US 5,468,630 AGrant

cDNA Clone for Human Inducible Nitric Oxide Synthase and Process for Preparing Same

Issue Date:1995-11-21

•23 Claims

•11 Drawing Sheets

Abstract

A human tissue inducible nitric oxide synthase cDNA clone is disclosed. A process for preparing this cDNA clone coding for human tissue inducible nitric oxide synthase and for expressing the human tissue inducible nitric oxide synthase protein are provided.

Metadata

Assignee

University of Pittsburg of the Commonwealth System of Higher Education

Inventors

Timothy R. Billiar
Andreas K. Nussler
David A. Geller
Richard L. Simmons

Application Information

Application Number:US 314917&

Filing Date:1994-09-28

Priority Date:1992-11-25

Art Unit:184

Classifications

IPC:

C12N 902C12N 1553C12N 1570C12N 1579

Field of Search:

43553669.1;189;252.3;252.33;320.123.2

Patent Drawings (11 sheets)

Description

The following microorganisms have been deposited by David A. Geller on behalf of the University of Pittsburgh of the Commonwealth System of Higher Education, Pittsburgh, Pa. 15260, USA, on Nov. 18, 1992, with and are available from the permanent collection of the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 202852-1776, USA:

ATCC 75358 Human Hepatocyte inducible Nitric Oxide Synthase cDNA in pBluescript (pHINOS)

ATCC 69126 Human Hepatocyte Inducible Nitric Oxide Synthase cDNA in pBluescript transformed in E. coli SOLR bacteria (plasmid HINOS cDNA)

The American Type Culture Collection has performed viability tests on each of the hereinbefore mentioned deposited microorganisms and has concluded on Nov. 20, 1992, that each of the hereinbefore mentioned deposited microorganisms is viable and capable of reproduction.

These deposits are available to the public upon the grant of a patent to the assignee, the University of Pittsburgh of the Commonwealth System of Higher Education, disclosing them. However, it should be understood that the availability of these deposits does not constitute a license to practice this invention in derogation of patent rights granted by governmental action.

FIELD OF THE INVENTION

This invention relates to a human tissue inducible nitric oxide synthase cDNA clone capable of expressing a human inducible nitric oxide synthase protein, and a process suitable for cloning a cDNA encoding amino acid sequences for the human inducible nitric oxide synthase. More specifically, this invention relates to a human hepatocyte inducible nitric oxide synthase cDNA clone and to a process for cloning and expression of the human hepatocyte inducible nitric oxide synthase cDNA to provide a source of the human hepatocyte inducible nitric oxide synthase enzyme.

This invention provides a process for cloning a cDNA having an amino acid sequence coding for the human hepatocyte inducible nitric oxide synthase. FIGS. 1A-G show and SEQ ID NO: 1 in the SEQUENCE LISTING contains the 4,145 nucleotide bases for the sense strand of cDNA for human hepatocyte inducible nitric oxide synthase and sets forth the base codes as triplets (codon) for the coding parts of the nucleotide sequence. FIGS. 1A-G snow and SEQ ID NOS: 1 and 2 sets forth the amino acid sequence for the cDNA clone for human hepatocyte inducible nitric oxide synthase encoding amino acids 1 through 1153 of the human hepatocyte inducible nitric oxide synthase enzyme.

BRIEF DESCRIPTION OF BACKGROUND ART

It is known by those skilled in the art that nitric oxide (NO) is a biologic mediator derived from the amino acid L-arginine. One of a family of enzymes, nitric oxide synthase (NOS), acts upon L-arginine to oxidize one of the guanidino nitrogens to NO while citrulline is formed from the remainder of the L-arginine molecule. Nitric oxide is a very short-lived free radical and is rapidly oxidized to nitrite (NO.sub.2.sup.-) and nitrate (NO.sub.3.sup.-) which is measured as the stable inactive end products of nitric oxide formation.

It is well known by those skilled in the art that multiple isoforms of the nitric oxide synthase enzyme exist and that they are generally classified into two broad categories: 1) constitutive and 2) inducible. These classes of NOS enzymes vary considerably in their size, amino acid sequence, activity and regulation. For example, cells such as neurons and vascular endothelial cells contain constitutive NOS isotypes while macrophages and vascular smooth muscle cells express an inducible NOS.

it is generally well known that small amounts of NO generated by a constitutive NOS appear to act as a messenger molecule by activating soluble guanylate cyclase and, thus, increasing intracellular guanosine, 3', 5'-cyclic monophosphate (cGMP) and the induction of biological responses that are dependent on cGMP as a secondary messenger. For example, through this mechanism, endothelial derived NO induces relaxation of vascular smooth muscle and is identified as endothelium derived relaxing factor (EDRF). Nature, Vol. 327, pp. 524-526 (1987) and Proc Natl. Acad Sci USA, Vol. 84, pp. 9265-9269 (1987). Another example includes, but is not limited by, neuronal nitric oxide which acts as a neuro transmitter by activating guanylate cyclase with important functions in the central nervous system and autonomic nervous systems. Proc Natl Acad Sci USA, Vol. 86, pp. 9030-9033 (1989) and Science, Vol. 257, p. 401 (1992).

It is generally known by those skilled in the art that the larger quantities of nitric oxide produced by the inducible nitric oxide synthase have antimicrobial and antitumor functions. J. Clin. Invest., Vol 81, pp. 1129-1136 (1989) and Science, Vol. 235, pp. 473-476 (1987), respectively. It is also known by those skilled in the art that when vascular smooth muscle cells are stimulated to express a NOS enzyme by inflammatory cytokines, the excess amounts of nitric oxides that are produced contribute to the vascular collapse seen in sepsis. FEBS Lett., Vol. 265, pp. 133-136, (1990).

Thus, it will be appreciated that nitric oxide has both normal physiologic intracellular and extracellular regulatory functions. However, excessive production of nitric oxide is detrimental. For example, stimulation of inducible nitric oxide synthesis in blood vessels by bacterial endotoxin such as for example bacterial lipopolysaccharide (LPS) and cytokines that are elevated in sepsis results in massive dilation of blood vessels and sustained hypotension commonly encountered in septic shock. Proc. Natl. Acad. Sci USA, Vol. 87, pp. 3629-32 (1990). It is known that overproduction of nitric oxide in the lungs stimulated by immune complexes directly damages the lung. J. Immunol., Vol. 148, p. 3086 (1992). Induction of nitric oxide synthase in pancreatic islets impairs insulin secretion and contributes to the onset of juvenile diabetes. J. Biol. Chem., Vol. 266, p. 21351 (1991).

It will be appreciated that there is a great need in the medical community for collective inhibition of the inducible form of NOS but not the constitutive types of NOS in humans because this would allow for a means of preventing, such as for example, the hypotensive shock seen in sepsis, without preventing the physiologic regulation of vasomotor tone or neuro transmission in the central nervous system.

We recently demonstrated that nitric oxide biosynthesis is induced in isolated human hepatocytes after stimulation with interleukin-1, tumor necrosis factor-alpha, interferon-gamma and bacterial lipopolysacharride (bacterial endotoxin). FASEB JOURNAL, Vol. 6, No. 5, page A1834 (April, 1992) and J. Exp. Med., Vol. 176, p. 261 (1992). Heretofore no human cell type was known to show increased production of nitrogen oxides when treated with cytokines. Res. Immunol., Vol. 142, p. 557 (1991). It is generally known by those skilled in the art that all attempts to induce nitric oxide synthase in human macrophages and related cells typical to those found in rodent macrophages have failed. Res. immunol., Vol. 142, p. 562, 589-90 (1991).

In spite of this background material, there remains a very real and substantial need for a cDNA clone for human tissue inducible nitric oxide synthase and a process for the molecular cloning of the same.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-G collectively show the cDNA sense sequence (top line of each horizontal row) and the amino acid sequence of amino acids 1-1153 (bottom line of each horizontal row) for the cDNA clone for human hepatocyte inducible nitric oxide synthase, SEQ ID NO: 1.

FIG. 2 shows a Northern Blot of a mouse macrophage NOS cDNA cross-hybridizing to human hepatocyte (HC) nitric oxide synthase mRNA.

FIG. 3 shows a Northern Blot of induced nitric oxide synthase mRNA isolated from three separate human liver samples using mouse macrophage cDNA.

FIG. 4 shows a Northern Blot of poly A mRNA purified from 2 separate human liver samples for the construction of the cDNA library for isolation of the cDNA clone for the human hepatocyte inducible nitric oxide synthase.

FIG. 5 shows a Northern Blot using cDNA isolated from human hepatocytes that sets forth the time course of induction of human nitric oxide synthase mRNA following cytokine and LPS stimulation.

SUMMARY OF THE INVENTION

The present invention has met the hereinbefore described needs. The present invention provides a cDNA clone for human tissue inducible nitric oxide synthase and a process for preparing the same.

More specifically, this invention provides a cDNA clone for human hepatocyte inducible nitric oxide synthase and a process for preparing the same. This process includes inducing nitric oxide synthase in human hepatocytes, identifying human hepatocyte nitric oxide synthase messenger RNA, isolating the human hepatocyte nitric oxide synthase messenger RNA, collecting the human hepatocyte nitric oxide synthase messenger RNA, separating human hepatocyte poly A messenger RNA from the human hepatocyte nitric oxide synthase messenger RNA, constructing a cDNA library for human hepatocyte nitric oxide synthase, screening this cDNA library for human hepatocyte inducible nitric oxide synthase cDNA clones, and converting the human hepatocyte inducible nitric oxide synthase cDNA clones to a plasmid vector for obtaining a substantially full length cDNA clone encoding human hepatocyte inducible nitric oxide synthase. This process further includes sequencing this cDNA, expressing the human hepatocyte inducible nitric oxide synthase cDNA protein in an expression system, and purifying the human hepatocyte inducible nitric oxide synthase cDNA protein.

It is an object of the present invention to provide for the molecular cloning and characterization of an inducible nitric oxide synthase in human tissues.

It is an object of the present invention to provide for the molecular cloning and characterization of an inducible nitric oxide synthase in human hepatocytes.

It is an object of the present invention to isolate a cDNA clone for human tissue inducible nitric oxide synthase.

It is an object of the present invention to isolate a cDNA clone for human hepatocyte inducible nitric oxide synthase.

It is an object of the present invention to provide a process for expressing and purifying human tissue inducible nitric oxide synthase enzyme.

It is an object of the present invention to provide a process for expressing and purifying human hepatocyte inducible nitric oxide synthase enzyme.

It is an object of this invention to provide for the regulation of gene expression for the human hepatocyte inducible nitric oxide synthase enzyme.

It is an object of this invention to provide for a protein including a human inducible nitric oxide synthase substantially free of other human proteins.

These and other objects of the invention will be more fully understood from the following description of the invention, the figures, the sequence listing and the claims appended hereto.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "patient" includes members of the animal kingdom including but not limited to human beings.

Nitric oxide is a biologic mediator derived from amino acid L-arginine. Nitric oxide synthase (NOS) acts upon L-arginine to oxidize one of the guanidino nitrogens to nitric oxide while citrulline is formed from the remainder of the L-arginine molecule. While it is understood by those skilled in the art that nitric oxide has both normal physiologic intracellular and extracellular regulatory functions, excessive production of nitric oxide is detrimental. It will be appreciated by those skilled in the art that there are no other readily available sources of human tissue inducible nitric oxide synthase. The present invention provides a cDNA clone for human tissue inducible nitric oxide synthase and a process for preparing the same. Therefore, the cloning and expression of a human tissue nitric oxide synthase cDNA of the present invention provides for a source of the enzyme for developing selective inhibitors of nitric oxide synthase.

The cloning and expression of a human tissue nitric oxide synthase cDNA of the present invention provides for a source of the enzyme in a sufficiently high concentration for providing a therapeutic purpose.

In one embodiment of this invention, a process for preparing a cDNA clone coding for a human tissue inducible nitric oxide synthase is provided. This process includes inducing the human tissue nitric oxide synthase in vitro, identifying the human tissue nitric oxide synthase messenger RNA (mRNA) by employing a cDNA probe capable of hybridizing with the human tissue inducible nitric oxide synthase mRNA, isolating the human tissue nitric oxide synthase mRNA, collecting the human tissue nitric oxide synthase mRNA, separating human tissue poly A mRNA from the human tissue nitric oxide synthase mRNA, constructing a human tissue inducible nitric oxide synthase cDNA library from the human tissue poly A mRNA using a reverse transcriptase enzyme and inserting a strand of the cDNA into a phage vector, screening the cDNA library for human tissue inducible nitric oxide synthase clones including incubating the phage vector containing the cDNA with a bacteria for forming at least one positive plaque containing the cDNA clone for human tissue inducible nitric oxide synthase, rescuing the cDNA clone from the phage vector by employing a helper phage, and converting the rescued cDNA clone to a plasmid vector for obtaining a substantially full length cDNA clone encoding human tissue inducible nitric oxide synthase.

In another embodiment of this invention, this process, as hereinbefore described, further includes excising cDNA inserts for human tissue inducible nitric oxide synthase from the plasmid vector. This process also includes confirming the cDNA inserts by employing a dideoxynucleotide DNA sequencing. Further, this process includes confirming the cDNA inserts by employing Southern blot hybridization.

In another embodiment of this Invention, the process, as hereinbefore described, includes expressing the human tissue inducible nitric oxide synthase cDNA protein in an expression system, such as for example, a bacterial expression system or a mammalian expression system.

It will be appreciated by those skilled in the art that the cloned human inducible nitric oxide synthase cDNA obtained through the methods described herein may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant inducible nitric oxide synthase. Techniques for such manipulations are fully described in Maniatis, et al., infra, and are well known in the art.

Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as for example bacteria, bluegreen algae, plant cells, insect cells and animal cells.

Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses. A variety of mammalian expression vectors may be used to express recombinant inducible nitric oxide synthase in mammalian cells.

Commercially available bacterial expression vectors which may be suitable for recombinant inducible nitric oxide synthase expression, include but are not limited to, pKC30 (ATCC 37286), pPLa2311 (ATCC 31694), pBR322 (ATCC 31344 and 37017), ptac12 (ATCC 37138), Lambda gt11 (ATCC 37194), pAS1 (ATCC39262), pLC24, pSB226, SV40 and pKK 223-3.

Commercially available mammalian expression vectors which may be suitable for recombinant inducible nitric oxide synthase expression, include but are not limited to, pBC12B1 (ATCC 67617), pMClneo (Stratagene), pXTI (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and lambda ZD35 (ATCC 37565).

DNA encoding inducible nitric oxide synthase may also be cloned into an expression vector for expression in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria, yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to drosophila derived cell lines. Cell lines derived from mammalian species which may be suitable and which are commercially available, include but are not limited to, CV-1 (ATCC CCL70), COS-1 (ATCC CRL1650), COS-7 (ATCC CRL1651), CHO-K1 (ATCC CCL61), 3T3 (ATCC CCL92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL2), C1271 (ATCC CRL1616), BS-C-1 (ATCC CCL26) and MRC-5 (ATCC CCL171). The bacterial cell most used for expression of recombinant protein is Escherichia coli. There are various strains of E. coli available and are well known in the art.

The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electropotation.

In a preferred embodiment of this invention, the process, as hereinbefore described, includes expressing the human tissue inducible nitric oxide synthase protein in a baculovirus expression system.

Another embodiment of this invention provides for a process, as hereinbefore described, including purifying the human tissue inducible nitric oxide synthase protein.

In a preferred embodiment of this invention, the process, as hereinbefore described, includes employing as the human tissue inducible nitric oxide synthase a human hepatocyte inducible nitric oxide synthase. This process further includes employing as the human tissue inducible nitric oxide synthase protein a human hepatocyte inducible nitric oxide synthase protein.

In another embodiment of this invention, a process is provided, as hereinbefore described, including inducing the human tissue nitric oxide synthase in vitro by stimulating a human tissue in vitro with at least one of the following (1) at least one cytokine, such as for example a cytokine selected from the group consisting of tissue necrosis factor (TNF), interleukin-1 (IL-1), and interferon-gamma (IFN-g), (2) at least one bacterial endotoxin including, such as for example, a bacterial lipopolysaccharide (LPS) and (3) combinations thereof.

A further preferred embodiment of this invention provides a process, as hereinbefore described, that includes constructing a human tissue inducible nitric oxide synthase cDNA library from the human tissue poly A mRNA using a reverse transcriptase enzyme and inserting a cDNA strand having a length of about at least 1,000 base pairs into the phage vector. In yet another preferred embodiment, a process is provided, as hereinbefore described, that includes employing lambda Zap II as the phage vector.

In another embodiment of this invention, a process is provided, as hereinbefore described, including screening the cDNA library including incubating the phage vector for about 6 to 24 hours with a bacteria at a temperature from about 34 to 40 degrees centigrade for effectuating phage lysis of the bacteria. This process further includes rescuing she cDNA clone from the phage vector by employing a helper phage such as for example ExAssist helper phage (Stratagene, La Jolla, Calif.).

In a preferred embodiment of this invention, a process, as hereinbefore described, is provided including converting the rescued cDNA clone to the plasmid vector for obtaining a substantially full length cDNA clone encoding the human tissue inducible nitric oxide synthase wherein the plasmid vector includes pBluescript (Stratagene, La Jolla, Calif.).

In another preferred embodiment of this invention, a process as hereinbefore described is provided including employing as the human tissue inducible nitric oxide synthase a human hepatocyte inducible nitric oxide synthase.

Another embodiment of this invention provides for a process for producing human hepatocyte inducible nitric oxide synthase protein comprising providing a replicatable DNA expression vector capable of expressing a DNA sequence encoding human hepatocyte inducible nitric oxide synthase in a suitable host, transforming the host for obtaining a recombinant host, and maintaining the recombinant host under conditions permitting expression of the DNA sequence to provide human hepatocyte inducible nitric oxide synthase.

Another embodiment of this invention provides a human tissue inducible nitric oxide synthase cDNA clone. A preferred embodiment of this invention provides a human hepatocyte inducible nitric oxide synthase cDNA clone. The human hepatocyte inducible nitric oxide synthase cDNA clone of this invention has a cDNA coding for the amino acid sequence, SEQ ID NOS: 1 and 2 shown in FIGS. 1A-G. FIGS. 1A-G show the cDNA sense sequence (top line of each horizontal row) and the deduced amino acid sequence of amino acids 1- 1153 (bottom line of each horizontal row) for the cDNA clone for the human hepatocyte inducible nitric oxide synthase of this invention. FIGS. 1A-G show that the cDNA sequence for the human hepatocyte inducible nitric oxide synthase of this invention is 4,145 nucleotide bases long with the start codon beginning at base number 207 and the stop codon ending at base number 3668. The cDNA double strand sequence was determined using the Sanger dideoxynucleotide sequence technique well known by those skilled in the art on a Genesis 2000 sequencing system (USB, Cleveland, Ohio). Proc. Natl. Acad. Sci. USA, Vol 74, p. 5463 (1977).

Another embodiment of this invention provides a human tissue inducible nitric oxide synthase recombinant protein expressed from a human tissue inducible nitric oxide synthase cDNA clone. In a preferred embodiment, a human hepatocyte inducible nitric oxide synthase recombinant protein expressed from a human hepatocyte inducible nitric oxide synthase cDNA clone is provided.

Another embodiment of this invention provides for a protein comprising a human inducible nitric oxide synthase substantially free of other human proteins.

Another embodiment of this invention provides for an isolated DNA sequence encoding human inducible nitric oxide synthase consisting essentially of an initiation codon positioned upstream and adjacent to an open reading frame consisting essentially of a DNA sequence encoding human inducible nitric oxide synthase.

A further embodiment of this invention provides for an isolated DNA sequence encoding human inducible nitric oxide synthase consisting essentially of an initiation codon positioned upstream and adjacent to an open reading frame consisting essentially of a DNA sequence encoding human inducible nitric oxide synthase protein. The human inducible nitric oxide synthase protein begins at the initiation codon and terminates at a stop codon.

In yet another embodiment of this invention a recombinant plasmid is provided containing a recombinant plasmid pHINOS having a deposit accession number ATCC 75358 deposited with the American Type Culture Collection. A further embodiment of this invention provides for bacteria transformed by the recombinant plasmid pHINOS.

In another embodiment of this invention a microorganism is provided containing a HINOS cDNA plasmid transformed in E. coli SOLR bacteria having a deposit accession number ATCC 69126 deposited with the American Type Culture Collection.

EXAMPLE 1

INDUCING HUMAN HEPATOCYTE INDUCIBLE NITRIC OXIDE SYNTHASE

mRNA is weakly induced following stimulation with cytokine signals such as for example tumor necrosis factor (TNF), interleukin-1 (IL-1) or interleukin-gamma (IFN-g). Cytokine signals synergize to further up-regulate mRNA levels and nitric oxide synthase activity. Maximum induction was achieved with a combination of TNF, IL-1, IFN-g and bacterial lipopolysaccharide (LPS). FASEB, Journal, Vol. 6, supra, and J. Exp. Med., Vol. 176, supra.

EXAMPLE 2

IDENTIFYING AND ISOLATING HUMAN HEPATOCYTE NITRIC OXIDE SYNTHASE mRNA

A cDNA probe capable of hybridizing with human hepatocyte inducible nitric oxide synthase mRNA was used for identifying and isolating the mRNA for human hepatocyte inducible nitric oxide synthase. The time-point of peak mRNA levels following cytokine and LPS [hereinafter cytokine mixture (CM)] stimulation was then determined.

Total RNA was extracted about 2-48 hours following CM-stimulation of cultured human hepatocytes using the RNAzol B modified method of Chomczynski and Sacchi. Anal Biochem., Vol 162; pp. 156-159 (1987). Northern blot analysis was performed on 20 microgram (ug) aliquots of total RNA using a murine macrophage cDNA probe, representing an excision fragment produced by Not I restriction enzyme [Proc. Natl. Acad. Sci. USA., Vol 89, pp. 6711-6715 (1992) GenBank Accession No. M92649] and cross-species hybridization. The human hepatocyte nitric oxide synthase mRNA was identified as a single band at about 4.5 kb (kilobase) with maximal mRNA levels seen about 8 hours after stimulation.

FIG. 2 shows the presence of the 4.5 kb message for human hepatocyte inducible nitric oxide synthase. Human hepatocytes (HC) that were freshly isolated were placed in cell culture and exposed to a combination of human recombinant tumor necrosis factor (500 units/milliliter), human recombinant interleukin-1 (5 units/milliliter), human recombinant interferon-gamma (100 units/milliliter), and lipopolysaccharide (10 micrograms/milliliter). FIG. 2 shows that at the indicated time points (2 hours, 4 hours, 6 hours and 8 hours) total RNA was isolated and that 20 micrograms per sample was subjected to Northern Blot analysis. A 2.7 Kb fragment of cDNA to murine macrophage inducible nitric oxide synthase was used to hybridize with the mRNA for human hepatocyte inducible nitric oxide synthase. FIG. 2 demonstrates that the 4.5 Kb message peaked at about 8 hours following stimulation. FIG. 2 shows that no mRNA signal was detected in control (unstimulated) hepatocytes. FIG. 3 shows the expression of the 4.5 Kb mRNA for human hepatocyte inducible nitric oxide synthase at about 8 hours after exposure to the above mentioned signals for hepatocytes isolated from three separate individuals [patient (Pt.) 1, 2, and 3]. FIG. 3 demonstrates that no signal was detected in control (unstimulated) hepatocytes.

Because the 8 hour time point yielded maximal mRNA levels, samples of RNA were isolated from two human livers about 8 hours following CM-stimulation in vitro and were pooled to obtain sufficient quantity for the cDNA library construction. The cDNA synthesis requires about from 10 to 20 micrograms of poly A mRNA rather than total RNA. To obtain purified poly A mRNA, poly A mRNA was separated from total RNA by elution through an oligo-dT cellulose column. The purity of the mRNA was assessed by repeat Northern blot analysis which included subjecting 0.5 micrograms of poly A RNA from each of the two human livers to Northern Blot analysis using the 2.7 Kb cDNA from murine macrophage inducible nitric oxide synthase. FIG. 4 shows strong nitric oxide synthase mRNA bands from 2 different patients without evidence of degraded poly A RNA.

FIG. 4 shows that the murine macrophage inducible nitric oxide synthase cross hybridizes with the human hepatocyte inducible nitric oxide synthase poly A RNA and effectively identifies the mRNA for human hepatocyte inducible nitric oxide synthase. These samples of poly A RNA were used to construct the cDNA library for isolation of the cDNA clone for the human hepatocyte inducible nitric oxide synthase.

EXAMPLE 3

CONSTRUCTING A HUMAN HEPATOCYTE INDUCIBLE NITRIC OXIDE SYNTHASE cDNA LIBRARY

Using about 20 micrograms of poly A RNA enriched for hepatocyte nitric oxide synthase mRNA by CM-stimulation, a cDNA library was constructed by Stratagene, La Jolla, Calif. The first strand cDNA was synthesized from the human hepatocyte poly A RNA using reverse transcriptase enzyme with random and oligo-dT primers. After size exclusion for a minimum of about 1000 nucleotide base pair length, the cDNA's were inserted into a lambda Zap II phage vector (Stratagene, La Jolla, Calif.) and was titered.

EXAMPLE 4

SCREENING THE cDNA LIBRARY FOR HUMAN HEPATOCYTE INDUCIBLE NITRIC OXIDE SYNTHASE cDNA CLONES

To screen the cDNA library, 1.times.10.sup.6 phage were incubated with bacteria (E. coli Sure strain) at about 34 to 40 degrees centigrade for about 15 to 30 minutes. This mixture was added to molten agarose and poured onto 20.times.20 centimeter agar plates at a density of about 2.times.10.sup.5 plaques/plate (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982). The plates were incubated from about 34 to 40 degrees centigrade overnight from about 6 to 24 hours to allow for phage lysis of bacteria. The plaques were then transferred to nylon filters and positive clones were identified by filter hybridization with .sup.32 P-labeled murine macrophage nitric oxide synthase cDNA probe. Positive clones were cored from the agar plates after localization by autoradiograph alignment. This procedure was repeated about 3 times until individual clones were isolated. The positive clones were rescued from the lambda Zap II phage vector using a helper phage ExAssist (Stratagene, La Jolla, Calif.), and then converted to the plasmid vector, pBluescript (Stratagene, La Jolla, Calif.). The cDNA inserts for human hepatocyte inducible nitric oxide synthase were excised from the Bluescript plasmid cloning sites by restriction analysis with EcoRI enzyme and then sized by gel electrophoresis. The cDNA insert identities were confirmed by DNA sequencing and by Southern blot hybridization with the murine macrophage cDNA clone. In addition, repeat Northern blot analysis was performed on cytokine-stimulated human hepatocytes in culture using the human nitric oxide synthase cDNA clone of this invention as probe. FIG. 5 shows a time course for mRNA expression for human hepatocyte inducible nitric oxide synthase. This RNA is from an individual patient different from the patients listed in FIGS. 2 and 3. The cells of the patient in FIG. 5 were exposed to the same agents as described for FIG. 2. FIG. 5 shows the human nitric oxide synthase cDNA identifying the same mRNA signal as the macrophage probe, thus, further confirming its identify. It is important to note that the isolated cDNA clone coding for human inducible nitric oxide synthase of this invention was used to hybridize with the mRNA, thus, confirming the capacity of the cDNA clone of this invention to identify the human hepatocyte inducible nitric oxide synthase mRNA.

EXAMPLE 5

cDNA SEQUENCING

The plasmid vector pBluescript contains universal primer regions which were used to facilitate double-stranded DNA sequencing. Positive clones were sequenced by using the dideoxynucleotide technique of Sanger, supra, with the Genesis 2000 sequencing system (USB, Cleveland, Ohio). Sequence analysis was done using Genbank DNA sequencing software programs available through the Pittsburgh Supercomputing Center (Billiar TR., Pittsburgh Supercomputing Center, Pittsburgh, Pa.).

EXAMPLE 6

EXPRESSING HUMAN HEPATOCYTE INDUCIBLE NITRIC OXIDE SYNTHASE

Verification of the full length cDNA identify was accomplished by expressing the recombinant human hepatocyte inducible nitric oxide synthase protein. The human hepatocyte inducible nitric oxide synthase clone was ligated into the pCIS expression vector (Genentech, Calif.) which utilizes a CMV promoter. Next the expression vector was transfected into human embryonic kidney 293 cells (ATCC, Maryland). Nitric oxide synthase activity was assessed by measuring the conversion of [.sup.3 H] arginine to [.sup.3 H] citrulline. It will be appreciated by those skilled in the art that this expression system was successfully used for expression of the cloned rat brain constitutive nitric oxide synthase, and there was negligible nitric oxide synthase activity in the unstimulated 293 kidney cells [Bredt et al., Nature, Vol 351, p. 714 (1991)]. After the identity of the human hepatocyte inducible nitric oxide synthase clone of this invention was verified as hereinbefore described, the cDNA was expressed in a baculovirus expression system (Invitrogen, San Diego, Calif.) which allowed for large scale enzyme production. Texas Agriculture Experiment Station Bulletin, No. 1555 (1988). More specifically, the human hepatocyte nitric oxide synthase cDNA was inserted into the baculovirus transfer vector and then co-transfected with wild type viral DNA into Sf9 insect cells (ATCC, Maryland). Recombinant viral plaques were isolated to allow for protein over-expression.

EXAMPLE 7

PURIFYING THE HUMAN HEPATOCYTE INDUCIBLE NITRIC OXIDE SYNTHASE PROTEIN

The resultant human hepatocyte inducible nitric synthase protein was purified using a two step procedure. First, the protein was passed through an anion-exchange column of DEAE cellulose. This was followed by affinity chromatography with 2', 5'-ADP Sepharose [Evans et al , Proc. Natl. Acad. Sci. USA, Vol 89, pp 5361-5365 (1992)] Purity was assessed by SDS-polyacrylamide gel electrophoresis. Activity was quantitated after each step by measuring the ability of the enzyme to generate NO.sub.2.sup.- and NO.sub.3.sup.- from L-arginine. NO.sub.2.sup.- and NO.sub.3.sup.- was measured using an automated colorimetric reaction based on the Greiss reaction [Green et al., Anal. Biochem., Vol. 126, p. 131 (1982)].

Whereas, particular embodiments of this invention have been described above for purposes of illustration, it will be evident to those persons skilled in the art that numerous variations of the details of the present invention may be made without departing from the invention as defined in the appended claims that follow the SEQUENCE LISTING.

Claims

What is claimed:

1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a human inducible nitric oxide synthase protein.

2. A DNA vector molecule comprising said nucleotide sequence of claim 1.

3. A host cell transformed with said DNA vector molecule of claim 2.

4. A host cell of claim 3 which is a bacterial host cell.

5. An isolated nucleic acid molecule comprising a nucleotide sequence expressing a human inducible protein which is SEQ ID NO:2, extending from the initiating methionine numbered amino acid residue 1 through amino acid residue number 1153.

6. A DNA vector molecule comprising said nucleotide sequence of claim 5.

7. A eukaryotic host cell transformed with said DNA vector molecule of claim 6.

8. A prokaryotic host cell transformed with said DNA vector molecule of claim 6.

9. An isolated nucleic acid molecule comprising the DNA sequence as set forth in SEQ ID NO: 1, a portion thereof extending from nucleotide 207 through nucleotide position 3665, inclusive, which encodes a human inducible nitric oxide synthase protein.

10. A DNA vector molecule comprising said nucleotide sequence of claim 9.

11. A host cell transformed with said DNA vector molecule of claim 10.

12. A host cell of claim 11 which is a bacterial host cell.

13. The isolated DNA molecule designated by SEQ ID NO:1, a portion thereof extending from nucleotide 207 through nucleotide position 3665, inclusive, which encodes the human inducible nitric oxide synthase protein.

14. A DNA vector molecule comprising said nucleotide sequence of claim 13.

15. A host cell transformed with said DNA vector molecule of claim 14.

16. A host cell of claim 15 which is a bacterial host cell.

17. The DNA vector molecule of claim 14 which is pHINOS, having the accession number ATCC 75358.

18. A bacterial host cell transformed with said pHINOS DNA vector molecule of claim 17.

19. The transformed bacterial host of claim 18 which is E. coli SOLR, having the accession number ATCC 69126.

20. The portion of said isolated DNA molecule of claim 13 encoding the human inducible nitric oxide synthase protein.

21. A DNA vector molecule comprising said nucleotide sequence of claim 20.

22. A host cell transformed with said DNA vector molecule of claim 21.

23. A host cell of claim 22 which is a bacterial host cell.

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