants retain activity as a serine protease.
- the polypeptide fragments of the first aspect of the invention may be polypeptide fragments that exhibit significant structural homology with the structure of the polypeptide fragment defined by the serine protease region of the SP2 polypeptide sequence, for example, as identified by the Inpharmatica Genome ThreaderTM. Accordingly, polypeptide fragments that are structural homologues of the polypeptide fragment defined by the serine protease region of the SP2 polypeptide sequence should adopt the same fold as that adopted by this polypeptide fragment, as this fold is defined above.
- Structural homologues of the polypeptide fragment defined by the serine protease region of the SP2 polypeptide sequence should also retain the catalytic triad (His64, AsplOl and Ser 178, or equivalent residues) that forms the active site.
- fragments may be "free-standing", i.e. not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide of which they form a part or region (a "fusion" polypeptide).
- the fragment of the invention When comprised within a larger polypeptide, the fragment of the invention most preferably forms a single continuous region.
- certain preferred embodiments relate to a fragment having a pre - and/or pro- polypeptide region fused to the amino terminus of the fragment and/or an additional region fused to the carboxyl terminus of the fragment.
- several fragments may be comprised within a single larger polypeptide.
- polypeptides of the present invention or their immunogenic fragments can be used to generate ligands, such as polyclonal or monoclonal antibodies, that are immunospecific for the polypeptides.
- ligands such as polyclonal or monoclonal antibodies
- Such antibodies may be employed to isolate or to identify clones expressing the polypeptides of the invention or to purify the polypeptides by affinity chromatography.
- the antibodies may also be employed as diagnostic or therapeutic aids, amongst other applications, as will be apparent to the skilled reader.
- immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
- antibody refers to intact molecules as well as to fragments thereof, such as Fab, F(ab') 2 and Fv, which are capable of binding to the antigenic determinant in question. Such antibodies thus bind to the polypeptides of the first aspect of the invention. If polyclonal antibodies are desired, a selected mammal, such as a mouse, rabbit, goat or horse, may be immunised with a polypeptide of the first aspect of the invention.
- the polypeptide used to immunise the animal can be derived by recombinant DNA technology or can be synthesized chemically. If desired, the polypeptide can be conjugated to a carrier protein. Commonly used carriers to which the polypeptides may be chemically coupled include bovine serum albumin, thyroglobulin and keyhole limpet haemocyanin.
- the coupled polypeptide is then used to immunise the animal. Serum from the immunised animal is collected and treated according to known procedures, for example by immunoaffinity chromatography. Monoclonal antibodies to the polypeptides of the first aspect of the invention can also be readily produced by one skilled in the art.
- Panels of monoclonal antibodies produced against the polypeptides of the first aspect of the invention can be screened for various properties, i.e., for isotype, epitope, affinity, etc. Monoclonal antibodies are particularly useful in purification of the individual polypeptides against which they are directed. Alternatively, genes encoding the monoclonal antibodies of interest may be isolated from hybridomas, for instance by PCR techniques known in the art, and cloned and expressed in appropriate vectors.
- Chimeric antibodies in which non-human variable regions are joined or fused to human constant regions (see, for example, Liu et al, Proc. Natl. Acad. Sci. USA, 84, 3439 (1987)), may also be of use.
- the antibody may be modified to make it less immunogenic in an individual, for example by humanisation (see Jones et al, Nature, 321, 522 (1986); Verhoeyen et al, Science, 239, 1534 (1988); Kabat et al, J. Immunol, 147, 1709 (1991); Queen et al, Proc. Natl Acad. Sci. USA, 86, 10029 (1989); Gorman et al, Proc. Natl Acad. Sci.
- humanised antibody refers to antibody molecules in which the CDR amino acids and selected other amino acids in the variable domains of the heavy and/or light chains of a non-human donor antibody have been substituted in place of the equivalent amino acids in a human antibody.
- the humanised antibody thus closely resembles a human antibody but has the binding ability of the donor antibody.
- the antibody may be a "bispecific" antibody, that is an antibody having two different antigen binding domains, each domain being directed against a different epitope.
- Phage display technology may be utilised to select genes which encode antibodies with binding activities towards the polypeptides of the invention either from repertoires of PCR amplified V-genes of lymphocytes from humans screened for possessing the relevant antibodies, 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).
- Antibodies generated by the above techniques have additional utility in that they may be employed as reagents in immunoassays, radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).
- the antibodies can be labelled with an analytically-detectable reagent such as a radioisotope, a fluorescent molecule or an enzyme.
- Preferred nucleic acid molecules of the second and third aspects of the invention are those which encode the polypeptide sequence recited in SEQ ID NO:2, and functionally equivalent polypeptides, including active fragments of the SP2 polypeptide, such as a fragment including the serine protease region of the SP2 polypeptide sequence, or a homologue thereof. These nucleic acid molecules may be used in the methods and applications described herein. Nucleic acid molecules encompassing this stretch of sequence form a preferred embodiment of this aspect of the invention.
- nucleic acid molecules of the invention preferably comprise at least n consecutive nucleotides from the sequences disclosed herein where, depending on the particular sequence, n is 10 or more (for example, 12, 14, 15, 18, 20, 25, 30, 35, 40 or more).
- nucleic acid molecules of the invention also include sequences that are complementary to nucleic acid molecules described above (for example, for antisense or probing purposes).
- Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance cDNA, synthetic DNA or genomic DNA. Such nucleic acid molecules may be obtained by cloning, by chemical synthetic techniques or by a combination thereof.
- the nucleic acid molecules can be prepared, for example, by chemical synthesis using techniques such as solid phase phosphoramidite chemical synthesis, from genomic or cDNA libraries or by separation from an organism. RNA molecules may generally be generated by the in vitro or in vivo transcription of DNA sequences.
- the nucleic acid molecules may be double-stranded or single-stranded.
- Single-stranded DNA may be the coding strand, also known as the sense strand, or it may be the non- coding strand, also referred to as the anti-sense strand.
- the term "nucleic acid molecule” also includes analogues of DNA and RNA, such as those containing modified backbones, and peptide nucleic acids (PNA).
- PNA peptide nucleic acids
- PNA refers to an antisense molecule or an anti-gene agent which comprises an oligonucleotide of at least five nucleotides in length linked to a peptide backbone of amino acid residues, which preferably ends in lysine.
- PNAs may be pegylated to extend their lifespan in a cell, where they preferentially bind complementary single stranded DNA and RNA and stop transcript elongation (Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63).
- a nucleic acid molecule which encodes the polypeptide of SEQ ID NO:2, or an active fragment thereof may be identical to the coding sequence of the nucleic acid molecule shown in SEQ ID NO:l. These molecules also may have a different sequence which, as a result of the degeneracy of the genetic code, encodes the polypeptide of SEQ ID NO:2, or an active fragment of the SP2 polypeptide, such as a fragment including the serine protease region of the SP2 polypeptide sequence, or a homologue thereof.
- the serine protease region of the SP2 polypeptide is considered to extend between, at the most, residue 44 and residue 212, and at the least, residue 54 and residue 197 of the SP2 polypeptide sequence.
- the serine protease region is encoded by, at the most, a nucleic acid molecule including nucleotide 130 to nucleotide 636 and, at the least, by a nucleic acid molecule including nucleotide 160 to nucleotide 591. Nucleic acid molecules encompassing this stretch of sequence, and homologues of this sequence, form a preferred embodiment of this aspect of the invention.
- nucleic acid molecules that encode the polypeptide of SEQ ID NO:2 or active fragments thereof may include, but are not limited to, the coding sequence for the mature polypeptide or polypeptide fragment by itself; the coding sequence for the mature polypeptide or polypeptide fragment and additional coding sequences, such as those encoding a leader or secretory sequence, such as a pro-, pre- or prepro- polypeptide sequence; the coding sequence of the mature polypeptide or polypeptide fragment, with or without the aforementioned additional coding sequences, together with further additional, non-coding sequences, including non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription (including termination signals), ribosome binding and mRNA stability.
- the nucleic acid molecules may also include additional sequences which encode additional amino acids, such as those which provide additional functionalities.
- nucleic acid molecules of the second and third aspects of the invention may encode the fragments or the functional equivalents of the polypeptides and fragments of the first aspect of the invention.
- a preferred fragment of the SP2 polypeptide is a fragment including the serine protease region of the SP2 polypeptide sequence, or a homologue thereof.
- the serine protease region is encoded by, at the most, a nucleic acid molecule including nucleotides 130 to 636 of SEQ ID NO:l and, at the least, by a nucleic acid molecule including nucleotides 160 to 591 of SEQ ID NO: 1
- Functionally equivalent nucleic acid molecules according to the invention may be naturally-occurring variants such as a naturally-occurring allelic variant, or the molecule may be a variant that is not known to occur naturally.
- Such non-naturally occurring variants of the nucleic acid molecule may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.
- variants in this regard are variants that differ from the aforementioned nucleic acid molecules by nucleotide substitutions, deletions or insertions.
- the substitutions, deletions or insertions may involve one or more nucleotides.
- the variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or insertions.
- the nucleic acid molecules of the invention can also be engineered, using methods generally known in the art, for a variety of reasons, including modifying the cloning, processing, and/or expression of the gene product (the polypeptide).
- DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides are included as techniques which may be used to engineer the nucleotide sequences.
- Site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations and so forth.
- Nucleic acid molecules which encode a polypeptide of the first aspect of the invention may be ligated to a heterologous sequence so that the combined nucleic acid molecule encodes a fusion protein.
- Such combined nucleic acid molecules are included within the second or third aspects of the invention.
- a fusion protein that can be recognised by a commercially-available antibody.
- a fusion protein may also be engineered to contain a cleavage site located between the sequence of the polypeptide of the invention and the sequence of a heterologous protein so that the polypeptide may be cleaved and purified away from the heterologous protein.
- the nucleic acid molecules of the invention also include antisense molecules that are partially complementary to nucleic acid molecules encoding polypeptides of the present invention and that therefore hybridize to the encoding nucleic acid molecules (hybridization).
- antisense molecules such as oligonucleotides, can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide of the invention, as will be known by those of ordinary skill in the art (see, for example, Cohen, J.S., Trends in Pharm. Sci., 10, 435 (1989), Okano, J. Neurochem. 56, 560 (1991); O'Connor, J. Neurochem 56, 560 (1991); Lee et al, Nucleic Acids Res 6, 3073 (1979); Cooney et al, Science 241, 456 (1988); Dervan et al, Science 251, 1360 (1991).
- hybridization refers to the association of two nucleic acid molecules with one another by hydrogen bonding. Typically, one molecule will be fixed to a solid support and the other will be free in solution. Then, the two molecules may be placed in contact with one another under conditions that favour hydrogen bonding. Factors that affect this bonding include: the type and volume of solvent; reaction temperature; time of hybridization; agitation; agents to block the non-specific attachment of the liquid phase molecule to the solid support (Denhardt's reagent or BLOTTO); the concentration of the molecules; use of compounds to increase the rate of association of molecules (dextran sulphate or polyethylene glycol); and the stringency of the washing conditions following hybridization (see Sambrook et al.
- Stringency refers to conditions in a hybridization reaction that favour the association of very similar molecules over association of molecules that differ.
- High stringency hybridisation conditions are defined as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C.
- Low stringency conditions involve the hybridisation reaction being carried out at 35°C (see Sambrook et al [supra]).
- the conditions used for hybridization are those of high stringency.
- Preferred embodiments of this aspect of the invention are nucleic acid molecules that are at least 70% identical over their entire length to a nucleic acid molecule encoding the SP2 polypeptide (SEQ ID NO:2) or encoding active fragments of this polypeptide, and nucleic acid molecules that are substantially complementary to such nucleic acid molecules.
- a preferred active fragment is a fragment that includes the serine protease region of the SP2 polypeptide sequence.
- preferred nucleic acid molecules include those that are at least 70% identical over their entire length to a nucleic acid molecule encoding the serine protease region of the SP2 polypeptide sequence. Percentage identity, as referred to herein, is as determined using BLAST version 2.1.3 using the default parameters specified by the NCBI (the National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/).
- a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to the nucleic acid molecule having the sequence given in SEQ ID NO:l, to a region including nucleotides 130-636 of this sequence, to a region including nucleotides 160-591 of this sequence, or a nucleic acid molecule that is complementary to any one of these regions of nucleic acid.
- nucleic acid molecules at least 90%, preferably at least 95%, more preferably at least 98% or 99% identical over their entire length to the same are particularly preferred.
- Preferred embodiments in this respect are nucleic acid molecules that encode polypeptides which retain substantially the same biological function or activity as the SP2 polypeptide.
- the invention also provides a process for detecting a nucleic acid molecule of the invention, comprising the steps of: (a) contacting a nucleic probe according to the invention with a biological sample under hybridizing conditions to form duplexes; and (b) detecting any such duplexes that are formed.
- a nucleic acid molecule as described above may be used as a hybridization probe for RNA, cDNA or genomic DNA, in order to isolate full-length cDNAs and genomic clones encoding the SP2 polypeptide and to isolate cDNA and genomic clones of homologous or orthologous genes that have a high sequence similarity to the gene encoding this polypeptide.
- the following techniques among others known in the art, may be utilised and are discussed below for purposes of illustration. Methods for DNA sequencing and analysis are well known and are generally available in the art and may, indeed, be used to practice many of the embodiments of the invention discussed herein.
- Such methods may employ such enzymes as the Klenow fragment of DNA polymerase I, Sequenase (US Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, IL), or combinations of polymerases and proof-reading exonucleases such as those found in the ELONGASE Amplification System marketed by Gibco/BRL (Gaithersburg, MD).
- the sequencing process may be automated using machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), the Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).
- One method for isolating a nucleic acid molecule encoding a polypeptide with an equivalent function to that of the SP2 polypeptide, particularly with an equivalent function to the serine protease region of the SP2 polypeptide is to probe a genomic or cDNA library with a natural or artificially-designed probe using standard procedures that are recognised in the art (see, for example, "Current Protocols in Molecular Biology", Ausubel et al. (eds). Greene Publishing Association and John Wiley Interscience, New York, 1989,1992).
- Probes comprising at least 15, preferably at least 30, and more preferably at least 50, contiguous bases that correspond to, or are complementary to, nucleic acid sequences from the appropriate encoding gene (SEQ ID NO:l), particularly a region from nucleotides 130-636, or from nucleotides 160-591 of SEQ ID NO. 1, are particularly useful probes.
- Such probes may be labelled with an analytically-detectable reagent to facilitate their identification.
- Useful reagents include, but are not limited to, radioisotopes, fluorescent dyes and enzymes that are capable of catalysing the formation of a detectable product.
- probes the ordinarily skilled artisan will be capable of isolating complementary copies of genomic DNA, cDNA or RNA polynucleotides encoding proteins of interest from human, mammalian or other animal sources and screening such sources for related sequences, for example, for additional members of the family, type and/or subtype.
- isolated cDNA sequences will be incomplete, in that the region encoding the polypeptide will be cut short, normally at the 5' end.
- Several methods are available to obtain full length cDNAs, or to extend short cDNAs. Such sequences may be extended utilising a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed is based on the method of Rapid Amplification of cDNA Ends (RACE; see, for example, Frohman et al, PNAS USA 85, 8998-9002, 1988).
- RACE Rapid Amplification of cDNA Ends
- Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al (1991) PCR Methods Applic, 1, 111-119). Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991); Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PromoterFinderTM libraries to walk genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
- libraries that have been size- selected to include larger cDNAs.
- random-primed libraries are preferable, in that they will contain more sequences that contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
- Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
- the nucleic acid molecules of the present invention may be used for chromosome localisation.
- a nucleic acid molecule is specifically targeted to, and can hybridize with, a particular location on an individual human chromosome.
- the mapping of relevant sequences to chromosomes according to the present invention is an important step in the confirmatory correlation of those sequences with the gene-associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, V. McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library).
- the relationships between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localised by genetic linkage to a particular genomic region, any sequences mapping to that area may represent associated or regulatory genes for further investigation.
- the nucleic acid molecule may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.
- the nucleic acid molecules of the present invention are also valuable for tissue localisation. Such techniques allow the determination of expression patterns of the polypeptide in tissues by detection of the mRNAs that encode them.
- the vectors of the present invention comprise nucleic acid molecules of the invention and may be cloning or expression vectors.
- the host cells of the invention which may be transformed, transfected or transduced with the vectors of the invention may be prokaryotic or eukaryotic.
- polypeptides of the invention may be prepared in recombinant form by expression of their encoding nucleic acid molecules in vectors contained within a host cell. Such expression methods are well known to those of skill in the art and many are described in detail by Sambrook et al (supra) and Fernandez & Hoeffler (1998, eds. "Gene expression systems. Using nature for the art of expression”. Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto).
- any system or vector that is suitable to maintain, propagate or express nucleic acid molecules to produce a polypeptide in the required host may be used.
- the appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well- known and routine techniques, such as, for example, those described in Sambrook et al, (supra).
- the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the transformed host cell.
- suitable expression systems include, for example, chromosomal, episomal and virus-derived systems, including, for example, vectors derived from: bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids.
- Human artificial chromosomes may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.
- Particularly suitable expression systems include microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (for example, baculovirus); plant cell systems transformed with virus expression vectors (for example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (for example, Ti or pBR322 plasmids); or animal cell systems.
- Cell-free translation systems can also be employed to produce the polypeptides of the invention.
- nucleic acid molecules encoding a polypeptide of the present invention into host cells can be effected by methods described in many standard laboratory manuals, such as Davis et al, Basic Methods in Molecular Biology (1986) and Sambrook et al, [supra]. Particularly suitable methods include calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction or infection (see Sambrook et al, 1989 [supra]; Ausubel et al, 1991 [supra]; Spector, Goldman & Leinwald, 1998). In eukaryotic cells, expression systems may either be transient (for example, episomal) or permanent (chromosomal integration) according to the needs of the system.
- the encoding nucleic acid molecule may or may not include a sequence encoding a control sequence, such as a signal peptide or leader sequence, as desired, for example, for secretion of the translated polypeptide into the lumen of the endoplasmic reticulum, into the periplasmic space or into the extracellular environment.
- a control sequence such as a signal peptide or leader sequence
- These signals may be endogenous to the polypeptide or they may be heterologous signals.
- Leader sequences can be removed by the bacterial host in post-translational processing.
- control sequences it may be desirable to add regulatory sequences that allow for regulation of the expression of the polypeptide relative to the growth of the host cell.
- regulatory sequences are those which cause the expression of a gene to be increased or decreased in response to a chemical or physical stimulus, including the presence of a regulatory compound or to various temperature or metabolic conditions.
- Regulatory sequences are those non-translated regions of the vector, such as enhancers, promoters and 5' and 3' untranslated regions. These interact with host cellular proteins to carry out transcription and translation. Such regulatory sequences may vary in their strength and specificity. Depending on the vector system and host utilised, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluescript phagemid (Sfratagene,
- LaJolla, CA) or pSportlTM plasmid (Gibco BRL) and the like may be used.
- the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (for example, heat shock, RUBISCO and storage protein genes) or from plant viruses (for example, viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence, vectors based on S V40 or EB V may be used with an appropriate selectable marker.
- An expression vector is constructed so that the particular nucleic acid coding sequence is located in the vector with the appropriate regulatory sequences, the positioning and orientation of the coding sequence with respect to the regulatory sequences being such that the coding sequence is transcribed under the "control" of the regulatory sequences, i.e., RNA polymerase which binds to the DNA molecule at the control sequences transcribes the coding sequence. In some cases it may be necessary to modify the sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the reading frame.
- the control sequences and other regulatory sequences may be ligated to the nucleic acid coding sequence prior to insertion into a vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site.
- cell lines which stably express the polypeptide of interest may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
- the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences.
- Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
- Mammalian cell lines available as hosts for expression are known in the art and include many immortalised cell lines available from the American Type Culture Collection
- ATCC Chinese hamster ovary
- HeLa Chinese hamster ovary
- BHK baby hamster kidney
- COS monkey kidney
- the materials for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA (the "MaxBac” kit). These techniques are generally known to those skilled in the art and are described fully in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Particularly suitable host cells for use in this system include insect cells such as Drosophila S2 and Spodoptera Sf9 cells.
- all plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be utilised, so that whole plants are recovered which contain the transferred gene.
- Practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugar cane, sugar beet, cotton, fruit and other trees, legumes and vegetables.
- Examples of particularly preferred bacterial host cells include streptococci, staphylococci, E. coli, Streptomyces and Bacillus subtilis cells.
- yeast cells for example, S. cerevisiae
- Aspergillus cells examples include yeast cells (for example, S. cerevisiae) and Aspergillus cells.
- any number of selection systems are known in the art that may be used to recover transformed cell lines. Examples include the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11 :223-32) and adenine phosphoribosylfransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes that can be employed in tk- or aprt ⁇ cells, respectively.
- antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dihydrofolate reductase (DHFR) that confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl Acad. Sci. 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively. Additional selectable genes have been described, examples of which will be clear to those of skill in the art.
- marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed.
- a marker gene can be placed in tandem with a sequence encoding a polypeptide of the invention under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
- host cells that contain a nucleic acid sequence encoding a polypeptide of the invention and which express said polypeptide may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA- DNA or DNA-RNA hybridizations and protein bioassays, for example, fluorescence activated cell sorting (FACS) or immunoassay techniques (such as the enzyme-linked immunosorbent assay [ELISA] and radioimmunoassay [RIA]), that include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein (see Hampton, R. et al.
- FACS fluorescence activated cell sorting
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- Means for producing labelled hybridization or PCR probes for detecting sequences related to nucleic acid molecules encoding polypeptides of the present invention include oligolabelling, nick translation, end-labelling or PCR amplification using a labelled polynucleotide.
- sequences encoding the polypeptide of the invention may be cloned into a vector for the production of an mRNA probe.
- a vector for the production of an mRNA probe Such vectors are known in the art, are commercially available, and may be used to synthesise RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labelled nucleotides. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, MI); Promega (Madison WI); and U.S. Biochemical Corp., Cleveland, OH)).
- Suitable reporter molecules or labels which may be used for ease of detection, include radionucleides, enzymes and fluorescent, chemiluminescent or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
- Nucleic acid molecules according to the present invention may also be used to create transgenic animals, particularly rodent animals. Such transgenic animals form a further aspect of the present invention. This may be done locally by modification of somatic cells, or by germ line therapy to incorporate heritable modifications. Such transgenic animals may be particularly useful in the generation of animal models for drug molecules effective as modulators of the polypeptides of the present invention.
- the polypeptide can be recovered and purified from recombinant cell cultures by well- known methods including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography is particularly useful for purification. Well known techniques for refolding proteins may be employed to regenerate an active conformation when the polypeptide is denatured during isolation and or purification.
- Specialised vector constructions may also be used to facilitate purification of proteins, as desired, by joining sequences encoding the polypeptides of the invention to a nucleotide sequence encoding a polypeptide domain that will facilitate purification of soluble proteins.
- purification-facilitating domains include metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilised metals, protein A domains that allow purification on immobilised immunoglobulin, and the domain utilised in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA).
- cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the polypeptide of the invention may be used to facilitate purification.
- One such expression vector provides for expression of a fusion protein containing the polypeptide of the invention fused to several histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilised metal ion affinity chromatography as described in Porath, J. et al. (1992), Prot. Exp. Purif.
- the polypeptide is to be expressed for use in screening assays, generally it is preferred that it be produced at the surface of the host cell in which it is expressed. In this event, the host cells may be harvested prior to use in the screening assay, for example using techniques such as fluorescence activated cell sorting (FACS) or immunoaffinity techniques. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the expressed polypeptide. If polypeptide is produced intracellularly, the cells must first be lysed before the polypeptide is recovered.
- the polypeptide of the invention can be used to screen libraries of compounds in any of a variety of drug screening techniques.
- Such compounds may activate (agonise) or inhibit (antagonise) the level of expression of the gene or the activity of the polypeptide of the invention and form a further aspect of the present invention.
- Preferred compounds are effective to alter the expression of a natural gene which encodes a polypeptide of the first aspect of the invention or to regulate the activity of a polypeptide of the first aspect of the invention.
- Agonist or antagonist compounds may be isolated from, for example, cells, cell-free preparations, chemical libraries or natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, enzymes, receptors or structural or functional mimetics. For a suitable review of such screening techniques, see Coligan et al, Current Protocols in Immunology l(2):Chapter 5 (1991).
- Compounds that are most likely to be good antagonists are molecules that bind to the polypeptide of the invention without inducing the biological effects of the polypeptide upon binding to it.
- Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby inhibit or extinguish its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be inhibited, such that the normal biological activity of the polypeptide is prevented.
- polypeptide of the invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly.
- such screening procedures may involve using appropriate cells or cell membranes that express the polypeptide that are contacted with a test compound to observe binding, or stimulation or inhibition of a functional response.
- the functional response of the cells contacted with the test compound is then compared with control cells that were not contacted with the test compound.
- Such an assay may assess whether the test compound results in a signal generated by activation of the polypeptide, using an appropriate detection system.
- Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the test compound is observed.
- simple binding assays may be used, in which the adherence of a test compound to a surface bearing the polypeptide is detected by means of a label directly or indirectly associated with the test compound or in an assay involving competition with a labelled competitor.
- competitive drug screening assays may be used, in which neutralising antibodies that are capable of binding the polypeptide specifically compete with a test compound for binding. In this manner, the antibodies can be used to detect the presence of any test compound that possesses specific binding affinity for the polypeptide.
- Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding the polypeptide in cells.
- an ELISA may be constructed that measures secreted or cell-associated levels of polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. The formation of binding complexes between the polypeptide and the compound being tested may then be measured.
- Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564).
- This method large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with the polypeptide of the invention and washed.
- One way of immobilising the polypeptide is to use non-neutralising antibodies. Bound polypeptide may then be detected using methods that are well known in the art. Purified polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques.
- the polypeptide of the invention may be used to identify membrane-bound or soluble receptors, through standard receptor binding techniques that are known in the art, such as ligand binding and crosslinking assays in which the polypeptide is labelled with a radioactive isotope, is chemically modified, or is fused to a peptide sequence that facilitates its detection or purification, and incubated with a source of the putative receptor (for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids).
- a source of the putative receptor for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids.
- the efficacy of binding may be measured using biophysical techniques such as surface plasmon resonance and spectroscopy.
- Binding assays may be used for the purification and cloning of the receptor, but may also identify agonists and antagonists of the polypeptide, that compete with the binding of the polypeptide to its receptor. Standard methods for conducting screening assays are well understood in the art.
- the invention also includes a screening kit useful in the methods for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, that are described above.
- the invention includes the agonists, antagonists, ligands, receptors, substrates and enzymes, and other compounds which modulate the activity or antigenicity of the polypeptide of the invention discovered by the methods that are described above.
- the invention also provides pharmaceutical compositions comprising a polypeptide, nucleic acid, ligand or compound of the invention in combination with a suitable pharmaceutical carrier. These compositions may be suitable as therapeutic or diagnostic reagents, as vaccines, or as other immunogenic compositions, as outlined in detail below.
- a composition containing a polypeptide, nucleic acid, ligand or compound [X] is "substantially free of impurities [herein, Y] when at least 85%) by weight of the total X+Y in the composition is X.
- X comprises at least about 90%) by weight of the total of X+Y in the composition, more preferably at least about 95%, 98% or even 99% by weight.
- compositions should preferably comprise a therapeutically effective amount of the polypeptide, nucleic acid molecule, ligand, or compound of the invention.
- therapeutically effective amount refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent a targetted disease or condition, or to exhibit a detectable therapeutic or preventative effect.
- the therapeutically effective dose can be estimated initially either in cell culture assays, for example, of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
- an effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.
- a pharmaceutical composition may also contain a pharmaceutically acceptable carrier, for administration of a therapeutic agent.
- a pharmaceutically acceptable carrier for administration of a therapeutic agent.
- Such carriers include antibodies and other polypeptides, genes and other therapeutic agents such as liposomes, provided that the carrier does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
- Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles.
- Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
- mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like
- organic acids such as acetates, propionates, malonates, benzoates, and the like.
- compositions of therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient. Once formulated, the compositions of the invention can be administered directly to the subject.
- the subjects to be treated can be animals; in particular, human subjects can be treated.
- compositions utilised in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, infra- arterial, intramedullary, intrathecal, intraventricular, transdermal or transcutaneous applications (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal means.
- Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention.
- the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
- Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue.
- the compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.
- One approach comprises administering to a subject an inhibitor compound (antagonist) as described above, along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
- an inhibitor compound as described above
- a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of ligands, substrates, enzymes, receptors, or by inhibiting a second signal, and thereby alleviating the abnormal condition.
- antagonists are antibodies.
- such antibodies are chimeric and/or humanised to minimise their immunogenicity, as described previously.
- soluble forms of the polypeptide that retain binding affinity for the ligand, substrate, enzyme, receptor, in question may be administered.
- the polypeptide may be administered in the form of fragments that retain the relevant portions.
- expression of the gene encoding the polypeptide can be inhibited using expression blocking techniques, such as the use of antisense nucleic acid molecules (as described above), either internally generated or separately administered. Modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions (signal sequence, promoters, enhancers and introns) of the gene encoding the polypeptide.
- triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
- triplex DNA Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al. (1994) In: Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Furura Publishing Co., Ml Kisco, NY).
- the complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Such oligonucleotides may be administered or may be generated in situ from expression in vivo.
- Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al, Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribozymes may be synthesised with non-natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases.
- RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, queosine and butosine, as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine and uridine which are not as easily recognised by endogenous endonucleases.
- One approach comprises administering to a subject a therapeutically effective amount of a compound that activates the polypeptide, i.e., an agonist as described above, to alleviate the abnormal condition.
- a therapeutic amount of the polypeptide in combination with a suitable pharmaceutical carrier may be administered to restore the relevant physiological balance of polypeptide.
- Gene therapy may be employed to effect the endogenous production of the polypeptide by the relevant cells in the subject.
- Gene therapy is used to treat permanently the inappropriate production of the polypeptide by replacing a defective gene with a corrected therapeutic gene.
- Gene therapy of the present invention can occur in vivo or ex vivo. Ex vivo gene therapy requires the isolation and purification of patient cells, the introduction of a therapeutic gene and introduction of the genetically altered cells back into the patient. In contrast, in vivo gene therapy does not require isolation and purification of a patient's cells.
- Gene delivery vehicles may be non- viral, such as liposomes, or replication-deficient viruses, such as adenovirus as described by Berkner, K.L., in Curr. Top. Microbiol Immunol, 158, 39-66
- AAV adeno-associated virus
- a nucleic acid molecule encoding a polypeptide of the invention may be engineered for expression in a replication-defective retroviral vector.
- This expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding the polypeptide, such that the packaging cell now produces infectious viral particles containing the gene of interest.
- producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo (see Chapter 20, Gene Therapy and other Molecular
- Another approach is the administration of "naked DNA" in which the therapeutic gene is directly injected into the bloodstream or muscle tissue.
- the invention provides that they can be used in vaccines to raise antibodies against the disease causing agent.
- Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat disease after infection).
- Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with pharmaceutically-acceptable carriers as described above, which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Additionally, these carriers may function as immunostimulating agents ("adjuvants").
- the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and other pathogens.
- vaccines comprising polypeptides are preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection).
- parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.
- the vaccine formulations of the invention may be presented in unit-dose or multi-dose containers.
- sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.
- the dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
- This invention also relates to the use of nucleic acid molecules according to the present invention as diagnostic reagents. Detection of a mutated form of the gene characterised by the nucleic acid molecules of the invention which is associated with a dysfunction will provide a diagnostic tool that can add to, or define, a diagnosis of a disease, or susceptibility to a disease, which results from under-expression, over-expression or altered spatial or temporal expression of the gene. Individuals carrying mutations in the gene may be detected at the DNA level by a variety of techniques.
- Nucleic acid molecules for diagnosis may be obtained from a subject's cells, such as from blood, urine, saliva, tissue biopsy or autopsy material.
- the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR, ligase chain reaction (LCR), strand displacement amplification (SDA), or other amplification techniques (see Saiki et al, Nature, 324, 163-166 (1986); Bej, et al, Crit. Rev. Biochem. Molec. Biol, 26, 301-334 (1991); Birkenmeyer et al, J. Virol. Meth., 35, 117-126 (1991); Van Brunt, J., Bio/Technology, 8, 291 -294 (1990)) prior to analysis.
- LCR ligase chain reaction
- SDA strand displacement amplification
- this aspect of the invention provides a method of diagnosing a disease in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to the invention and comparing said level of expression to a control level, wherein a level that is different to said control level is indicative of disease.
- the method may comprise the steps of: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the invention and the probe; b) contacting a control sample with said probe under the same conditions used in step a); and c) detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of disease.
- a further aspect of the invention comprises a diagnostic method comprising the steps of: a) obtaining a tissue sample from a patient being tested for disease; b) isolating a nucleic acid molecule according to the invention from said tissue sample; and c) diagnosing the patient for disease by detecting the presence of a mutation in the nucleic acid molecule which is associated with disease.
- an amplification step for example using PCR, may be included. Deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labelled RNA of the invention or alternatively, labelled antisense DNA sequences of the invention. Perfectly-matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperatures.
- the presence or absence of the mutation in the patient may be detected by contacting DNA with a nucleic acid probe that hybridises to the DNA under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation in the corresponding portion of the DNA strand.
- Such diagnostics are particularly useful for prenatal and even neonatal testing.
- Point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by other well-known techniques, such as direct DNA sequencing or single-strand conformational polymorphism, (see Orita et al, Genomics, 5, 874-879 (1989)).
- a sequencing primer may be used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
- the sequence determination is performed by conventional procedures with radiolabelled nucleotides or by automatic sequencing procedures with fluorescent-tags.
- Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR.
- point mutations and other sequence variations, such as polymorphisms can be detected as described above, for example, through the use of allele-specific oligonucleotides for PCR amplification of sequences that differ by single nucleotides.
- DNA sequence differences may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et al, Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (see Cotton et al, Proc. Natl Acad. Sci. USA (1985) 85: 4397-4401).
- mutations such as microdeletions, aneuploidies, translocations, inversions, can also be detected by in situ analysis (see, for example, Keller et al, DNA Probes, 2nd Ed., Stockton Press, New York, N.Y., USA (1993)), that is, DNA or RNA sequences in cells can be analysed for mutations without need for their isolation and/or immobilisation onto a membrane.
- FISH Fluorescence in situ hybridization
- an array of oligonucleotide probes comprising a nucleic acid molecule according to the invention can be constructed to conduct efficient screening of genetic variants, mutations and polymorphisms.
- Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M. Chee et al, Science (1996), Vol 274, pp 610-613).
- the array is prepared and used according to the methods described in PCT application WO95/11995 (Chee et al); Lockhart, D. J. et al. (1996) Nat. Biotech. 14: 1675-1680); and Schena, M. et al. (1996) Proc. Natl Acad. Sci. 93: 10614-10619).
- Oligonucleotide pairs may range from two to over one million.
- the oligomers are synthesized at designated areas on a substrate using a light-directed chemical process.
- the substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support.
- an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et al).
- a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures.
- An array such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number between two and over one million which lends itself to the efficient use of commercially-available instrumentation.
- diseases may be diagnosed by methods comprising determining, from a sample derived from a subject, an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
- nucleic acid amplification for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.
- Assay techniques that can be used to determine levels of a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and are discussed in some detail above (including radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays).
- This aspect of the invention provides a diagnostic method which comprises the steps of: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand- polypeptide complex; and (b) detecting said complex.
- Protocols such as ELISA, RIA, and FACS for measuring polypeptide levels may additionally provide a basis for diagnosing altered or abnormal levels of polypeptide expression.
- Normal or standard values for polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably humans, with antibody to the polypeptide under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, such as by photometric means.
- Antibodies which specifically bind to a polypeptide of the invention may be used for the diagnosis of conditions or diseases characterised by expression of the polypeptide, or in assays to monitor patients being treated with the polypeptides, nucleic acid molecules, ligands and other compounds of the invention.
- Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for the polypeptide include methods that utilise the antibody and a label to detect the polypeptide in human body fluids or extracts of cells or tissues.
- the antibodies may be used with or without modification, and may be labelled by joining them, either covalently or non-covalently, with a reporter molecule.
- reporter molecules A wide variety of reporter molecules known in the art may be used, several of which are described above. Quantities of polypeptide expressed in subject, control and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
- Diagnostic assays may be used to distinguish between absence, presence, and excess expression of polypeptide and to monitor regulation of polypeptide levels during therapeutic intervention. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.
- a diagnostic kit of the present invention may comprise:
- a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease.
- the kit may further comprise a third container holding an agent for digesting unhybridised RNA.
- a diagnostic kit may comprise an array of nucleic acid molecules, at least one of which may be a nucleic acid molecule according to the invention.
- a diagnostic kit may comprise one or more antibodies that bind to a polypeptide according to the invention; and a reagent useful for the detection of a binding reaction between the antibody and the polypeptide.
- kits will be of use in diagnosing a disease or susceptibility to disease, particularly bacterial infections.
- Various aspects and embodiments of the present invention will now be described in more detail by way of example, with particular reference to the SP2 polypeptide. It will be appreciated that modification of detail may be made without departing from the scope of the invention.
- Figure 1 Front page of the BiopendiumTM. Search initiated using 1PJP:A.
- Figure 2 A Inpharmatica Genome ThreaderTM results of search using 1PJP:A.
- the arrow points to CAB46586.1 (SP2), a hypothetical protein.
- Figure 2B PSI-BLAST results from search using 1PJP:A.
- FIG. 3 Redundant Sequence Display page for CAB46586.1 (SP2).
- Figure 4A NCBI protein report for CAB46586.1 (SP2).
- Figure 4B FEMS Microbiol Lett 1995 article pertaining to virulence associated with a Yersinia gene cluster, to which CAB46586.1 (SP2) belongs.
- Figure 5 PFAM search results for CAB46586.1 (SP2).
- Figure 6A Inpharmatica Genome ThreaderTM results of search using CAB46586.1 (SP2).
- the arrows points to 1PJP:A, 1BIO, 1DSU:B, and 1HFD.
- FIGB PSI-BLAST results from search using CAB46586.1 (SP2).
- Figure 7 A Genome ThreaderTM alignment of CAB46586.1 (SP2) with Human Chymase (1PJP:A) and Complement Factor D (1BIO, 1DSU:B, and 1HFD).
- FIG. 7B Genome ThreaderTM alignment of CAB46586.1 (SP2) with Elastase (1B0F:A) and Trypsin (2STA:E).
- Figure 8 A LigEye for 1PJP:A which illustrates the sites of interaction of the peptide inhibitor Succinyl-Ala-Ala-Pro-Phe-Chloromethylketone with human Chymase.
- Figure 8B RasMol view of 1PJP:A, human Chymase in complex with Succinyl-Ala-Ala- Pro-Phe-Chloromethylketone.
- the coloured balls represent the amino acids in Chymase that directly interact with the inhibitor and are conserved in Chymase.
- Figure 10 NCBI CDD search results for CAB46586.1 (SP2) as of 24 th July 2001.
- Figure 11 InterPro search results for residues 44-212 of CAB46586.1 (SP2) as of 24 th July 2001.
- Figure 12 NCBI CDD search results for residues 44-212 of CAB46586.1 (SP2) as of 24 th July 2001.
- the 955 Genome ThreaderTM results include examples of typical serine proteases, such as Trypsin, Elastase, and Chymotrypsin.
- serine proteases appears a protein of apparently unknown function, annotated as hypothetical protein, CAB46586.1 (SP2) ( Figure 2 A, arrow).
- the Inpharmatica Genome ThreaderTM has identified residues 51-197 of a sequence, CAB46586.1 (SP2), as having an equivalent structure to residues 32-199 of Human Chymase (PDB code 1PJP:A), a serine protease. Having a structure similar to a serine protease suggests that residues 51-197 of CAB46586.1 (SP2) is a serine protease.
- the Inpharmatica Genome ThreaderTM identifies this with 80% confidence.
- PSI-BLAST Figure 2B
- PSI-BLAST does identify Chymase itself and other related serine proteases with varying degrees of probability (E value) as would be expected.
- CAB46586.1 is a Yersinia enterocolitica sequence, its Genbank protein ID is CAB46586.1 and it is 383 amino acids in length. There are no associated PROSITE or PRINTS hits for this sequence. Returning zero hits from both databases means that CAB46586.1 (SP2) is unidentifiable as a serine protease using PROSITE or PRINTS.
- CAB46586.1 was cloned by several groups of scientists. There is no annotation for CAB46586.1 (SP2), except that it is a hypothetical protein and was cloned from a region of the Yersinia enterocolitica genome that is responsible for pathogenicity in humans ( Figure 4B). As such, this gene may be responsible for the pathogenicity of this bacterial strain.
- the public domain information for this gene does not annotate it as a serine protease.
- CAB46586.1 (SP2) is searched against the Protein Family Database of Alignment and HMM's (PFAM) database ( Figure 5). The results identify that CAB46586.1 has no hits, hence no identifiable domains. PFAM does not identify CAB46586.1 as a serine protease. Therefore using the best public domain annotation tools available, CAB46586.1 (SP2) is not annotated as a serine protease. Only the Inpharmatica Genome ThreaderTM is able to annotate this protein as a serine protease.
- PFAM Protein Family Database of Alignment and HMM's
- CAB46586.1 is now used as the query sequence in the BiopendiumTM database.
- the Inpharmatica Genome ThreaderTM identifies 23 hits (Figure 6A) while Reverse-Maximised PSI-BLASTTM returns 4 hits ( Figure 6B).
- the Inpharmatica Genome ThreaderTM ( Figure 6 A, arrow ⁇ ) identifies residues 51-197 of CAB46586.1 (SP2) as having a structure that is similar to residues 32-199 of Human Chymase (1PJP:A) with 80% confidence.
- a range of other SI serine proteases are also identified in the Genome ThreaderTM results.
- the Inpharmatica Genome ThreaderTM ( Figure 6 A, arrow ®) also identifies residues 54-212 of CAB46586.1 (SP2) as having a structure that is similar to residues 31-215 of the serine protease Human Complement Factor D (1HFD, 1DSU:B and 1BIO) with 89% confidence.
- the Inpharmatica Genome ThreaderTM ( Figure 6A, arrow ®) also identifies residues 47- 212 of CAB46586.1 (SP2) as having a structure that is similar to residues 22-205 of the serine protease Human Neutrophil Elastase (1B0F:A) with 89% confidence.
- the Inpharmatica Genome ThreaderTM ( Figure 6 A, arrow ®) also identifies residues 44-197 of CAB46586.1 (SP2) as having a structure that is similar to residues 16-193 of the serine protease Trypsin (2STA:E) with 88% confidence.
- SP2 residues 44-197 of CAB46586.1
- 2STA:E serine protease Trypsin
- a region including from residues 44-54 to residues 197-212 of CAB46586.1 (SP2) has been identified as adopting an equivalent fold to a range of other serine proteases including Chymase, Complement Factor D, Elastase and Trypsin.
- Forward PSI-BLAST does not return this result. It is only the Inpharmatica Genome ThreaderTM that is able to identify this relationship.
- Figure 7A illustrates the point that the catalytic triad (Histidine, Aspartate, and Serine) possessed by all known serine proteases is conserved as His64, AsplOl and Serl78 in CAB46586.1 (SP2). This indicates that CAB46586.1 (SP2) is a serine protease.
- FIG. 7A shows conservation of the HIS57, ASP102, and SER195 of the serine protease's catalytic triad between residues 51-197 of CAB46586.1.
- Figure 7B shows the Genome ThreaderTM alignment of CAB46586.1 (SP2) with Human Neutrophil Elastase (1B0F:A) and Trypsin (2STA:E). This illustrates that the catalytic triad of Human Neutrophil Elastase and Trypsin are also conserved as His64, AsplOl and Serl78 in CAB46586.1 (SPG2).
- the peptide inhibitor sees 3 different amino acids in the catalytic domain of human Chymase. Of the three amino acids, two are conserved in between residues 51-197 of CAB46586.1. These are His57 and Gly216. This indicates that indeed as predicted by the Inpharmatica Genome ThreaderTM residues 51-197 of CAB46586.1 (SP2) folds in a similar manner to human Chymase and as such is identified as a serine protease.
- SEQ ID NO: 2 (Protein CAB46586.1; SP2)
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PCT/GB2001/003357 WO2002008393A2 (en) | 2000-07-24 | 2001-07-24 | Serine protease from yersinia enterocolitica |
Country Status (3)
Country | Link |
---|---|
AU (1) | AU2002224552A1 (en) |
GB (1) | GB0018125D0 (en) |
WO (1) | WO2002008393A2 (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998014461A1 (en) * | 1996-09-30 | 1998-04-09 | Smithkline Beecham Plc | NOVEL spsB |
-
2000
- 2000-07-24 GB GBGB0018125.5A patent/GB0018125D0/en not_active Ceased
-
2001
- 2001-07-24 WO PCT/GB2001/003357 patent/WO2002008393A2/en active Application Filing
- 2001-07-24 AU AU2002224552A patent/AU2002224552A1/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998014461A1 (en) * | 1996-09-30 | 1998-04-09 | Smithkline Beecham Plc | NOVEL spsB |
Non-Patent Citations (6)
Also Published As
Publication number | Publication date |
---|---|
AU2002224552A1 (en) | 2002-02-05 |
WO2002008393A3 (en) | 2002-06-20 |
GB0018125D0 (en) | 2000-09-13 |
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