WO1999023110A1 - Hptlg gene from human hypothalamus - Google Patents

Abstract

The invention provides isolated nucleic acid compounds encoding the HPTLG protein of human hypothalamus. Also provided are vectors and transformed heterologous host cells for expressing the HPTLG protein and a method for identifying compounds that bind said protein.

Description

HPTLG GENE FROM HUMAN HYPOTHALAMUS

BACKGROUND OF THE INVENTION

This invention relates to recombinant DNA technology. In particular the invention pertains to a gene isolated from human hypothalamus and to a protein product encoded thereby. The hypothalamus exercises considerable influence on behavioral, autonomic, visceral, and endocrine functions via its interaction with the pituitary gland. For example, hypothalmus function is implicated in the control of water balance, ingestion of food, body temperature and circadian rhythms, sleep, and emotional state. (See generally Functional Anatomy of the Neuroendocrine Hypothalamus, John Wiley & Sons 1992; Progress in Brain Research Vol 93 : The Human Hypothalamus in Health and Disease, Eds. D.F. Swaab et al . Elsevier, 1992). The hypothalamus comprises a number of cytologically and anatomically distinct cell types. The discovery of neuropeptides as secretory products of neurons of the hypothalamus has led to substantial advancements in understanding the complexities of cell communication within

the nervous system. Neuroendocrine cells of the hypothalamus produce more than 20 neuropeptides including, for example, hypophysiotropic hormones such as thyrotropin-releasing hormone and corticotropin-releasing factor, brain-borne anterior pituitary hormones, opioid peptides, brain-borne gastrointestinal peptides such as neuropeptide Y and tachy inins , and brain-borne vasoactive peptides such as neurotensin and bradykinin The central role played by the hypothalamus in regulating mammalian endocrine, autonomic, and behavioral systems is now well recognized. Indeed, this diminutive region of the brain is sometimes referred to as the "sovereign center of centers."

In humans, a number of syndromes have been linked with abnormal function of the hypothalamus. For example, sexual abnormalities, diabetes, psychic disturbances, obesity, somnolence, anorexia and disorders of temperature regulation are all correlated with hypothalamic dysfunction. Treatment of one or more of these conditions would be facilitated by a better understanding of hypothalamic function.

BRIEF SUMMARY OF THE INVENTION The present invention provides isolated nucleic acid molecules that encode a testican-like protein ("HPTLG") from human hypothalmus . The invention also provides the protein product of said nucleic acid, in substantially purified form.

Having the cloned HPTLG gene enables the production of recombinant HPTLG protein, the isolation of homologous genes from other organisms, and/or related genes from the same organism, chromosome mapping studies, and the implementation of large scale screens to identify compounds that bind said protein and modulate the activity thereof . The proteins disclosed herein are also useful, among other things, as feed additives . In one embodiment the present invention relates to an isolated nucleic acid molecule encoding HPTLG protein. In another embodiment, the invention relates to a nucleic acid molecule comprising the nucleotide sequence identified as SEQ ID NO:l or SEQ ID NO: 3. In another embodiment, the present invention relates to a nucleic acid that encodes SEQ ID NO: 2 or fragment thereof. In another embodiment, the present invention relates to a nucleic acid that is at least 75% identical to a nucleic acid that encodes SEQ ID NO: 2 or fragment thereof. In another embodiment, the present invention relates to a nucleic acid that hybridizes to SEQ ID NO:l under high stringency conditions.

In another embodiment, the present invention relates to a nucleic acid that hybridizes to SEQ ID NO:l under low stringency conditions.

In another embodiment the present invention relates to an isolated protein molecule, or functional fragment thereof, wherein said protein molecule comprises the sequence identified as SEQ ID NO: 2. In yet another embodiment, the present invention relates to a recombinant DNA vector which incorporates the

HPTLG gene in operable linkage to gene expression sequences enabling the gene to be transcribed and translated in a host cell . In still another embodiment the present invention relates to host cells that have been transformed or transfected with the cloned HPTLG gene such that the HPTLG gene is expressed in the host cell. This invention also provides a method of determining whether a nucleic acid sequence of the present invention, or fragment thereof, is present within a nucleic acid- containing sample, comprising contacting the sample under suitable hybridization conditions with a nucleic acid probe of the present invention.

In a still further embodiment, the present invention relates to a method for identifying compounds that bind the HPTLG protein.

DETAILED DESCRIPTION OF THE INVENTION

Definitions "HPTLG" refers to a novel proteoglycan disclosed herein that is related to testican, a multifunctional protein associated with cell adhesion, cell migration, proliferation, and counter adhesion in neural development. The terms "complementary" or "complementarity" as used herein refer to the capacity of purine and pyrimidine nucleotides to associate through hydrogen bonding to form double stranded nucleic acid molecules. The following base pairs are related by complementarity: guanine and cytosine; adenine and thymine; and adenine and uracil . As used herein, "complementary" means that the aforementioned relationship applies to substantially all base pairs comprising two single-stranded nucleic acid molecules over the entire length of said molecules. "Partially complementary" refers to the aforementioned relationship in which one of two single-stranded nucleic acid molecules is shorter in length than the other such that a portion of one of the molecules remains single-stranded. "Conservative substitution" or "conservative amino acid substitution" refers to a replacement of one or more amino acid residue (s) in a protein or peptide as stipulated in Table 1.

"Fragment thereof" refers to a fragment, piece, or sub-region of a nucleic acid or protein molecule whose sequence is disclosed herein, such that the fragment comprises 5 or more amino acids, or 10 or more nucleotides that are contiguous in the parent protein or nucleic acid molecule . Fragment thereof may or may not retain biological activity. For example, a fragment of a protein disclosed herein could be used as an antigen to raise a specific antibody against the parent protein molecule. When referring to a nucleic acid molecule, "fragment thereof" refers to 10 or more contiguous nucleotides, derived from the parent nucleic acid, and also, owing to the genetic code, to the complementary sequence. For example if the fragment entails the sequence 5' -AGCTAGCTAG-3 ' , then "fragment thereof" would also include the complementary sequence, 3 ' -TCGATCGATC-5' .

The term "fusion protein" denotes a hybrid protein molecule not found in nature comprising a translational fusion or enzymatic fusion in which two or more different proteins or fragments thereof are covalently linked on a single polypeptide chain.

"Functional fragment" or "functionally equivalent fragment", as used herein, refers to a region, or fragment of a full length protein, or sequence of amino acids that, for example, comprises an active site, or any other conserved motif, relating to biological function. Functional fragments are capable of providing a substantially similar biological activity as a full length protein disclosed herein, in vivo or in vitro, viz . the capacity to promote tissue differentiation. Functional fragments may be produced by cloning technology, or as the natural products of alternative splicing mechanisms. "Host cell" refers to any eucaryotic or procaryotic cell that is suitable for propagating and/or expressing a cloned gene contained on a vector that is introduced into said host cell by, for example, transformation or transfection, or the like. "HPTLG" refers to a gene and a protein or amino acid sequence encoded thereby that is related to a mesodermal tissue differentiation factor, designated "lunatic fringe" in Xenopus laevis, and "fringe" in Drosophila melanogaster . The term "homolog" or "homologous" describes the relationship between different nucleic acid molecules or amino acid sequences such that said sequences or molecules are related by partial identity or similarity at one or more regions within said molecules or sequences. The term "hybridization" as used herein refers to a process in which a single- stranded nucleic acid molecule joins with a complementary strand through nucleotide base pairing. "Selective hybridization" refers to hybridization under conditions of high stringency. The degree of hybridization depends upon, for example, the degree of homology, the stringency of hybridization, and the length of hybridizing strands.

"Isolated nucleic acid compound" refers to any RNA or DNA sequence, however constructed or synthesized, which is locationally distinct from its natural location.

A "nucleic acid probe" or "probe" as used herein is a labeled nucleic acid compound which hybridizes with another nucleic acid compound. "Nucleic acid probe" means a single stranded nucleic acid sequence that will combine with a complementary or partially complementary single stranded target nucleic acid sequence to form a double-stranded molecule. A nucleic acid probe may be an oligonucleotide or a nucleotide polymer. A probe will usually contain a detectable moiety which may be attached to the end(s) of the probe or be internal to the sequence of the probe .

The term "plasmid" refers to an extrachromosomal genetic element. The plasmids disclosed herein are commercially available, publicly available on an unrestricted basis, or can be constructed from readily available plasmids in accordance with published procedures. A "primer" is a nucleic acid fragment which functions as an initiating substrate for enzymatic or synthetic elongation of, for example, a nucleic acid molecule . The term "promoter" refers to a nucleic acid sequence that directs transcription, for example, of DNA to RNA. An inducible promoter is one that is regulatable by environmental signals, such as carbon source, heat, or metal ions, for example. A constitutive promoter generally operates at a constant level and is not regulatable. "Recombinant DNA cloning vector" as used herein refers to any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been incorporated.

The term "recombinant DNA expression vector" or "expression vector" as used herein refers to any recombinant DNA cloning vector, for example a plasmid or phage, in which a promoter and other regulatory elements are present thereby enabling transcription of an inserted DNA, which may encode a protein.

The term "stringency" refers to hybridization conditions. High stringency conditions disfavor non- homologous basepairing. Low stringency conditions have the opposite effect. Stringency may be altered, for example, by temperature and salt concentration.

"Low stringency" conditions comprise, for example, a temperature of about 37° C or less, a formamide concentration of less than about 50%, and a moderate to low salt (SSC) concentration; or, alternatively, a temperature of about 50° C or less, and a moderate to high salt (SSPE) concentration, for example 1M NaCl.

"High stringency" conditions comprise, for example, a temperature of about 42° C or less, a formamide concentration of less than about 20%, and a low salt (SSC) concentration; or, alternatively, a temperature of about 65° C, or less, and a low salt (SSPE) concentration. For example, high stringency conditions comprise hybridization in 0.5 M NaHP0₄, 7% sodium dodecyl sulfate (SDS) , 1 mM EDTA at 65°C (Ausubel, F.M. et al . Current Protocols in Molecular Biology, Vol. I, 1989; Green Inc. New York, at 2.10.3) .

"SSC" comprises a hybridization and wash solution. A stock 2OX SSC solution contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0.

"SSPE" comprises a hybridization and wash solution. A IX SSPE solution contains 180 mM NaCl, 9mM Na₂HP0₄, 0.9 mM NaH₂P0₄ and 1 mM EDTA, pH 7.4.

"Substantially pure" used in reference to a peptide or protein means that said peptide or protein is separated from a large fraction of all other cellular and non-cellular molecules, including other protein molecules. A substantially pure preparation would be about at least 85% pure; preferably about at least 95% pure. For example, a "substantially pure" protein as described herein could be prepared by the IMAC protein purification method.

The term "vector" as used herein refers to a nucleic acid compound used for introducing exogenous or endogenous DNA into host cells . A vector comprises a nucleotide sequence which may encode one or more protein molecules. Plasmids, cosmids, viruses, and bacteriophages, in the natural state or which have undergone recombinant engineering, are examples of commonly used vectors.

The various restriction enzymes disclosed and described herein are commercially available and the manner of use of said enzymes including reaction conditions, cofactors, and other requirements for activity are well known to one of ordinary skill in the art . Reaction conditions for particular enzymes were carried out according to the manufacturer's recommendation. The HPTLG gene of human hypothalamus encodes a novel protein that is related to testican. Testican was first identified in human testis as a proteoglycan molecule having domains characteristic of molecules involved in cell adhesion, migration, and proliferation (P.M. Alliel et al . Eur. J. Biochem. 214, 347-50 (1993) . Subsequently, it was determined that a testican homolog exists in mouse brain, primarily in the CA3 area of the hippocampus, and that expression of said homolog appears most predominantly in the post-synaptic region of pyramidal neurons (F. Bonnet et al . J. Biol . Chem . 271, 4373- 4380 (1996) . These findings suggest that testican may play a role in binding ligands to target cells, perhaps serving in the brain as a receptor for growth factors. The HPTLG cDNA comprises a DNA sequence designated herein as SEQ ID NO:l. Those skilled in the art will recognize that owing to the degeneracy of the genetic code (i.e. 64 codons which encode 20 amino acids), numerous "silent" substitutions of nucleotide base pairs could be introduced into the sequence identified as SEQ ID NO:l without altering the identity of the encoded amino acid(s) or protein product. All such substitutions are intended to be within the scope of the invention.

Also contemplated by the present invention are HPTLG proteins and related functional fragments such as, for example, smaller alternatively spliced forms, or substitutions in which the primary sequence disclosed in SEQ ID NO: 2 is altered at one or more amino acid positions such that biological function is maintained. Several structural motifs have been identified within the primary sequence of HPTLG that are thought to be important for biological function. For example, amino acid residues 136 through 182 of SEQ ID NO: 2 comprise a consensus "Kazal-type" domain, thought to impart protease inhibitor activity to proteins such as QR1, SCI, and follistatin (Guermah et.al. Proc. Nat . Acad . Sci . 88, 4503-4507, 1991). A second domain comprising an about 46 amino acid motif, referred to herein as the "CWCV" domain, is located about between amino acid residues 332 through 381 of SEQ ID NO: 2. The CWCV domain is considered to be a signature of an emerging family of binding proteins. For example, the CWCV domain has been implicated in binding insulin-like growth factors (Huhtala et.al. Biochem. Biophys . Res . Commun . 141, 263-270, 1986). Functional analogs of the HPTLG protein (s) are typically generated by deletion, insertion, or substitutions of a single (or few) amino acid residues. Conservative amino acid substitutions typically do not alter function and can generally be made in accordance with Table 1.

Fragments of proteins

One embodiment of the instant invention provides fragments of the proteins disclosed that may or may not be biologically active. Such fragments are useful, for example, in providing an antigen for producing an antibody to said proteins . Fragments of proteins disclosed herein may be generated by any number of suitable techniques, including chemical synthesis of any portion of SEQ ID NO: 2, proteolytic digestion of SEQ ID NO: 2, or most preferably, by recombinant DNA mutagenesis techniques, well known to the skilled artisan. See. e . g. K. Struhl, "Reverse biochemistry: Methods and applications for synthesizing yeast proteins in vitro, " Meth . Enzymol . 194, 520-535. For example, in a preferred method, a nested set of deletion mutations are introduced into the intact gene (SEQ ID NO:l or SEQ ID NO: 3) encoding the native HPTLG protein such that varying amounts of the protein coding region are deleted, either from the amino terminal end, or from the carboxyl end of the protein molecule. This method can also be used to create internal fragments of the intact protein in which both the carboxyl and amino terminal ends are removed. Several appropriate nucleases can be used to create such deletions, for example Bal 31, or in the case of a single stranded nucleic acid molecule, mung bean nuclease . For simplicity in generating deletions, it is preferred that the intact HPTLG gene be cloned into a single-stranded cloning vector, such as bacteriophage M13, or equivalent. If desired, the resulting gene deletion fragments can be subcloned into any suitable vector for propagation and expression of said fragments in any suitable host cell .

The present invention also provides fragments of the intact proteins disclosed herein wherein said fragments retain biological activity. As used herein, "functional fragments" refer to fragments of SEQ ID NO: 2 that exhibit, under the appropriate conditions, some measurable biological activity, for example, protease inhibitor activity, or insulin-like growth factor binding.

Functional fragments of the intact proteins disclosed herein may be produced as described above for fragments of the intact protein, preferably using cloning techniques to produce fragments of the intact gene. Fragments may be tested for biological activity using any suitable assay, for example, the ability of a fragment to act as a protease inhibitor, or the ability of a fragment to bind an insulin- like growth factor. The Kazal-type motif, which mediates protease inhibitor activity, resides about between amino acid residues 136 through 182 of SEQ ID N0:2. Therefore, any fragment lacking the Kazal region will be unable to function as a protease inhibitor molecule. Similarly, any fragment lacking the CWCV domain, from about amino acid residues from about 332 through about 381 of SEQ ID NO: 2 would not be expected to bind insulin-like growth factors.

Gene Isolation Procedures Those skilled in the art will recognize that the HPTLG gene may be obtained by a plurality of applicable recombinant DNA techniques including, for example, polymerase chain reaction (PCR) amplification, or de novo DNA synthesis. (See e . g. , J. Sambrook et al . Molecular Cloning, 2d Ed. Chap. 14 (1989)).

Methods for constructing cDNA libraries in a suitable vector such as a plasmid or phage for propagation in procaryotic or eucaryotic cells are well known to those skilled in the art. [See e . g. J. Sambrook et al . Supra] . Suitable cloning vectors are widely available . Skilled artisans will recognize that the HPTLG gene or fragment thereof could be isolated by PCR amplification from a human cDNA library prepared from a tissue in which said gene is expressed, using oligonucleotide primers targeted to any suitable region of SEQ ID NO:l. Methods for PCR amplification are widely known in the art. See e . g. PCR Protocols: A Guide to Method and Application, Ed. M. Innis et al . , Academic Press (1990). The amplification reaction comprises template DNA, suitable enzymes, primers, and buffers, and is conveniently carried out in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CT) . A positive result is determined by detecting an appropriately-sized DNA fragment following agarose gel electrophoresis .

Protein Production Methods

One embodiment of the present invention relates to the substantially purified protein encoded by the HPTLG gene.

Skilled artisans will recognize that the proteins of the present invention can be synthesized by a number of different methods, such as chemical methods well known in the art, including solid phase peptide synthesis or recombinant methods. Both methods are described in U.S. Patent 4,617,149, incorporated herein by reference. The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts in the area. See, e . g. , H. Dugas and C. Penney, Bioorganic Chemistry (1981) Springer-Verlag, New York, 54-92. For example, peptides may be synthesized by solid-phase methodology utilizing an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, CA) and synthesis cycles supplied by Applied Biosystems.

The protein of the present invention can also be produced by recombinant DNA methods using the cloned HPTLG gene. Recombinant methods are preferred if a high yield is desired. Expression of the cloned gene can be carried out in a variety of suitable host cells well known to those skilled in the art. For this purpose, the HPTLG gene is introduced into a host cell by any suitable means, well known to those skilled in the art. While chromosomal integration of the cloned gene is within the scope of the present invention, it is preferred that the gene be cloned into a suitable extra-chromosomally maintained expression vector so that the coding region of the HPTLG gene is operably-linked to a constitutive or inducible promoter.

The basic steps in the recombinant production of the HPTLG protein are: a) constructing a natural, synthetic or semi-synthetic DNA encoding HPTLG protein;

b) integrating said DNA into an expression vector in a manner suitable for expressing the HPTLG protein, either alone or as a fusion protein;

c) transforming or otherwise introducing said vector into an appropriate eucaryotic or prokaryotic host cell forming a recombinant host cell, d) culturing said recombinant host cell in a manner to express the HPTLG protein; and

e) recovering and substantially purifying the HPTLG protein by any suitable means, well known to those skilled in the art .

Expressing Recombinant HPTLG Protein in Procaryotic and Eucaryotic Host Cells Procaryotes may be employed in the production of the HPTLG protein. For example, the Escherichia coli K12 strain 294 (ATCC No. 31446) is particularly useful for the prokaryotic expression of foreign proteins. Other strains of E. coli , bacilli such as Bacillus subtilis, enterobacteriaceae such as Salmonella typhimurium or

Serratia marcescans, various Pseudomonas species and other bacteria, such as Strepto/nyces, may also be employed as host cells in the cloning and expression of the recombinant proteins of this invention. Promoter sequences suitable for driving the expression of genes in procaryotes include b -lactamase [e . g. vector pGX2907, ATCC 39344, contains a replicon and b -lactamase gene], lactose systems [Chang et al . , Nature (London), 275:615 (1978); Goeddel et al . , Nature (London), 281:544 (1979)], alkaline phosphatase, and the tryptophan (trp) promoter system [vector pATHl (ATCC 37695) which is designed to facilitate expression of an open reading frame as a trpE fusion protein under the control of the trp promoter] . Hybrid promoters such as the tac promoter (isolatable from plasmid pDR540, ATCC-37282) are also suitable. Still other bacterial promoters, whose nucleotide sequences are generally known, enable one of skill in the art to ligate such promoter sequences to DNA encoding the proteins of the instant invention using linkers or adapters to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence operably linked to the DNA encoding the desired polypeptides . These examples are illustrative rather than limiting. The protein (s) of this invention may be synthesized either by direct expression or as a fusion protein comprising the protein of interest as a translational fusion with another protein or peptide which may be removable by enzymatic or chemical cleavage. It is often observed in the production of certain peptides in recombinant systems that expression as a fusion protein prolongs the lifespan, increases the yield of the desired peptide, or provides a convenient means of purifying the protein. A variety of peptidases (e.g. enterokinase and thrombin) which cleave a polypeptide at specific sites or digest the peptides from the amino or carboxy termini (e.g. diaminopeptidase) of the peptide chain are known. Furthermore, particular chemicals (e.g. cyanogen bromide) will cleave a polypeptide chain at specific sites. The skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi-synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See e . g. , P. Carter, "Site Specific Proteolysis of Fusion Proteins", Chapter 13, in Protein Purification: From Molecular Mechanisms to Large Scale Processes, American Chemical Society, Washington, D.C. (1990) .

In addition to procaryotes, a variety of mammalian cell systems can be used. The choice of a particular host cell depends to some extent on the particular expression vector used. Exemplary mammalian host cells suitable for use in the present invention include HepG-2 (ATCC HB 8065) , CV-1 (ATCC CCL 70) , LC-MK2 (ATCC CCL 7.1), 3T3 (ATCC CCL 92), CHO-K1 (ATCC CCL 61), HeLa (ATCC CCL 2), RPMI8226 (ATCC CCL 155), H4IIEC3 (ATCC CCL 1600),

C127I (ATCC CCL 1616) , HS-Sultan (ATCC CCL 1484) , and BHK-21 (ATCC CCL 10) , for example.

A wide variety of vectors are suitable for transforming mammalian host cells. For example, the pSV2- type vectors comprise segments of the simian virus 40 (SV40) genome required for transcription and polyadenylation. A large number of plasmid pSV2-type vectors have been constructed, such as pSV2-gpt, pSV2-neo, pSV2-dhfr, pSV2- hyg, and pSV2-b-globin, in which the SV40 promoter drives transcription of an inserted gene . These vectors are widely available from sources such as the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland, 20852, or the Northern Regional Research Laboratory (NRRL) , 1815 N. University Street, Peoria, Illinois, 61604.

Promoters suitable for expression in mammalian cells include the SV40 late promoter, promoters from eukaryotic genes, such as, for example, the estrogen- inducible chicken ovalbumin gene, the interferon genes, the gluco-corticoid-inducible tyrosine aminotransferase gene, the thymidine kinase gene promoter, and the promoters of the major early and late adenovirus genes.

Plasmid pRSVcat (ATCC 37152) comprises portions of a long terminal repeat of the Rous Sarcoma virus, a virus known to infect chickens and other host cells. This long terminal repeat contains a promoter which is suitable for use in the vectors of this invention. H. Gorman et al . , Proc. Nat . Acad. Sci . (USA) , 79, 6777 (1982). The plasmid pMSVi (NRRL B-15929) comprises the long terminal repeats of the Murine Sarcoma virus, a virus known to infect mouse and other host cells. The mouse metallothionein promoter has also been well characterized for use in eukaryotic host cells and is suitable for use in the present invention. This promoter is present in the plasmid pdBPV-MMTneo (ATCC 37224) which can serve as the starting material for the construction of other plasmids of the present invention.

Transfection of mammalian cells with vectors can be performed by a plurality of well known processes including, but not limited to, protoplast fusion, calcium phosphate co-precipitation, electroporation and the like. See, e . g. , Maniatis et al . , supra .

Some viruses also make appropriate vectors. Examples include the adenoviruses, the adeno-associated viruses, the vaccinia virus, the herpes viruses, the baculoviruses, and the rous sarcoma virus, as described in U.S. Patent 4,775,624, incorporated herein by reference.

Eucaryotic microorganisms such as yeast and other fungi are also suitable host cells. The yeast Saccharomyces cerevisiae is the preferred eucaryotic microorganism. Other yeasts such as Kluyveromyces lactis are also suitable. For expression in Saccharomyces , the plasmid YRp7 (ATCC-40053) , for example, may be used. See, e . g. , L. Stinchcomb et al . , Nature, 282, 39 (1979); J. Kingsman et al . , Gene, 7, 141 (1979); S. Tschemper et al . , Gene, 10, 157 (1980). Plasmid YRp7 contains the TRP1 gene which provides a selectable marker for use in a trpl auxotrophic mutant .

Purification of Recombinantly-Produced HPTLG Protein

An expression vector carrying the cloned HPTLG gene of human hypothalamus is transformed or transfected into a suitable host cell using standard methods. Cells which contain the vector are propagated under conditions suitable for expression of the HPTLG protein. If the recombinant gene has been placed under the control of an inducible promoter then suitable growth conditions would incorporate the appropriate inducer . The recombinantly- produced protein may be purified from cellular extracts of transformed cells by any suitable means.

In a preferred process for protein purification the HPTLG gene is modified at the 5 ' end to incorporate several histidine residues at the amino terminus of the HPTLG protein product. This "histidine tag" enables a single-step protein purification method referred to as "immobilized metal ion affinity chromatography" (IMAC) , essentially as described in U.S. Patent 4,569,794 which hereby is incorporated by reference. The IMAC method enables rapid isolation of substantially pure HPTLG protein starting from a crude cellular extract . Production of Antibodies The proteins of this invention and fragments thereof may be used in the production of antibodies. The term "antibody" as used herein describes antibodies, fragments of antibodies (such as, but not limited, to Fab, Fab', Fab2 ' , and Fv fragments), and chimeric, humanized, veneered, resurfaced, or CDR-grafted antibodies capable of binding antigens of a similar nature as the parent antibody molecule from which they are derived. The instant invention also encompasses single chain polypeptide binding molecules. The production of antibodies, both monoclonal and polyclonal, in animals, especially mice, is well known in the art. See, e . g. , C. Milstein, Handbook of Experimental Immunology, (Blackwell Scientific Pub., 1986); J. Goding, Monoclonal Antibodies: Principles and Practice, (Academic Press, 1983) . For the production of monoclonal antibodies the basic process begins with injecting a mouse, or other suitable animal, with an immunogen. The mouse is subsequently sacrificed and cells taken from its spleen are fused with myeloma cells, resulting in a hybridoma that reproduces in vi tro . The population of hybridomas is screened to isolate individual clones, each of which secretes a single antibody species, specific for the immunogen. Each antibody obtained in this way is the clonal product of a single B cell . Chimeric antibodies are described in U.S. Patent

No. 4,816,567, the entire contents of which is herein incorporated by reference. This reference discloses methods and vectors for the preparation of chimeric antibodies. An alternative approach is provided in U.S. Patent No. 4,816,397, the entire contents of which is herein incorporated by reference . This patent teaches co- expression of the heavy and light chains of an antibody in the same host cell .

The approach of U.S. Patent 4,816,397 has been further refined in European Patent Publication No. 0 239

400. The teachings of this European patent publication are a preferred format for genetic engineering of monoclonal antibodies. In this technology the complementarity determining regions (CDRs) of a human antibody are replaced with the CDRs of a murine monoclonal antibody, thereby converting the specificity of the human antibody to the specificity of a murine antibody.

Single chain antibodies and libraries thereof are yet another variety of genetically engineered antibody technology that is well known in the art. (See, e . g. R.E. Bird, et al . , Science 242:423-426 (1988); PCT Publication Nos. WO 88/01649, WO 90/14430, and WO 91/10737. Single chain antibody technology involves covalently joining the binding regions of heavy and light chains to generate a single polypeptide chain. The binding specificity of the intact antibody molecule is thereby reproduced on a single polypeptide chain.

The antibodies contemplated by this invention are useful in diagnostics, therapeutics or in diagnostic/therapeutic combinations.

The proteins of this invention or suitable fragments thereof can be used to generate polyclonal or monoclonal antibodies, and various inter-species hybrids, or humanized antibodies, or antibody fragments, or single-chain antibodies. The techniques for producing antibodies are well known to skilled artisans. (See e . g. A.M. Campbell, Monoclonal Antibody Technology: Laboratory Techniques in Biochemsitry and Molecular Biology, Elsevier Science Publishers, Amsterdam (1984) ; Kohler and Milstein, Nature 256, 495-497 (1975) ; Monoclonal Antibodies: Principles & Applications Ed. J.R. Birch & E.S. Lennox, Wiley-Liss, 1995.

A protein used as an immunogen may be modified or administered in an adjuvant, by subcutaneous or intraperitoneal injection into, for example, a mouse or a rabbit. For the production of monoclonal antibodies, spleen cells from immunized animals are removed, fused with myeloma cells, such as SP2/0-Agl4 cells, and allowed to become monoclonal antibody producing hybridoma cells in the manner known to the skilled artisan. Hybridomas that secrete a desired antibody molecule can be screened by a variety of well known methods, for example ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al . Exp . Cell Res . 175, 109-124 (1988) ; Monoclonal Antibodies : Principles & Applications Ed. J.R. Birch & E.S. Lennox, Wiley-Liss, 1995). For some applications labeled antibodies are desirable. Procedures for labeling antibody molecules are widely known, including for example, the use of radioisotopes, affinity labels, such as biotin or avidin, enzymatic labels, for example horseradish peroxidase, and fluorescent labels, such as FITC or rhodamine (See e . g. Enzyme-Mediated Immunoassay, Ed. T. Ngo, H. Lenhoff, Plenum Press 1985; Principles of Immunology and Immunodiagnostics, R.M. Aloisi, Lea & Febiger, 1988) .

Labeled antibodies are useful for a variety of diagnostic applications. In one embodiment the present invention relates to the use of labeled antibodies to detect the presence of HPTLG. Alternatively, the antibodies could be used in a screen to identify potential modulators of HPTLG. For example, in a competitive displacement assay, the antibody or compound to be tested is labeled by any suitable method. Competitive displacement of an antibody from an antibody-antigen complex by a test compound such that a test compound-antigen complex is formed provides a method for identifying compounds that bind HPTLG. Other embodiments of the present invention comprise isolated nucleic acid sequences which encode SEQ ID NO: 2. As skilled artisans will recognize, the amino acid compounds of the invention can be encoded by a multitude of different nucleic acid sequences because most of the amino acids are encoded by more than one codon. Because these alternative nucleic acid sequences would encode the same amino acid sequences, the present invention further comprises these alternate nucleic acid sequences. Also contemplated are related nucleic acids that are at least about 75% identical to SEQ ID NO:l or SEQ ID NO: 3, or to their complementary sequence, or nucleic acids that hybridize to SEQ ID NO:l or SEQ ID NO: 3 under low stringency or high stringency conditions. Such sequences may come, for example, from other related genes, or comprise fragments of SEQ ID NO:l or SEQ ID NO: 3. Fragments of the nucleic acids disclosed herein could be useful, for example, as hybridization probes or primers.

The HPTLG gene, which comprises nucleic acid encoding SEQ ID NO: 2, may be produced using synthetic methodology. The synthesis of nucleic acids is well known in the art. See, e . g. , E.L. Brown, R. Belagaje, M.J. Ryan, and H.G. Khorana, Methods in Enzymology, 68:109-151 (1979) . The DNA segments corresponding to the HPTLG gene could be generated using a conventional DNA synthesizing apparatus, such as the Applied Biosystems Model 380A or 380B DNA synthesizers (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404) which employ phosphoramidite chemistry. Alternatively, phosphotriester chemistry may be employed to synthesize the nucleic acids of this invention. [See, e . g. , M.J. Gait, ed., Oligonucleotide Synthesis, A Practical Approach, (1984).]

In an alternative methodology, namely PCR, the DNA sequence comprising a portion or all of SEQ ID NO:l can be generated from a suitable cDNA library, or cDNA pool wherein said cDNA molecules are derived from a tissue that expresses the HPTLG gene. For this purpose, suitable oligonucleotide primers complementary to SEQ ID NO:l or region therein are prepared, as described in U.S. Patent No. 4,889,818, which hereby is incorporated by reference. Protocols for performing the PCR are disclosed in, for example, PCR Protocols: A Guide to Method and Applications, Ed. Michael A. Innis et al . , Academic Press, Inc. (1990).

The ribonucleic acids of the present invention may be prepared using the polynucleotide synthetic methods discussed supra, or they may be prepared enzymatically using RNA polymerase to transcribe a HPTLG DNA template.

The most preferred systems for preparing the ribonucleic acids of the present invention employ the RNA polymerase from the bacteriophage T7 or the bacteriophage SP6. These RNA polymerases are highly specific, requiring the insertion of bacteriophage-specific sequences at the 5' end of the template to be transcribed. See, J. Sambrook, et al . , supra, at 18.82-18.84.

This invention also provides nucleic acids, RNA or DNA, which are complementary to SEQ ID NO:l or SEQ ID NO: 3. Nucleic Acid Probes

The present invention also provides probes and primers useful for a variety of molecular biology techniques including, for example, hybridization screens of genomic, subgenomic, or cDNA libraries. Such hybridization screens are useful as methods to identify homologous and/or functionally related sequences from the same or other organisms. A nucleic acid compound comprising SEQ ID NO: 1, SEQ ID NO: 3 or a complementary sequence thereof, or a fragment thereof, which is at least 14 base pairs in length, and which will selectively hybridize to human DNA or mRNA encoding HPTLG protein or fragment thereof, or a functionally related protein, is provided. Preferably, the 14 or more base pair compound is DNA. See e . g. B. Wallace and G. Miyada, "Oligonucleotide Probes for the Screening of Recombinant DNA Libraries," In Met . Enzym. , 152, 432-442, Academic Press (1987) .

Probes and primers can be prepared by enzymatic or recombinant methods, well known to those skilled in the art (See e . g. Sambrook et al . supra) . A probe may be a single stranded nucleic acid sequence that is complementary in some particular degree to a nucleic acid sequence sought to be detected ("target sequence") . A probe may be labeled with a detectable moiety such as a radio-isotope, antigen, or chemiluminescent moiety. A description of the use of nucleic acid hybridization as a procedure for the detection of particular nucleic acid sequences is presented in U.S. Patent No. 4,851,330 to Kohne, entitled "Method for Detection, Identification and Quantitation of Non-Viral Organisms."

DNA sequence information provided by the present invention allows for the preparation of relatively short DNA (or RNA) sequences having the ability to specifically hybridize to gene sequences disclosed herein. In these aspects, nucleic acid probes of an appropriate length are prepared. The ability of such nucleic acid probes to specifically hybridize to a polynucleotide encoding a HPTLG gene or related sequence lends particular utility in a variety of embodiments. Most importantly, the probes may be used in a variety of assays for detecting the presence of complementary sequences in a given sample .

In certain embodiments, it is advantageous to use oligonucleotide primers. The sequence of such primers is designed using a polynucleotide of the present invention for use in detecting, amplifying or mutating a defined segment of a gene or polynucleotide that encodes a HPTLG polypeptide.

Preferred nucleic acid sequences employed for hybridization studies, or assays, include probe molecules that are complementary to at least an about

14 to an about 70-nucleotide long stretch of a polynucleotide that encodes a HPTLG polypeptide, such as the nucleotide base sequences designated as SEQ ID NO:l or SEQ ID NO: 3. A length of at least 14 nucleotides helps to ensure that the fragment is of sufficient length to form a duplex molecule that is both stable and selective . In order to increase stability and selectivity of the hybrid, molecules having complementary sequences over stretches greater than 14 bases in length are generally preferred. One will generally prefer to design nucleic acid molecules having gene-complementary stretches of 25 to 40 nucleotides, 55 to 70 nucleotides, or even longer where desired. Such fragments may be readily prepared by, for example, directly synthesizing the fragment by chemical means, by application of nucleic acid reproduction technology, such as the PCR TM technology of U.S. Pat. No. 4,603,102, herein incorporated by reference, or by excising selected DNA fragments from recombinant plasmids containing appropriate inserts and suitable restriction enzyme sites. The following guidelines are useful for designing probes with desirable characteristics. The extent and specificity of hybridization reactions are affected by a number of factors that determine the sensitivity and specificity of a particular probe, whether perfectly complementary to its target or not. The affect of various experimental parameters and conditions are well known to those skilled in the art.

First, the stability of the probe: target nucleic acid hybrid should be chosen to be compatible with the assay conditions. This may be accomplished by avoiding long A and T rich sequences, by terminating the hybrids with G:C base pairs and by designing a probe with an appropriate Tm (i.e. melting temperature) . The melting profile, including the Tm of a hybrid comprising an oligonucleotide and target sequence, may be determined using a Hybridization Protection Assay. The probe should be chosen so that the length and % GC content result in a Tm about 2°-10° C higher than the temperature at which the final assay will be performed. The base composition of the probe is also a significant factor because G-C base pairs exhibit greater thermal stability as compared to A-T base pairs. Thus, hybridization involving complementary nucleic acids of higher G-C content will be more stable at higher temperatures .

The ionic strength and incubation temperature under which a probe will be used should also be taken into account. It is known that hybridization will increase as the ionic strength of the reaction mixture increases, and that the thermal stability of molecular hybrids will increase with increasing ionic strength. On the other hand, chemical reagents such as formamide, urea, DMSO and alcohols, which disrupt hydrogen bonds, increase the stringency of hybridization. Destabilization of hydrogen bonds by such reagents can greatly reduce the Tm. In general, optimal hybridization for synthetic oligonucleotide probes of about 10-50 bases in length occurs approximately 5° C below the melting temperature for a given duplex. Incubation at temperatures below the optimum may allow mismatched base sequences to hybridize and can therefore result in reduced specificity. The length of the target nucleic acid sequence and, accordingly, the length of the probe sequence can also be important. In some cases, there may be several sequences from a particular region, varying in location and length, which will yield probes with the desired hybridization characteristics. In other cases, one sequence may be significantly better than another even though the one sequence differs merely by a single base. Finally, there can be intramolecular and intermolecular hybrids formed within a probe if there is sufficient self-complementarity. Such structures can be avoided through careful probe design.

Computer programs are available to search for this type of interaction.

A probe molecule may be used for hybridizing to a sample suspected of possessing a HPTLG or HPTLG-related nucleotide sequence. The hybridization reaction is carried out under suitable conditions of stringency.

Alternatively, such DNA molecules may be used in a number of techniques including their use as: (1) diagnostic tools to detect polymorphisms in DNA samples from a human or other mammal; (2) means for detecting and isolating homologs of HPTLG and related polypeptides from a DNA library potentially containing such sequences; (3) primers for hybridizing to related sequences for the purpose of amplifying those sequences; and (4) primers for altering the native HPTLG DNA sequences; as well as other techniques which rely on the similarity of the DNA sequences to those of the HPTLG DNA segments herein disclosed.

Once synthesized, oligonucleotide probes may be labeled by any of several well known methods. See e . g. Maniatis et.al., Molecular Cloning (2d ed. 1989). Useful labels include radioisotopes, as well as non-radioactive reporting groups. Isotopic labels include H³, S³⁵, P³², I¹²⁵, Cobalt, and C¹⁴. Most methods of isotopic labeling involve the use of enzymes and include methods such as nick-translation, end- labeling, second strand synthesis, and reverse transcription. When using radio-labeled probes, hybridization can be detected by autoradiography, scintillation counting, or gamma counting. The detection method selected will depend upon the hybridization conditions and the particular radio isotope used for labeling.

Non-isotopic materials can also be used for labeling, and may be introduced internally into the sequence or at the end of the sequence. Modified nucleotides may be incorporated enzymatically or chemically, and chemical modifications of the probe may be performed during or after synthesis of the probe, for example, by the use of non- nucleotide linker groups. Non-isotopic labels include fluorescent molecules, chemiluminescent molecules, enzymes, cofactors, enzyme substrates, haptens or other ligands.

In a preferred embodiment of the invention, the length of an oligonucleotide probe is greater than or equal to about 18 nucleotides and less than or equal to about 50 nucleotides. Labeling of an oligonucleotide of the present invention may be performed enzymatically using [³²P] -labeled ATP and the enzyme T4 polynucleotide kinase.

Vectors

Another aspect of the present invention relates to recombinant DNA cloning vectors and expression vectors comprising the nucleic acids of the present invention. The preferred nucleic acid vectors are those which comprise DNA. The most preferred recombinant DNA vectors comprise the isolated DNA sequence, SEQ ID NO:l. The skilled artisan understands that choosing the most appropriate cloning vector or expression vector depends upon a number of factors including the availability of restriction enzyme sites, the type of host cell into which the vector is to be transfected or transformed, the purpose of the transfection or transformation (e.g., stable transformation as an extrachromosomal element, or integration into the host chromosome) , the presence or absence of readily assayable or selectable markers (e.g., antibiotic resistance and metabolic markers of one type and another) , and the number of copies of the gene to be present in the host cell.

Vectors suitable to carry the nucleic acids of the present invention comprise RNA viruses, DNA viruses, lytic bacteriophages, lysogenic bacteriophages, stable bacteriophages, plasmids, viroids, and the like. The most preferred vectors are plasmids .

When preparing an expression vector the skilled artisan understands that there are many variables to be considered, for example, whether to use a constitutive or inducible promoter. Inducible promoters are preferred because they enable high level, regulatable expression of an operably-linked gene. The skilled artisan will recognize a number of inducible promoters which respond to a variety of inducers, for example, carbon source, metal ions, and heat. The practitioner also understands that the amount of nucleic acid or protein to be produced dictates, in part, the selection of the expression system. The addition of certain nucleotide sequences is useful for directing the localization of a recombinant protein. For example, a sequence encoding a signal peptide preceding the coding region of a gene, is useful for directing the extra-cellular export of a resulting polypeptide.

Host cells harboring the nucleic acids disclosed herein are also provided by the present invention. Any host that can be transfected or transformed with a vector that comprises a nucleic acid of the present invention, and that will express said nucleic acid is suitable.

The present invention also provides a method for constructing a recombinant host cell capable of expressing SEQ ID NO: 2, said method comprising transforming or otherwise introducing into a host cell a recombinant DNA vector that comprises an isolated DNA sequence which encodes SEQ ID NO: 2. Any host cell that can accomodate high level expression of an exogenously introduced gene is suitable. Preferred vectors for expression are those which comprise SEQ ID NO:l. An especially preferred expression vector for use in E. coli is a plasmid that comprises SEQ ID NO:l. Transformed host cells may be cultured under conditions well known to skilled artisans such that SEQ ID NO: 2 is expressed, thereby producing HPTLG protein in the recombinant host cell.

For the purpose of identifying or developing inhibitors or other modifiers of the proteins disclosed herein, it would be desirable to identify compounds that bind the HPTLG protein. A method for determining agents that will bind the HPTLG protein comprises contacting the HPTLG protein with a test compound and monitoring binding by any suitable means. The instant invention provides such a screening system useful for discovering compounds which bind the HPTLG protein, said screening system comprising the steps of:

a) preparing HPTLG protein;

b) exposing said HPTLG protein to a test compound;

c) quantifying the binding of said compound to HPTLG.

Utilization of the screening system described above provides a means to determine compounds which may alter the biological function of HPTLG. This screening method may be adapted to automated procedures such as a

PANDEX (Baxter-Dade Diagnostics) system, allowing for efficient high-volume screening of potential therapeutic agents.

In such a screening protocol, HPTLG is prepared as described herein, preferably using recombinant DNA technology. A test compound is introduced into the reaction vessel containing HPTLG. In one method radioactively or chemically-labeled compound is used and the binding of said compound to HPTLG is assayed by any suitable means . The absence or diminution of the chemical label or radioactivity indicates the degree to which the reaction is inhibited.

In such a screening protocol HPTLG is prepared as described herein, preferably using recombinant DNA technology. A test compound is introduced into a reaction vessel containing the HPTLG protein, or fragment thereof, and binding of HPTLG is determined by any suitable means. For example, in one method radioactively-labeled or chemically-labeled test compound may be used. Binding of the protein by the compound is assessed, for example, by quantifying bound label versus unbound label using any suitable method. Binding of a test compound may also be carried out by a method disclosed in U.S. Patent 5,585,277, which hereby is incorporated by reference. In this method, binding of a test compound to a protein is assessed by monitoring the ratio of folded protein to unfolded protein, for example, by monitoring sensitivity of said protein to a protease, or amenability to binding of said protein by an antibody specific for the folded state of the protein.

The foregoing screening methods are useful for identifying a ligand of a HPTLG protein, perhaps as a lead to a pharmaceutical compound. A ligand that binds HPTLG or related fragment thereof is identified by combining a test ligand with HPTLG under conditions that cause the protein to exist in a ratio of folded to unfolded states. If the test ligand binds the folded state of the protein, the relative amount of folded protein will be higher than in the case of a test ligand that does not bind the protein. A similar result would be expected in a control reaction in which test ligand is left out of the reaction mix. The ratio of protein in folded versus unfolded states is easily determinable by, for example, susceptibility to digestion by a protease, binding to a specific antibody, or binding to chaperonin protein, or binding to a suitable surface.

Skilled artisans will recognize that IC5₀ values are dependent on the selectivity of the compound tested. For example, a compound with an IC5₀ which is less than 10 nM is generally considered an excellent candidate for drug therapy. However, a compound which has a lower affinity, but is selective for a particular target, may be an even better candidate. The skilled artisan will recognize that any information regarding inhibitory activity or selectivity of a particular compound is beneficial in the pharmaceutical arts.

The following examples more fully describe the present invention. Those skilled in the art will recognize that the particular reagents, equipment, and procedures described are merely illustrative and are not intended to limit the present invention in any manner.

EXAMPLE 1 RT-PCR Amplification of HPTLG Gene from mRNA

The HPTLG gene is isolated by reverse transcriptase PCR (RT-PCR) using conventional methods. Total RNA from a tissue that expresses the HPTLG gene, for example, human hypothalamus, is prepared and first strand cDNA is obtained by any suitable method. The HPTLG gene is amplified directly by the PCR using specific primers directed at any suitable region of SEQ ID NO:l.

Amplification is carried out by adding to the first strand cDNA (dried under vacuum) : 8 ul of 10X synthesis buffer (200 mM Tris-HCl, pH 8.4; 500 mM KCl, 25 mM MgCl₂, 1 ug/ul BSA) ; 68 ul distilled water; 1 ul each of a 10 uM solution of each primer; and 1 ul Taq DNA polymerase (2 to 5 U/ul) . The reaction is heated at 94° C for 5 min. to denature the RNA/cDNA hybrid, and then 15 to 30 cycles of PCR amplification are performed using any suitable thermal cycle apparatus. The amplified sample may be analyzed by agarose gel electrophoresis to check for the presence of an appropriately sized-fragment .

EXAMPLE 2

Production of a Vector for Expressing HPTLG in a Host Cell An expression vector suitable for expressing HPTLG or fragment thereof in a variety of procaryotic host cells, such as E. coli is easily made. The vector contains an origin of replication (Ori) , an ampicillin resistance gene

(Amp) useful for selecting cells which have incorporated the vector following a tranformation procedure, and further comprises the T7 promoter and T7 terminator sequences in operable linkage to a HPTLG coding region. Plasmid pETHA (obtained from Novogen, Madison WI) is a suitable parent plasmid. pETHA is linearized by restriction with endonucleases Ndel and BamHI . Linearized pETHA is ligated to a DNA fragment bearing Ndel and BamHI sticky ends and comprising the coding region of the HPTLG gene as disclosed by SEQ ID NO:l, or fragment thereof.

The HPTLG gene used in this construction may be slightly modified at the 5' end (amino terminus of encoded protein) in order to simplify purification of the encoded protein product. For this purpose, an oligonucleotide encoding 8 histidine residues is inserted after the ATG start codon. Placement of the histidine residues at the amino terminus of the encoded protein serves to enable the IMAC one-step protein purification procedure.

EXAMPLE 3 Recombinant Expression and Purification of HPTLG Protein An expression vector that carries an ORF encoding HPTLG, or fragment thereof, and which ORF is operably-linked to an expression promoter, is transformed into E. coli BL21 (DE3) (hsdS gal lclts857 indlSam7nin51acUV5-T7gene 1) using standard methods. Transformants, selected for resistance to ampicillin, are chosen at random and tested for the presence of the vector by agarose gel electrophoresis using quick plasmid preparations. Colonies which contain the vector are grown in L broth and the protein product encoded by the vector-borne ORF is purified by immobilized metal ion affinity chromatography (IMAC) , essentially as described in US Patent 4,569,794.

Briefly, the IMAC column is prepared as follows. A metal-free chelating resin (e.g. Sepharose 6B IDA, Pharmacia) is washed in distilled water to remove preservative substances and infused with a suitable metal ion [e.g. Ni(II), Co (II), or Cu(II)] by adding a 50mM metal chloride or metal sulfate aqueous solution until about 75% of the interstitial spaces of the resin are saturated with colored metal ion. The column is then ready to receive a crude cellular extract containing the recombinant protein product .

After removing unbound proteins and other materials by washing the column with any suitable buffer, pH 7.5, the bound protein is eluted in any suitable buffer at pH 4.3, or preferably with an imidizole-containing buffer at pH 7.5.

EXAMPLE 4 Tissue Distributuion of HPTLG mRNA The presence of HPTLG mRNA in a variety of human tissues was analyzed by Northern analysis. Total RNA from different tissues or cultured cells was isolated by a standard guanidine chloride/phenol extraction method, and poly-A⁺ RNA was isolated using oligo (dT) -cellulose type 7 (Pharmacia) . Electrophoresis of RNA samples was carried out in formaldehyde followed by capillary transfer to Zeta-Probe TM nylon membranes (Bio-Rad, Hercules, Calif.). SEQ ID NO:l was the template for generating probes using a MultiPrime™ random priming kit (Amersham, Arlington Heights, 111.). The efficiency of the labeling reaction was approximately 4 x 10¹⁰ cpm incorporated per μg of template. The hybridization buffer contained 0.5M sodium phosphate, 7% SDS (wt/vol), 1% BSA (wt/vol) , and 1 mM EDTA. Prehybridization was carried out in hybridization buffer at 65° C for 2 h and ³²P-labeled probe was added and incubation continued overnight. The filters were washed in Buffer A (40 mM sodium phosphate pH 7.2, 5% SDS [wt/vol], 0.5% BSA [wt/vol], and 1 mM EDTA) at 65° C for 1 h, and then in Buffer B (40 mM sodium phosphate, pH 7.2, 1% SDS [wt/vol], and 1 mM EDTA) at 65° C for 20 min. The filters were air-dried and exposed to Kodak X-OMAT AR film at -80° C with an intensifying screen.

EXAMPLE 5 Detecting Ligands that Bind HPTLG Using a Chaperonin

Protein Assay

The wells of an ELISA plate are coated with chaperonin by incubation for several hours with a 4 ug/ml solution of the protein in Tris-buffered Saline (TBS: 10 mM Tris-HCl, pH7.5, 0.2M NaCl) . The plates are then washed 3 times with TBS containing 0.1% Tween-20 (TBST) . Then, a mixture of HPTLG protein (sufficient amount to saturate about 50% of the binding sites on chaperonin) and test compound (10^~9 to 10^"5 M) in about 50 μl volume is added to each well of the plate for an incubation of about 60 minutes. Aliquots of the well solutions are then transferred to the wells of fresh plates and incubated for 60 minutes at room temperature, followed by 3 washes with TBST. Next, about 50 μl of an antibody specific for HPTLG plus 5% nonfat dry milk are added to each well for a 30 minute incubation at room temperature. After washing, about 50 μl of goat anti-rabbit IgG alkaline phosphatase conjugate at an appropriate dilution in TBST plus 5% nonfat dry milk are added to each will and incubated 30 minutes at room temperature. The plates are washed again with TBST and 0.1 ml of 1 mg/ml p- nitrophenylphosphate in 0.1% diethanolamine is added. Color development (proportional to bound alkaline phosphatase antibody conjugate) is monitored with an ELISA plate reader. When test ligand binding has occurred, ELISA analysis reveals HPTLG in solution at higher concentrations than in the absence of test ligand.

EXAMPLE 6 Production of an Antibody to a HPTLG Protein

Substantially pure protein or fragment thereof is isolated from transfected or transformed cells using any of the well known methods in the art, or by a method specifically disclosed herein. Concentration of protein in a final preparation is adjusted, for example, by filtration through an Amicon filter device such that the level is about 1 to 5 ug/ml . Monoclonal or polyclonal antibody can be prepared as follows .

Monoclonal antibody can be prepared from murine hybridomas according to the method of Kohler and Milstein (Nature, 256, 495, 1975), or a modified method thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the protein or fragment thereof, or fusion peptide thereof, over a period of a few weeks. The mouse is then sacrificed and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells. Fused cells that produce antibody are identified by any suitable immunoassay, for example, ELISA, as described in E. Engvall, Meth . Enzy ol . , 70, 419, 1980.

Polyclonal antiserum can be prepared by well known methods (See e . g. J. Vaitukaitis et.al. Clin . Endocirnol . Metab. 33, 988, 1971) that involve immunizing suitable animals with the proteins, fragments thereof, or fusion proteins thereof, disclosed herein. Small doses (e.g. nanogram amounts) of antigen administered at multiple intradermal sites appears to be the most reliable method.

Claims

WE CLAIM :

1. A substantially pure HPTLG protein having the amino acid sequence which is SEQ ID NO: 2.

2. An isolated nucleic acid compound encoding the protein of Claim 1, said protein having the amino acid sequence which is SEQ ID NO: 2.

3. An isolated nucleic acid compound encoding the protein of Claim 1 wherein said compound has a sequence selected from the group consisting of :

(a) SEQ ID NO:l;

(b) SEQ ID NO: 3; or (c) a nucleic acid compound complementary to (a) or (b) .

4. An isolated nucleic acid compound of Claim 3 wherein the sequence of said compound is SEQ ID NO:l or a sequence complementary to SEQ ID NO : 1.

5. An isolated nucleic acid compound of Claim 3 wherein the sequence of said compound is SEQ ID NO: 3 or a sequence complementary to SEQ ID NO: 3.

6. A vector comprising an isolated nucleic acid compound of Claim 3.

7. A vector, as in Claim 6, wherein said isolated nucleic acid compound is SEQ ID NO:l operably-linked to a promoter sequence .

8. A host cell containing the vector of Claim 7.

9. A method for constructing a recombinant host cell having the potential to express SEQ ID NO: 2, said method comprising introducing into said host cell by any suitable means a vector of Claim 7.

10. A method for expressing SEQ ID NO: 2 in the recombinant host cell of Claim 9, said method comprising culturing said recombinant host cell under conditions suitable for gene expression.

11. An antibody that selectively binds to a protein identified herein as SEQ ID NO: 2, or fragment thereof.

12. A substantially pure peptide wherein said peptide comprises residues 1 through 182 of SEQ ID NO:2.

13. A substantially pure peptide wherein said peptide comprises residues 183 through 433 of SEQ ID NO:2.

14. A substantially pure peptide consisting of at least any 50 or more contiguous amino acid residues of SEQ ID N0:2.

15. A substantially pure fragment of a protein identified herein as SEQ ID NO: 2, wherein said fragment functions as an protease inhibitor.

16. A substantially pure fragment, as in Claim 15, wherein said fragment comprises amino acid residues 136 through 182 of SEQ ID NO:2.

17. A substantially pure fragment of a protein identified herein as SEQ ID NO: 2, wherein said fragment functions as a mediator of cell surface adhesion.

18. A substantially pure fragment , as in Claim 17, wherein said fragment comprises amino acid residues 332 through 381 of SEQ ID NO: 2.

19. A nucleic acid molecule comprising a fragment of SEQ ID NO:l, wherein said fragment consists of at least 14 contiguous base pairs therefrom.

20. An isolated nucleic acid compound that encodes a protein having HPTLG activity wherein said nucleic acid hybridizes to SEQ ID NO:l under low stringency conditions.

21. A substantially pure protein encoded by the nucleic acid of Claim 20.

22. An isolated nucleic acid compound that encodes a protein having HPTLG activity wherein said nucleic acid hybridizes to SEQ ID NO:l under high stringency conditions.

23. A substantially pure protein encoded by the nucleic acid of Claim 22.

-1 - SEQUENCE LISTING

<110> Sankhavaram, Patanjali R. Little, Rebecca A. Basinski, Margret B. Rosteck Jr., Paul R. Burgett , Stanley G.

<120> HPTLG Gene From Human Hypothalamus

<130> X-11328

<140> <141>

<160> 3

<170> Patentln Ver. 2.0

<210> 1

<211> 1299

<212> DNA

<213> Homo sapiens

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tct cag tat gac aag gaa gtc gga cag tgg aac aaa ttc cga gac gat 192 Ser Gin Tyr Asp Lys Glu Val Gly Gin Trp Asn Lys Phe Arg Asp Asp 50 55 60

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gtt gga cag tgc tgg tgt gtt gac aga tat gga aat gaa gtc atg gga 1104 Val Gly Gin Cys Trp Cys Val Asp Arg Tyr Gly Asn Glu Val Met Gly 355 360 365

tec aga ata aat ggt gtt gca gat tgt get ata gat ttt gag ate tec 1152 Ser Arg lie Asn Gly Val Ala Asp Cys Ala lie Asp Phe Glu lie Ser 370 375 380

gga gat ttt get agt ggc gat ttt cat gaa tgg act gat gat gag gat 1200 Gly Asp Phe Ala Ser Gly Asp Phe His Glu Trp Thr Asp Asp Glu Asp 385 390 395 400

gat gaa gac gat att atg aat gat gaa gat gaa att gaa gat gat gat 1248 Asp Glu Asp Asp lie Met Asn Asp Glu Asp Glu lie Glu Asp Asp Asp 405 410 415

gaa gat gaa ggg gat gat gat gat ggt ggt gat gac cat gat gta tac 1296 Glu Asp Glu Gly Asp Asp Asp Asp Gly Gly Asp Asp His Asp Val Tyr 420 425 430 - 6

att 1299

He

<210> 2

<211> 433

<212> PRT

<213> Homo sapiens

<400> 2

Met Leu Lys Val Ser Ala Val Leu Cys Val Cys Ala Ala Ala Trp Cys 1 5 10 15

Ser Gin Ser Leu Ala Ala Ala Ala Ala Val Ala Ala Ala Gly Gly Arg 20 25 30

Ser Asp Gly Gly Asn Phe Leu Asp Asp Lys Gin Trp Leu Thr Thr He 35 40 45

Ser Gin Tyr Asp Lys Glu Val Gly Gin Trp Asn Lys Phe Arg Asp Asp 50 55 60

Asp Tyr Phe Arg Thr Trp Ser Pro Gly Lys Pro Phe Asp Gin Ala Leu 65 70 75 80

Asp Pro Ala Lys Asp Pro Cys Leu Lys Met Lys Cys Ser Arg His Lys -7 - 85 90 95

Val Cys He Ala Gin Asp Ser Gin Thr Ala Val Cys He Ser His Arg 100 105 110

Arg Leu Thr His Arg Met Lys Glu Ala Gly Val Asp His Arg Gin Trp 115 120 125

Arg Gly Pro He Leu Ser Thr Cys Lys Gin Cys Pro Val Val Tyr Pro 130 135 140

Ser Pro Val Cys Gly Ser Asp Gly His Thr Tyr Ser Phe Gin Cys Lys 145 150 155 160

Leu Glu Tyr Gin Ala Cys Val Leu Gly Lys Gin He Ser Val Lys Cys 165 170 175

Glu Gly His Cys Pro Cys Pro Ser Asp Lys Pro Thr Ser Thr Ser Arg 180 185 190

Asn Val Lys Arg Ala Cys Ser Asp Leu Glu Phe Arg Glu Val Ala Asn 195 200 205

Arg Leu Arg Asp Trp Phe Lys Ala Leu His Glu Ser Gly Ser Gin Asn 210 215 220

Lys Lys Thr Lys Thr Leu Leu Arg Pro Glu Arg Ser Arg Phe Asp Thr -8 - 225 230 235 240

Ser He Leu Pro He Cys Lys Asp Ser Leu Gly Trp Met Phe Asn Arg 245 250 255

Leu Asp Thr Asn Tyr Asp Leu Leu Leu Asp Gin Ser Glu Leu Arg Ser 260 265 270

He Tyr Leu Asp Lys Asn Glu Gin Cys Thr Lys Ala Phe Phe Asn Ser 275 280 285

Cys Asp Thr Tyr Lys Asp Ser Leu He Ser Asn Asn Glu Trp Cys Tyr 290 295 300

Cys Phe Gin Arg Gin Gin Asp Pro Pro Cys Gin Thr Glu Leu Ser Asn 305 310 315 320

He Gin Lys Arg Gin Gly Val Lys Lys Leu Leu Gly Gin Tyr He Pro 325 330 335

Leu Cys Asp Glu Asp Gly Tyr Tyr Lys Pro Thr Gin Cys His Gly Ser 340 345 350

Val Gly Gin Cys Trp Cys Val Asp Arg Tyr Gly Asn Glu Val Met Gly 355 360 365

Ser Arg He Asn Gly Val Ala Asp Cys Ala He Asp Phe Glu He Ser - 9 - 370 375 380

Gly Asp Phe Ala Ser Gly Asp Phe His Glu Trp Thr Asp Asp Glu Asp 385 390 395 400

Asp Glu Asp Asp He Met Asn Asp Glu Asp Glu He Glu Asp Asp Asp 405 410 415

Glu Asp Glu Gly Asp Asp Asp Asp Gly Gly Asp Asp His Asp Val Tyr 420 425 430

He

<210> 3

<211> 1299

<212> RNA

<213> Homo sapiens

<400> 3 augcueaagg ugucagecgu aeugugugug ugugeagccg cuuggugcag ueagucucuc 60

geagcugceg eggcgguggc ugcageeggg gggcgguegg acggcgguaa uuuucuggau 120

gauaaacaau ggcueaccac aaucueucag uaugaeaagg aagucggaca guggaacaaa 180 -10 - uuccgagacg augauuauuu eegcacuugg aguccaggaa aaeccuucga ucaggeuuua 240

gauccagcua aggauccaug cuuaaagaug aaauguaguc gccauaaagu augcauugcu 300

caagauucuc agacugcagu cugcauuagu caccggaggc uuacacacag gaugaaagaa 360

gcaggaguag accauaggca guggaggggu cccauauuau ccaccugcaa gcagugccca 420

guggueuauc ccagcecugu uugugguuca gauggucaua eeuacucuuu ucagugeaaa 480

cuagaauauc aggcaugugu cuuaggaaaa cagaucucag ucaaauguga aggacauugc 540

ccauguccuu cagauaagcc caccaguaca agcagaaaug uuaagagagc augcagugac 600

cuggaguuca gggaaguggc aaacagauug cgggacuggu ucaaggcccu ucaugaaagu 660

ggaagucaaa acaagaagac aaaaacauug cugaggccug agagaagcag auucgauacc 720

agcaucuugc caauuugcaa ggacucacuu ggcuggaugu uuaacagacu ugauacaaac 780

uaugaccugc uauuggacca gucagagcuc agaagcauuu accuugauaa gaaugaacag 840

uguaccaagg cauucuucaa uucuugugac acauacaagg acaguuuaau aucuaauaau 900

gaguggugcu acugcuucca gagacagcaa gacccaccuu gccagacuga gcucagcaau 960

auueagaagc ggcaaggggu aaagaagcuc cuaggacagu auaucccceu gugugaugaa 1020 -11

gaugguuacu aeaagccaae acaaugucau ggeaguguug gaeagugcug guguguugac 1080

agauauggaa augaagueau gggauccaga auaaauggug uugcagauug ugeuauagau 1140

uuugagaucu ecggagauuu ugcuaguggc gauuuucaug aauggacuga ugaugaggau 1200

gaugaagacg auauuaugaa ugaugaagau gaaauugaag augaugauga agaugaaggg 1260

gaugaugaug auggugguga ugaccaugau guauacauu 1299