5-lipoxygenase-activating protein II

Disclosed is a human FLAP II polypeptide and DNA (RNA) encoding such polypeptide. Also provided is a procedure for producing such polypeptide by recombinant techniques. Further, antagonists against such polypeptide are disclosed. Such antagonists may be used for therapeutic proposes, for example, for treating inflamation, bronchial asthma and may also be used as gastric cytoprotective agents and to treat human glomerulonephritis. Diagnostic assays for identifying mutations in nucleic acid sequences encoding a polypeptide of the present invention and for detecting altered levels of the polypeptide of the present invention are also disclosed.

This invention relates to newly identified polynucleotides, polypeptides 
encoded by such polynucleotides, the use of such polynucleotides and 
polypeptides, as well as the production of such polynucleotides and 
polypeptides. More particularly, the polypeptide of the present invention 
is 5-lipoxygenase-activating protein II "FLAP II". The invention also 
relates to inhibiting the action of such polypeptides. 
Leukotrienes (LTs), formed in granulocytes, monocytes/macrophages and mast 
cells, mediate immunological and inflammatory responses. Increased levels 
of LTs in clinical samples implicate these compounds in a number of 
hypersensitivity and inflammatory diseases, including asthma and 
inflammatory bowel disease (Ford-Hutchinson, et al., in: Leukotrienes and 
Lipoxygenases, J. Rokach, ed., Elsevier Science Publishing, New York, 
405-425 (1989); Konig, et al., Eicosanoids 3: 1-22; Robinson and Holgate 
Adv. Prostaglandin Thromboxane Leukotriene Res. 20:209-216, (1990). 
Recently, much attention has been given to the LTs as the major 
pathophysiologic mediators of the inflammatory response since they are 
much more potent than the prostaglandins (PGs) with regard to increasing 
vascular permeability, adhesion of leukocytes to the vessel wall, and 
edema production. Inhibitors of LT synthesis are currently being developed 
for possible clinical applications as anti-inflammatory agents. Recent 
studies appear to place the LTs rather than PGs as the most central agents 
in the etiologic genesis of bronchial asthma. They have been identified as 
the agents formerly known as slow-reacting substance and have 200 to 
20,000 times the bronchoconstrictor activity as histamine. It is currently 
believed that an LT antagonist or synthesis inhibitor holds great promise 
in the treatment of bronchial asthma. LTs have been shown to increase 
insulin secretion and an alternate current hypothesis is that carbohydrate 
intolerance in some patients with diabetes mellitus may result from an 
imbalance in the PG to LT ratio in the islet cell. 
The first two steps in the biosynthesis of LTs are catalyzed by the 
Ca.sup.2+ and ATP-dependent enzyme 5-lipoxygenase (5-LO) which catalyzes 
the conversion of arachidonic acid to 
5-hydroperoxy-6,8,11,14-eicosatetraeonic acid (5HPETE), and subsequently 
to leukotriene A.sub.4 (Samuelson, et al., Science 237:1171-1176, (1987)). 
Prostaglandins are also synthesized from arachidonic acid precursors. 
Aspirin-like drugs, and other enzymes, are efficient at preventing 
prostaglandin synthesis from arachidonic acid to prevent inflammation and 
generally relieve pain. However, these aspirin-like drugs and other 
enzymes are ineffective for preventing the synthesis of LTs from 
arachidonic acid. The Ca.sup.2+ -dependent translocation of 5-LO from the 
cytosolic to a membrane fraction appears to be a critical step in the 
activation of the enzyme (Rouzer and Kargman, J. Biol. Chem. 
263:10980-10988, Wong, et al., Biochemistry 27:6763-6769, (1988)). Indole 
and quinoline classes of LT biosynthesis inhibitors and a series of 
structural hybrids of these compounds block this membrane association but 
have no significant inhibitory effect on 5-LO in cell free assays. MK-886 
(Gillard, et al., Can. J. Physiol. Pharmacol. 67:456-464, (1989)) and 
MK-0591 (Brideau, et al., Ca. J. Physiol. Pharmacol. 70:799-807, (1992)) 
are potent members of these inhibitors. FLAP has been identified as the 
cellular target of this class of inhibitors (Miller, et al., Nature, 
343:278-281, (1990)). Inhibitors which bind to FLAP may directly compete 
with 5-LO for binding to the protein or may cause a conformational change 
in FLAP leading to a decreased affinity of 5-LO for its membrane binding 
site. 
cDNA clones for FLAP have been isolated from several species (human, mouse, 
horse, pig, sheep, rabbit, rat and mouse, see: Vickers, et al., Mol. 
Pharmacology 42:1014-1019 (1992)). The deduced amino acid sequences 
correspond to hydrophobic proteins with three potential membrane-spanning 
domains. 
In accordance with one aspect of the present invention, there is provided a 
novel mature polypeptide which is FLAP II, as well as fragments, analogs 
and derivatives thereof. The polypeptide of the present invention is of 
human origin. 
In accordance with another aspect of the present invention, there are 
provided polynucleotides (DNA or RNA) which encode such polypeptides. 
In accordance with yet a further aspect of the present invention, there is 
provided a process for producing such polypeptide by recombinant 
techniques. 
In accordance with yet a further aspect of the present invention, there is 
provided a process for utilizing such polypeptide, or polynucleotide 
encoding such polypeptide to identify substances preventing the 
interaction of FLAP II with 5-lipoxygenase and to develop inhibitors for 
the biosynthesis of LTs. 
In accordance with yet a further aspect of the present invention, there is 
provided an antibody against such polypeptides. 
In accordance with yet another aspect of the present invention, there are 
provided antagonists to such polypeptides, which may be used to inhibit 
the action of such polypeptides, for example, in the treatment of angina, 
endotoxic shock, inflammatory conditions, such as psoriasis, atopic 
eczema, rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, 
tendinitis, bursitis, ulcerative colitis and other immediate 
hypersensitive reactions, and LT-mediated naso-bronchial obstructive 
air-passageway conditions, such as allergic bronchoasthma, allergic 
rhinitis, allergic conjunctivitis, for the treatment of human 
glomerulonephritis, migraine headaches and as a gastric cytoprotective 
agent. 
In accordance with yet a further aspect of the present invention, there is 
also provided nucleic acid probes comprising nucleic acid molecules of 
sufficient length to specifically hybridize to a nucleic acid sequence of 
the present invention. 
In accordance with still another aspect of the present invention, there are 
provided diagnostic assays for detecting diseases or susceptibility to 
diseases related to mutations in the nucleic acid sequences encoding a 
polypeptide of the present invention. 
In accordance with yet a further aspect of the present invention, there is 
provided a process for utilizing such polypeptides, or polynucleotides 
encoding such polypeptides, for in vitro purposes related to scientific 
research, for example, synthesis of DNA and manufacture of DNA vectors. 
These and other aspects of the present invention should be apparent to 
those skilled in the art from the teachings herein.

In accordance with an aspect of the present invention, there is provided an 
isolated nucleic acid (polynucleotide) which encodes for the mature 
polypeptide having the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2) 
or for the mature polypeptide encoded by the cDNA of the clone deposited 
as ATCC Deposit No. 75771 on May 12, 1994 with the American Type Culture 
Collection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209, USA. 
BRIEF DESCRIPTION OF THE DRAWINGS 
The polynucleotide of this invention was discovered in a cDNA library 
derived from aorta endothelial cells induced with tumor necrosis factor a. 
It is structurally related to the FLAP family. It contains an open reading 
frame encoding a protein of 147 amino acid residues. The protein exhibits 
the highest degree of homology to the human FLAP protein with 34% identity 
and 51% similarity over the entire coding sequence. Further, there is a 
highly conserved region of FLAP I across many different species (residues 
42-61) (Vickers, P. J., et al., J. Lipid Mediat., 6:31-42 (1993)). The 
sequences of the present invention show significant homology to this 
conserved region (55%). 
The polynucleotide of the present invention may be in the form of RNA or in 
the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. 
The DNA may be double stranded or single-stranded, and if single stranded 
may be the coding strand or non-coding (anti-sense) strand. The coding 
sequence which encodes the mature polypeptide may be identical to the 
coding sequence shown in FIG. 1 (SEQ ID NO:1) or that of the deposited 
clone or may be a different coding sequence which coding sequence, as a 
result of the redundancy or degeneracy of the genetic code, encodes the 
same mature polypeptide as the DNA of FIG. 1 (SEQ ID NO:1) or the 
deposited cDNA. 
The above-referenced deposit was deposited with the ATCC, 12301 Parklawn 
Drive, Rockville, Md. 20852. Since the strain referred to is being 
maintained under the terms of the Budapest Treaty, it will be made 
available to a patent office signatory to the Budapest Treaty. If a patent 
should issue which is directed to the present invention, upon the issuance 
of such a patent the deposited strain of ATCC 75771 will be irrevocably 
and without restriction released to the public, excepting for those 
restrictions permitted by enforcement of the patent. 
The polynucleotide which encodes for the mature polypeptide of FIG. 1 (SEQ 
ID NO:2) or for the mature polypeptide encoded by the deposited cDNA may 
include, but is not limited to: only the coding sequence for the mature 
polypeptide; the coding sequence for the mature polypeptide and additional 
coding sequence such as a leader or secretory sequence or a proprotein 
sequence; the coding sequence for the mature polypeptide (and optionally 
additional coding sequence) and non-coding sequence, such as introns or 
noncoding sequence 5' and/or 3' of the coding sequence for the mature 
polypeptide. 
Thus, the term "polynucleotide encoding a polypeptide" encompasses a 
polynucleotide which includes only coding sequence for the polypeptide as 
well as a polynucleotide which includes additional coding and/or 
non-coding sequence. 
The present invention further relates to variants of the hereinabove 
described polynucleotides which encode for fragments, analogs and 
derivatives of the polypeptide having the deduced amino acid sequence of 
FIG. 1 (SEQ ID NO:2) or the polypeptide encoded by the cDNA of the 
deposited clone. The variant of the polynucleotide may be a naturally 
occurring allelic variant of the polynucleotide or a nonnaturally 
occurring variant of the polynucleotide. 
Thus, the present invention includes polynucleotides encoding the same 
mature polypeptide as shown in FIG. 1 (SEQ ID NO:2) or the same mature 
polypeptide encoded by the cDNA of the deposited clone as well as variants 
of such polynucleotides which variants encode for a fragment, derivative 
or analog of the polypeptide of FIG. 1 (SEQ ID NO:2) or the polypeptide 
encoded by the cDNA of the deposited clone. Such nucleotide variants 
include deletion variants, substitution variants and addition or insertion 
variants. 
As hereinabove indicated, the polynucleotide may have a coding sequence 
which is a naturally occurring allelic variant of the coding sequence 
shown in FIG. 1 (SEQ ID NO:1) or of the coding sequence of the deposited 
clone. As known in the art, an allelic variant is an alternate form of a 
polynucleotide sequence which may have a substitution, deletion or 
addition of one or more nucleotides, which does not substantially alter 
the function of the encoded polypeptide. 
The present invention also includes polynucleotides, wherein the coding 
sequence for the mature polypeptide may be fused in the same reading frame 
to a polynucleotide sequence which aids in expression and secretion of a 
polypeptide from a host cell, for example, a leader sequence which 
functions as a secretory sequence for controlling transport of a 
polypeptide from the cell. The polypeptide having a leader sequence is a 
preprotein and may have the leader sequence cleaved by the host cell to 
form the mature form of the polypeptide. The polynucleotides may also 
encode for a proprotein which is the mature protein plus additional 5' 
amino acid residues. A mature protein having a prosequence is a proprotein 
and is an inactive form of the protein. Once the prosequence is cleaved an 
active mature protein remains. 
Thus, for example, the polynucleotide of the present invention may encode 
for a mature protein, or for a protein having a prosequence or for a 
protein having both a prosequence and a presequence (leader sequence). 
The polynucleotides of the present invention may also have the coding 
sequence fused in frame to a marker sequence which allows for purification 
of the polypeptide of the present invention. The marker sequence may be a 
hexahistidine tag supplied by a pQE-9 vector to provide for purification 
of the mature polypeptide fused to the marker in the case of a bacterial 
host, or, for example, the marker sequence may be a hemagglutinin (HA) tag 
when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds 
to an epitope derived from the influenza hemagglutinin protein (Wilson, 
I., et al., Cell, 37:767 (1984)). 
The term "gene" means the segment of DNA involved in producing a 
polypeptide chain; it includes regions preceding and following the coding 
region (leader and trailer) as well as intervening sequences (introns) 
between individual coding segments (exons). 
Fragments of the full length gene of the invention may be used as a 
hybridization probe for a cDNA library to isolate the full length cDNA and 
to isolate other cDNAs which have a high sequence similarity to the gene 
or similar biological activity. Probes of this type preferably have at 
least 30 bases and may contain, for example, 50 or more bases. The probe 
may also be used to identify a cDNA clone corresponding to a full length 
transcript and a genomic clone or clones that contain the complete gene of 
the invention including regulatory and promotor regions, exons, and 
introns. An example of a screen comprises isolating the coding region of 
the gene by using the known DNA sequence to synthesize an oligonucleotide 
probe. Labeled oligonucleotides having a sequence complementary to that of 
the gene of the present invention are used to screen a library of human 
cDNA, genomic DNA or mRNA to determine which members of the library the 
probe hybridizes to. 
The present invention further relates to polynucleotides which hybridize to 
the hereinabove-described sequences if there is at least 70%, preferably 
at least 90%, and more preferably at least 95% identity between the 
sequences. The present invention particularly relates to polynucleotides 
which hybridize under stringent conditions to the hereinabove-described 
polynucleotides. As herein used, the term "stringent conditions" means 
hybridization will occur only if there is at least 95% and preferably at 
least 97% identity between the sequences. The polynucleotides which 
hybridize to the hereinabove described polynucleotides in a preferred 
embodiment encode polypeptides which either retain substantially the same 
biological function or activity as the mature polypeptide encoded by the 
cDNAs of FIG. 1 (SEQ ID NO:1) or the deposited cDNA(s). 
Alternatively, the polynucleotide may have at least 20 bases, preferably 30 
bases, and more preferably at least 50 bases which hybridize to a 
polynucleotide of the present invention and which has an identity thereto, 
as hereinabove described, and which may or may not retain activity. For 
example, such polynucleotides may be employed as probes for the 
polynucleotide of SEQ ID NO:1, for example, for recovery of the 
polynucleotide or as a diagnostic probe or as a PCR primer. 
Thus, the present invention is directed to polynucleotides having at least 
a 70% identity, preferably at least 90% and more preferably at least a 95% 
identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 
as well as fragments thereof, which fragments have at least 30 bases and 
preferably at least 50 bases and to polypeptides encoded by such 
polynucleotides. 
The deposit(s) referred to herein will be maintained under the terms of the 
Budapest Treaty on the International Recognition of the Deposit of 
Micro-organisms for purposes of Patent Procedure. These deposits are 
provided merely as convenience to those of skill in the art and are not an 
admission that a deposit is required under 35 U.S.C. .sctn.112. The 
sequence of the polynucleotides contained in the deposited materials, as 
well as the amino acid sequence of the polypeptides encoded thereby, are 
incorporated herein by reference and are controlling in the event of any 
conflict with any description of sequences herein. A license may be 
required to make, use or sell the deposited materials, and no such license 
is hereby granted. 
The present invention further relates to a polypeptide which has the 
deduced amino acid sequence of FIG. 1 (SEQ ID NO:2) or which has the amino 
acid sequence encoded by the deposited cDNA, as well as fragments, analogs 
and derivatives of such polypeptide. 
The terms "fragment," "derivative" and "analog" when referring to the 
polypeptide of FIG. 1 (SEQ ID NO:2) or that encoded by the deposited cDNA, 
means a polypeptide which retains essentially the same biological function 
or activity as such polypeptide. Thus, an analog includes a proprotein 
which can be activated by cleavage of the proprotein portion to produce an 
active mature polypeptide. 
The polypeptide of the present invention may be a recombinant polypeptide, 
a natural polypeptide or a synthetic polypeptide, preferably a recombinant 
polypeptide. 
The fragment, derivative or analog of the polypeptide of FIG. 1 (SEQ ID 
NO:2) or that encoded by the deposited cDNA may be (i) one in which one or 
more of the amino acid residues are substituted with a conserved or 
non-conserved amino acid residue (preferably a conserved amino acid 
residue) and such substituted amino acid residue may or may not be one 
encoded by the genetic code, or (ii) one in which one or more of the amino 
acid residues includes a substituent group, or (iii) one in which the 
mature polypeptide is fused with another compound, such as a compound to 
increase the half-life of the polypeptide (for example, polyethylene 
glycol), or (iv) one in which the additional amino acids are fused to the 
mature polypeptide, such as a leader or secretory sequence or a sequence 
which is employed for purification of the mature polypeptide or a 
proprotein sequence. Such fragments, derivatives and analogs are deemed to 
be within the scope of those skilled in the art from the teachings herein. 
The polypeptides and polynucleotides of the present invention are 
preferably provided in an isolated form, and preferably are purified to 
homogeneity. 
The term "isolated" means that the material is removed from its original 
environment (e.g., the natural environment if it is naturally occurring). 
For example, a naturallyoccurring polynucleotide or polypeptide present in 
a living animal is not isolated, but the same polynucleotide or 
polypeptide, separated from some or all of the coexisting materials in the 
natural system, is isolated. Such polynucleotides could be part of a 
vector and/or such polynucleotides or polypeptides could be part of a 
composition, and still be isolated in that such vector or composition is 
not part of its natural environment. 
The polypeptides of the present invention include the polypeptide of SEQ ID 
NO:2 (in particular the mature polypeptide) as well as polypeptides which 
have at least 70% similarity (preferably at least 70% identity) to the 
polypeptide of SEQ ID NO:2 and more preferably at least 90% similarity 
(more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 
and still more preferably at least 95% similarity (still more preferably 
at least 95% identity) to the polypeptide of SEQ ID NO:2 and also include 
portions of such polypeptides with such portion of the polypeptide 
generally containing at least 30 amino acids and more preferably at least 
50 amino acids. 
As known in the art "similarity" between two polypeptides is determined by 
comparing the amino acid sequence and its conserved amino acid substitutes 
of one polypeptide to the sequence of a second polypeptide. 
Fragments or portions of the polypeptides of the present invention may be 
employed for producing the corresponding full-length polypeptide by 
peptide synthesis; therefore, the fragments may be employed as 
intermediates for producing the full-length polypeptides. Fragments or 
portions of the polynucleotides of the present invention may be used to 
synthesize full-length polynucleotides of the present invention. 
The present invention also relates to vectors which include polynucleotides 
of the present invention, host cells which are genetically engineered with 
vectors of the invention and the production of polypeptides of the 
invention by recombinant techniques. 
Host cells are genetically engineered (transduced or transformed or 
transfected) with the vectors of this invention which may be, for example, 
a cloning vector or an expression vector. The vector may be, for example, 
in the form of a plasmid, a viral particle, a phage, etc. The engineered 
host cells can be cultured in conventional nutrient media modified as 
appropriate for activating promoters, selecting transformants or 
amplifying the genes of the present invention. The culture conditions, 
such as temperature, pH and the like, are those previously used with the 
host cell selected for expression, and will be apparent to the ordinarily 
skilled artisan. 
The polynucleotides of the present invention may be employed for producing 
polypeptides by recombinant techniques. Thus, for example, the 
polynucleotide may be included in any one of a variety of expression 
vectors for expressing a polypeptide. Such vectors include chromosomal, 
nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; 
bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors 
derived from combinations of plasmids and phage DNA, viral DNA such as 
vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other 
vector may be used as long as it is replicable and viable in the host. 
The appropriate DNA sequence may be inserted into the vector by a variety 
of procedures. In general, the DNA sequence is inserted into an 
appropriate restriction endonuclease site(s) by procedures known in the 
art. Such procedures and others are deemed to be within the scope of those 
skilled in the art. 
The DNA sequence in the expression vector is operatively linked to an 
appropriate expression control sequence(s) (promoter) to direct mRNA 
synthesis. As representative examples of such promoters, there may be 
mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda 
P.sub.L promoter and other promoters known to control expression of genes 
in prokaryotic or eukaryotic cells or their viruses. The expression vector 
also contains a ribosome binding site for translation initiation and a 
transcription terminator. The vector may also include appropriate 
sequences for amplifying expression. 
In addition, the expression vectors preferably contain one or more 
selectable marker genes to provide a phenotypic trait for selection of 
transformed host cells such as dihydrofolate reductase or neomycin 
resistance for eukaryotic cell culture, or such as tetracycline or 
ampicillin resistance in E. coli. 
The vector containing the appropriate DNA sequence as hereinabove 
described, as well as an appropriate promoter or control sequence, may be 
employed to transform an appropriate host to permit the host to express 
the protein. 
As representative examples of appropriate hosts, there may be mentioned: 
bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; 
fungal cells, such as yeast; insect cells such as Drosophila S2 and 
Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; 
adenoviruses; plant cells, etc. The selection of an appropriate host is 
deemed to be within the scope of those skilled in the art from the 
teachings herein. 
More particularly, the present invention also includes recombinant 
constructs comprising one or more of the sequences as broadly described 
above. The constructs comprise a vector, such as a plasmid or viral 
vector, into which a sequence of the invention has been inserted, in a 
forward or reverse orientation. In a preferred aspect of this embodiment, 
the construct further comprises regulatory sequences, including, for 
example, a promoter, operably linked to the sequence. Large numbers of 
suitable vectors and promoters are known to those of skill in the art, and 
are commercially available. The following vectors are provided by way of 
example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10, phagescript, 
psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A 
(Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); 
Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, 
pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used 
as long as they are replicable and viable in the host. 
Promoter regions can be selected from any desired gene using CAT 
(chloramphenicol transferase) vectors or other vectors with selectable 
markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named 
bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P.sub.R, 
P.sub.L and trp. Eukaryotic promoters include CMV immediate early, HSV 
thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse 
metallothionein-I. Selection of the appropriate vector and promoter is 
well within the level of ordinary skill in the art. 
In a further embodiment, the present invention relates to host cells 
containing the above-described constructs. The host cell can be a higher 
eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, 
such as a yeast cell, or the host cell can be a prokaryotic cell, such as 
a bacterial cell. Introduction of the construct into the host cell can be 
effected by calcium phosphate transfection, DEAE-Dextran mediated 
transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic 
Methods in Molecular Biology, (1986)). 
The constructs in host cells can be used in a conventional manner to 
produce the gene product encoded by the recombinant sequence. 
Alternatively, the polypeptides of the invention can be synthetically 
produced by conventional peptide synthesizers. 
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or 
other cells under the control of appropriate promoters. Cell-free 
translation systems can also be employed to produce such proteins using 
RNAs derived from the DNA constructs of the present invention. Appropriate 
cloning and expression vectors for use with prokaryotic and eukaryotic 
hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory 
Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure 
of which is hereby incorporated by reference. 
Transcription of the DNA encoding the polypeptides of the present invention 
by higher eukaryotes is increased by inserting an enhancer sequence into 
the vector. Enhancers are cis-acting elements of DNA, usually about from 
10 to 300 bp that act on a promoter to increase its transcription. 
Examples include the SV40 enhancer on the late side of the replication 
origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the 
polyoma enhancer on the late side of the replication origin, and 
adenovirus enhancers. 
Generally, recombinant expression vectors will include origins of 
replication and selectable markers permitting transformation of the host 
cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae 
TRP1 gene, and a promoter derived from a highly-expressed gene to direct 
transcription of a downstream structural sequence. Such promoters can be 
derived from operons encoding glycolytic enzymes such as 
3-phosphoglycerate kinase (PGK), .alpha.-factor, acid phosphatase, or heat 
shock proteins, among others. The heterologous structural sequence is 
assembled in appropriate phase with translation initiation and termination 
sequences, and preferably, a leader sequence capable of directing 
secretion of translated protein into the periplasmic space or 
extracellular medium. Optionally, the heterologous sequence can encode a 
fusion protein including an N-terminal identification peptide imparting 
desired characteristics, e.g., stabilization or simplified purification of 
expressed recombinant product. 
Useful expression vectors for bacterial use are constructed by inserting a 
structural DNA sequence encoding a desired protein together with suitable 
translation initiation and termination signals in operable reading phase 
with a functional promoter. The vector will comprise one or more 
phenotypic selectable markers and an origin of replication to ensure 
maintenance of the vector and to, if desirable, provide amplification 
within the host. Suitable prokaryotic hosts for transformation include E. 
coli, Bacillus subtilis, Salmonella typhimurium and various species within 
the genera Pseudomonas, Streptomyces, and Staphylococcus, although others 
may also be employed as a matter of choice. 
As a representative but nonlimiting example, useful expression vectors for 
bacterial use can comprise a selectable marker and bacterial origin of 
replication derived from commercially available plasmids comprising 
genetic elements of the well known cloning vector pBR322 (ATCC 37017). 
Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine 
Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA). 
These pBR322 "backbone" sections are combined with an appropriate promoter 
and the structural sequence to be expressed. 
Following transformation of a suitable host strain and growth of the host 
strain to an appropriate cell density, the selected promoter is induced by 
appropriate means (e.g., temperature shift or chemical induction) and 
cells are cultured for an additional period. 
Cells are typically harvested by centrifugation, disrupted by physical or 
chemical means, and the resulting crude extract retained for further 
purification. 
Microbial cells employed in expression of proteins can be disrupted by any 
convenient method, including freeze-thaw cycling, sonication, mechanical 
disruption, or use of cell lysing agents, such methods are well known to 
those skilled in the art. 
Various mammalian cell culture systems can also be employed to express 
recombinant protein. Examples of mammalian expression systems include the 
COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 
23:175 (1981), and other cell lines capable of expressing a compatible 
vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. 
Mammalian expression vectors will comprise an origin of replication, a 
suitable promoter and enhancer, and also any necessary ribosome binding 
sites, polyadenylation site, splice donor and acceptor sites, 
transcriptional termination sequences, and 5' flanking nontranscribed 
sequences. DNA sequences derived from the SV40 splice, and polyadenylation 
sites may be used to provide the required nontranscribed genetic elements. 
The polypeptide can be recovered and purified from recombinant cell 
cultures by methods including ammonium sulfate or ethanol precipitation, 
acid extraction, anion or cation exchange chromatography, phosphocellulose 
chromatography, hydrophobic interaction chromatography, affinity 
chromatography, hydroxylapatite chromatography and lectin chromatography. 
Protein refolding steps can be used, as necessary, in completing 
configuration of the mature protein. Finally, high performance liquid 
chromatography (HPLC) can be employed for final purification steps. 
The polypeptides of the present invention may be a naturally purified 
product, or a product of chemical synthetic procedures, or produced by 
recombinant techniques from a prokaryotic or eukaryotic host (for example, 
by bacterial, yeast, higher plant, insect and mammalian cells in culture). 
Depending upon the host employed in a recombinant production procedure, 
the polypeptides of the present invention may be glycosylated or may be 
non-glycosylated. Polypeptides of the invention may also include an 
initial methionine amino acid residue. 
The polynucleotides and polypeptides of the present invention may be 
employed as research reagents and materials for discovery of treatments 
and diagnostics to human disease. 
The present invention is also directed to an assay which measures the 
ability of compounds to inhibit the interaction of FLAP II with 5-LO. 
Human osteosarcoma cell lines are transfected with DNA for FLAP II and 
5-LO. The cells are then treated with the Ca.sup.2+ ionophore A23187 
resulting in significant production of 5-LO products. Cells are then 
transfected in the presence of potential antagonist/inhibitor compounds 
and a comparison is done to determine if the level of 5-LO products is 
reduced. If so, then the compound is an effective antagonist/inhitor of 
FLAP II by preventing the interaction of FLAP II with 5-LO. 
An example is an antibody against the polypeptide, or in some cases an 
oligonucleotide, which binds to the polypeptide. Peptide derivatives of 
FLAP II which have no biological function will recognize and bind to the 
substrate and thereby prevent the action of FLAP II. 
Another potential antagonist is an antisense construct prepared using 
antisense technology. Antisense technology can be used to control gene 
expression through triple-helix formation or antisense DNA or RNA, both of 
which methods are based on binding of a polynucleotide to DNA or RNA. For 
example, the 5' coding portion of the polynucleotide sequence, which 
encodes for the mature polypeptides of the present invention, is used to 
design an antisense RNA oligonucleotide of from about 10 to 40 base pairs 
in length. A DNA oligonucleotide is designed to be complementary to a 
region of the gene involved in transcription (triple helix see Lee et al., 
Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); 
and Dervan et al., Science, 251: 1360 (1991)), thereby preventing 
transcription and the production of FLAP II. The antisense RNA 
oligonucleotide hybridizes to the mRNA in vivo and blocks translation of 
the mRNA molecule into FLAP II polypeptide (Antisense-Okano, J. 
Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors 
of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). The 
oligonucleotides described above can also be delivered to cells such that 
the antisense RNA or DNA may be expressed in vivo to inhibit production of 
FLAP II. 
Another example of an inhibitor is a small molecule which binds to the 
active receptor site of FLAP II thereby making it inaccessible to 5-LO 
such that 5-LO is not activated and does not catalyze the production of 
LTs. Examples of small molecules include but are not limited to small 
peptides or peptide-like molecules. 
The antagonists may, therefore, be employed to treat angina, endotoxic 
shock,inflammatory conditions, such as psoriasis, atopic eczema, 
rheumatoid arthritis, ulcerative colitis and other immediate 
hypersensitive reactions. These antagonist/inhibitors may also be used to 
treat LT-mediated naso-bronchial obstructive air-passageway conditions, 
such as allergic bronchial asthma, allergic rhinitis and allergic 
conjunctivitis. They may also be employed as gastric cytoprotective 
agents. 
The antagonists of the present invention may also be employed to treat 
migraine headaches and glomerulonephritis, since LTs cause diffuse 
inflammatory changes in the glomeruli which leads to proteinuria, 
hypertension and edema. Diabetes mellitus may also be treated with the 
antagonists since carbohydrate intolerance in patients with diabetes 
mellitus may result from an excessive imbalance of LT to PG in the islet 
cell. 
The antagonists may be employed in a composition with a pharmaceutically 
acceptable carrier, e.g., as hereinabove described. 
When the antagonist compounds of the invention are employed in the 
treatment of allergic airway disorders, as anti-inflammatory agents and/or 
as cytoprotective agents, they can be formulated into oral dosage forms 
such as tablets, capsules and the like. The compounds can be administered 
alone or by combining them with conventional carriers, such as magnesium 
carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, 
starch, gelatin, tragacanth, methylcellulose, sodium 
carboxymethyl-cellulose, low melting wax, cocoa butter and the like. 
Diluents, flavoring agents, solubilizers, lubricants, suspending agents, 
binders, tablet-disintegrating agents and the like may be employed. The 
compounds may be encapsulated with or without other carriers. In all 
cases, the proportion of active ingredients in said compositions both 
solid and liquid will be at least to impart the desired activity thereto 
on oral administration. The compounds may also be injected parenterally, 
in which case they are used in the form of a sterile solution containing 
other solutes, for example, enough saline or glucose to make the solution 
isotonic. For administration by inhalation or insufflation, the compounds 
may be formulated into an aqueous or partially aqueous solution, which can 
then be utilized in the form of an aerosol. 
The dosage requirements vary with the particular compositions employed, the 
route of administration, the severity of the symptoms presented and the 
particular subject being treated. Treatment will generally be initiated 
with small dosages less than the optimum dose of the compound. Thereafter, 
the dosage is increased until the optimum effect under the circumstances 
is reached. In general, the compounds of the invention are most desirably 
administered at a concentration that will generally afford effective 
results without causing any harmful or deleterious side effects, and can 
be administered either as a single unit dose, or if desired, the dosage 
may be divided into convenient subunits administered at suitable times 
throughout the day. 
The antagonists which are polypeptides may also be employed in accordance 
with the present invention by expression of such polypeptides in vivo, 
which is often referred to as "gene therapy." 
Thus, for example, cells from a patient may be engineered with a 
polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the 
engineered cells then being provided to a patient to be treated with the 
polypeptide. Such methods are well-known in the art and are apparent from 
the teachings herein. For example, cells may be engineered by the use of a 
retroviral plasmid vector containing RNA encoding a polypeptide of the 
present invention. 
Similarly, cells may be engineered in vivo for expression of a polypeptide 
in vivo by, for example, procedures known in the art. For example, a 
packaging cell is transduced with a retroviral plasmid vector containing 
RNA encoding a polypeptide of the present invention such that the 
packaging cell now produces infectious viral particles containing the gene 
of interest. These producer cells may be administered to a patient for 
engineering cells in vivo and expression of the polypeptide in vivo. These 
and other methods for administering a polypeptide of the present invention 
by such method should be apparent to those skilled in the art from the 
teachings of the present invention. 
Retroviruses from which the retroviral plasmid vectors hereinabove 
mentioned may be derived include, but are not limited to, Moloney Murine 
Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma 
Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia 
virus, human immunodeficiency virus, adenovirus, Myeloproliferative 
Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral 
plasmid vector is derived from Moloney Murine Leukemia Virus. 
The vector includes one or more promoters. Suitable promoters which may be 
employed include, but are not limited to, the retroviral LTR; the SV40 
promoter; and the human cytomegalovirus (CMV) promoter described in 
Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other 
promoter (e.g., cellular promoters such as eukaryotic cellular promoters 
including, but not limited to, the histone, pol III, and .beta.-actin 
promoters). Other viral promoters which may be employed include, but are 
not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and 
B19 parvovirus promoters. The selection of a suitable promoter will be 
apparent to those skilled in the art from the teachings contained herein. 
The nucleic acid sequence encoding the polypeptide of the present invention 
is under the control of a suitable promoter. Suitable promoters which may 
be employed include, but are not limited to, adenoviral promoters, such as 
the adenoviral major late promoter; or hetorologous promoters, such as the 
cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) 
promoter; inducible promoters, such as the MMT promoter, the 
metallothionein promoter; heat shock promoters; the albumin promoter; the 
ApoAI promoter; human globin promoters; viral thymidine kinase promoters, 
such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs 
(including the modified retroviral LTRs hereinabove described); the 
.beta.-actin promoter; and human growth hormone promoters. The promoter 
also may be the native promoter which controls the gene encoding the 
polypeptide. 
The retroviral plasmid vector is employed to transduce packaging cell lines 
to form producer cell lines. Examples of packaging cells which may be 
transfected include, but are not limited to, the PE501, 17, .psi.-2, 
.psi.-AM, 2, T19-14X, VT-19-17-H2, .psi.CRE, .psi.CRIP, GP+E-86, 
GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy, 
Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its 
entirety. The vector may transduce the packaging cells through any means 
known in the art. Such means include, but are not limited to, 
electroporation, the use of liposomes, and CaPO.sub.4 precipitation. In 
one alternative, the retroviral plasmid vector may be encapsulated into a 
liposome, or coupled to a lipid, and then administered to a host. 
The producer cell line generates infectious retroviral vector particles 
which include the nucleic acid sequence(s) encoding the polypeptides. Such 
retroviral vector particles then may be employed, to transduce eukaryotic 
cells, either in vitro or in vivo. The transduced eukaryotic cells will 
express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic 
cells which may be transduced include, but are not limited to, embryonic 
stem cells, embryonic carcinoma cells, as well as hematopoietic stem 
cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial 
cells, and bronchial epithelial cells. 
The present invention also relates to a diagnostic assay for detecting 
altered levels of FLAP II protein in various tissues since an 
over-expression of the proteins compared to normal control tissue samples 
can detect the presence of conditions associated with FLAP II activity. 
Assays used to detect levels of FLAP II protein in a sample derived from a 
host are well-known to those of skill in the art and include 
radioimmunoassays, competitive-binding assays, Western Blot analysis and 
preferably an ELISA assay. An ELISA assay initially comprises preparing an 
antibody specific to the FLAP II antigen, preferably a monoclonal 
antibody. In addition a reporter antibody is prepared against the 
monoclonal antibody. To the reporter antibody is attached a detectable 
reagent such as radioactivity, fluorescence or in this example a 
horseradish peroxidase enzyme. A sample is now removed from a host and 
incubated on a solid support, e.g. a polystyrene dish, that binds the 
proteins in the sample. Any free protein binding sites on the dish are 
then covered by incubating with a non-specific protein such as bovine 
serum albumin. Next, the monoclonal antibody is incubated in the dish 
during which time the monoclonal antibodies attach to any FLAP II proteins 
attached to the polystyrene dish. All unbound monoclonal antibody is 
washed out with buffer. The reporter antibody linked to horseradish 
peroxidase is now placed in the dish resulting in binding of the reporter 
antibody to any monoclonal antibody bound to FLAP II. Unattached reporter 
antibody is then washed out. Peroxidase substrates are then added to the 
dish and the amount of color developed in a given time period is a 
measurement of the amount of FLAP II protein present in a given volume of 
patient sample when compared against a standard curve. 
A competition assay may be employed wherein antibodies specific to FLAP II 
are attached to a solid support and labeled FLAP II and a sample derived 
from the host are passed over the solid support and the amount of label 
detected attached to the solid support can be correlated to a quantity of 
FLAP II in the sample. 
The present invention will be further described with reference to the 
following examples; however, it is to be understood that the present 
invention is not limited to such examples. All parts or amounts, unless 
otherwise specified, are by weight. 
In order to facilitate understanding of the following examples certain 
frequently occurring methods and/or terms will be described. 
"Plasmids" are designated by a lower case p preceded and/or followed by 
capital letters and/or numbers. The starting plasmids herein are either 
commercially available, publicly available on an unrestricted basis, or 
can be constructed from available plasmids in accord with published 
procedures. In addition, equivalent plasmids to those described are known 
in the art and will be apparent to the ordinarily skilled artisan. 
"Digestion" of DNA refers to catalytic cleavage of the DNA with a 
restriction enzyme that acts only at certain sequences in the DNA. The 
various restriction enzymes used herein are commercially available and 
their reaction conditions, cofactors and other requirements were used as 
would be known to the ordinarily skilled artisan. For analytical purposes, 
typically 1 .mu.g of plasmid or DNA fragment is used with about 2 units of 
enzyme in about 20 .mu.l of buffer solution. For the purpose of isolating 
DNA fragments for plasmid construction, typically 5 to 50 .mu.g of DNA are 
digested with 20 to 250 units of enzyme in a larger volume. Appropriate 
buffers and substrate amounts for particular restriction enzymes are 
specified by the manufacturer. Incubation times of about 1 hour at 
37.degree. C. are ordinarily used, but may vary in accordance with the 
supplier's instructions. After digestion the reaction is electrophoresed 
directly on a polyacrylamide gel to isolate the desired fragment. 
Size separation of the cleaved fragments is performed using 8 percent 
polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 
8:4057 (1980). 
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide 
or two complementary polydeoxynucleotide strands which may be chemically 
synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus 
will not ligate to another oligonucleotide without adding a phosphate with 
an ATP in the presence of a kinase. A synthetic oligonucleotide will 
ligate to a fragment that has not been dephosphorylated. 
"Ligation" refers to the process of forming phosphodiester bonds between 
two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 
146). Unless otherwise provided, ligation may be accomplished using known 
buffers and conditions with 10 units to T4 DNA ligase ("ligase") per 0.5 
.mu.g of approximately equimolar amounts of the DNA fragments to be 
ligated. 
Unless otherwise stated, transformation was performed as described in the 
method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973). 
EXAMPLE 1 
Cloning and Expression of FLAP II Using E.coli 
The DNA sequence encoding the FLAP II protein, ATCC # 75771, was amplified 
using PCR oligonucleotide primers corresponding to the 5' and 3' sequences 
of the gene: The forward primer has the sequence: 
CGCGGGATCCGCCGGGAACTCGATCCTGCTGGCTGCT (SEQ ID NO:3). 
It contains a recognition site for the restriction endonuclease BamHI 
followed by 27 nucleotides of the FLAP II gene encoding amino acids 2-10. 
The AUG codon encoding the first methionine is omitted. An initiation 
codon is provided by the vector pQE-9 (Qiagen, Inc., 9259 Eton Avenue, 
Chatsworth, Calif. 91311). 
The reverse primer has the sequence: GCGCAAGCTTAGAATTGCCGCCTCAGTTTCTTGGC 
(SEQ ID NO:4). It contains the last 24 nucleotides complementary to the 3' 
end of the FLAP II gene followed by a translational stop codon 
(underlined) and a recognition site for the restriction endonuclease 
HindIII (in bold). 
The amplified sequences were isolated from a 1% agarose gel using a 
commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Calif.). 
The fragment was then digested with the endonucleases BamHI and Asp 718 
and then purified again by isolation on a 1% agarose gel. This fragment is 
designated F1. 
pQE-9 encodes antibiotic resistance (Amp.sup.r), a bacterial origin of 
replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome 
binding site (RBS), a 6-His-tag and unique restriction enzyme cleavage 
sites. 
4 .mu.g of the plasmid pD10 (Qiagen) were digested with the enzymes BamHI 
and HindIII and then dephosphorylated using calf intestinal phosphatase 
using protocols known in the art. 
The plasmid was then isolated from a 1% agarose gel using the commercially 
available kit ("Geneclean"). The dephosphorylated vector DNA is designated 
V1. 
The dephosphorylated vector V1 was ligated with the fragment F1 using T4 
DNA ligase using procedures known in the art. The ligation mixture was 
then transformed into E.coli M15 (described as strain OZ 291 by Villarejo 
et al. in J. Bacteriol. 210:466-474 [1974] containing the repressor 
plasmid pDMI.1 (Certa et al. 1986, EMBO Journal 5:30513056). M15/pDMI.1 
contains multiple copies of the plasmid pDMI.1, which expresses the laci 
repressor and also confers kanamycin resistance (Kan.sup.r). 
Plasmids of transformed bacteria were then isolated and characterized for 
the correct insertion of the FLAP II gene using the restriction enzymes 
BamHI and HindIII. A plasmid was isolated containing the correct insert 
and called pHIS-FLAPII. 
E.coli M15 cells containing pDMI.1 were transformed with pHIS-FLAP II and 
subsequently grown at 37.degree. C. in LB medium (10 g bacto tryptone, 5 g 
yeast extract, 5 g NaCl per liter) containing 100 mg/l ampicillin and 25 
mg/l kanamycin. At an optical density at 600 nm of 0.8 IPTG was added to a 
final concentration of 2 mM. After additional 2.5 hours at 37.degree. C. 
the cells were harvested by centrifugation. 
The FLAP II protein expressed in E.coli was purified by Ni-chelate affinity 
chromatography. The E.coli cells of 1 liter induced culture were lysed by 
adding buffer A (6 M guanidine-hydrochloride, 0.1 M sodium phosphate, pH 
8.0) and stirring the suspension for 2 hours (100 rpm). The suspension was 
then centrifuged for 10 minutes at 100000.times.g. The supernatant was 
loaded onto a column containing 3 ml of the NTA-resin (Qiagen Inc.). Then, 
the column was washed with 30 ml of buffer A. Subsequently, the column was 
washed with 20 ml of buffer B (8 M urea, 0.1 M sodium phosphate, 0.01 M 
Tris, pH 8.0), and then with 20 ml of buffer B, pH 6.5. Finally, the FLAP 
II protein was eluted with buffer B, pH 4.5. The presence of the FLAP II 
protein was confirmed by SDS-PAGE, (Laemmli, Nature 227, 680-685 (1970). 
Descriptions for the purification of various His-tagged proteins can be 
found in Hochuli et al., J. Chromatography 411:177-184 (1984), Hochuli et 
al. Bio/Technology 11:1321-1325 and Gentz et al. (1989) Proc. Natl. Acad. 
Sci. USA 86:821-824. 
EXAMPLE 2 
Cloning and Expression of FLAP II Using the Baculovirus Expression System 
The DNA sequence encoding the full length FLAP II protein, ATCC # 75771, 
was amplified using PCR oligonucleotide primers corresponding to the 5' 
and 3' sequences of the gene: 
The 5' primer has the sequence CCGGATCCGCCACCATGGCCGGGAACTCGATCCT (SEQ ID 
NO:5), and contains a BamHI restriction enzyme site (in bold) followed by 
6 nucleotides resembling an efficient signal for the initiation of 
translation in eukaryotic cells (J. Mol. Biol. 1987, 196, 947-950, Kozak, 
M.), and just behind the first 20 nucleotides of the FLAP II gene (the 
initiation codon for translation "ATG" is underlined). 
The 3' primer has the sequence CACAGGTACCAGCTTCTGCAAGCATTAAAG (SEQ ID 
NO:6), and contains the cleavage site for the restriction endonuclease 
Asp718 and 20 nucleotides complementary to the 3' non-translated sequence 
of the FLAP II gene. The amplified sequences were isolated from a 1% 
agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc., 
La Jolla, Calif.). The fragment was then digested with the endonucleases 
BamHI and Asp 718 and then purified as described in Example 1. This 
fragment is designated F2. 
The vector pRG1 (modification of pVL941 vector, discussed below) is used 
for the expression of the FLAP II protein using the baculovirus expression 
system (for review see: Summers, M. D. and Smith, G. E. 1987, A manual of 
methods for baculovirus vectors and insect cell culture procedures, Texas 
Agricultural Experimental Station Bulletin No. 1555). This expression 
vector contains the strong polyhedrin promoter of the Autographa 
californica nuclear polyhedrosis virus (AcMNPV) followed by the 
recognition sites for the restriction endonucleases BamHI and Asp718. The 
polyadenylation site of the simian virus (SV)40 is used for efficient 
polyadenylation. For an easy selection of recombinant viruses the 
beta-galactosidase gene from E.coli is inserted in the same orientation as 
the polyhedrin promoter followed by the polyadenylation signal of the 
polyhedrin gene. The polyhedrin sequences are flanked at both sides by 
viral sequences for the cell-mediated homologous recombination of 
cotransfected wild-type viral DNA. Many other baculovirus vectors could be 
used in place of pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V.A. and 
Summers, M. D., Virology, 170:31-39). 
The plasmid was digested with the restriction enzymes BamHI and Asp718 and 
then dephosphorylated using calf intestinal phosphatase by procedures 
known in the art. The DNA was then isolated from a 1% agarose gel as 
described in Example 1. This vector DNA is designated V2. 
Fragment F2 and the dephosphorylated plasmid V2 were ligated with T4 DNA 
ligase. E.coli HB101 cells were then transformed and bacteria identified 
that contained the plasmid (pBacFLAPII) with the FLAP II gene using the 
enzymes BamHI and Asp718. The sequence of the cloned fragment was 
confirmed by DNA sequencing. 
5 .mu.g of the plasmid pBacFLAPII were cotransfected with 1.0 .mu.g of a 
commercially available linearized baculovirus ("BaculoGold.TM. baculovirus 
DNA", Pharmingen, San Diego, Calif.) using the lipofection method (Felgner 
et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)). 
1 .mu.g of BaculoGold.TM. virus DNA and 5 .mu.g of the plasmid pBacFLAP II 
were mixed in a sterile well of a microtiter plate containing 50 .mu.l of 
serum free Grace's medium (Life Technologies Inc., Gaithersburg, Md.). 
Afterwards 10 .mu.l Lipofectin plus 90 .mu.l Grace's medium were added, 
mixed and incubated for 15 minutes at room temperature. Then the 
transfection mixture was added dropwise to the Sf9 insect cells (ATCC CRL 
1711) seeded in a 35 mm tissue culture plate with lml Grace' medium 
without serum. The plate was rocked back and forth to mix the newly added 
solution. The plate was then incubated for 5 hours at 27.degree. C. After 
5 hours the transfection solution was removed from the plate and 1 ml of 
Grace's insect medium supplemented with 10% fetal calf serum was added. 
The plate was put back into an incubator and cultivation continued at 
27.degree. C. for four days. 
After four days the supernatant was collected and a plaque assay performed 
similar as described by Summers and Smith (supra). As a modification an 
agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) was 
used which allows an easy isolation of blue stained plaques. (A detailed 
description of a "plaque assay" can also be found in the user's guide for 
insect cell culture and baculovirology distributed by Life Technologies 
Inc., Gaithersburg, page 9-10). 
Four days after the serial dilution of the viruses was added to the cells, 
blue stained plaques were picked with the tip of an Eppendorf pipette. The 
agar containing the recombinant viruses was then resuspended in an 
Eppendorf tube containing 200 .mu.l of Grace's medium. The agar was 
removed by a brief centrifugation and the supernatant containing the 
recombinant baculoviruses was used to infect Sf9 cells seeded in 35 mm 
dishes. Four days later the supernatants of these culture dishes were 
harvested and then stored at 4.degree. C. 
Sf9 cells were grown in Grace's medium supplemented with 10% 
heat-inactivated FBS. The cells were infected with the recombinant 
baculovirus V-FLAP II at a multiplicity of infection (MOI) of 2. Six hours 
later the medium was removed and replaced with SF900 II medium minus 
methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours 
later 5 .mu.Ci of .sup.35 S-methionine and 5 .mu.Ci .sup.35 S cysteine 
(Amersham) were added. The cells were further incubated for 16 hours 
before they were harvested by centrifugation and the labelled proteins 
visualized by SDS-PAGE and autoradiography. 
EXAMPLE 3 
Expression of FLAP II in Mammalian Cells 
Fragment F2 described in example 2 was used for the insertion into the 
mammalian expression vector pCMV11. 
Plasmid pCMV11 contains the strong promoter and enhancer of the "major 
immediate-early" gene of human cytomegalovirus ("HCMV"; Boshart et al., 
Cell, 41:521-530 (1985)) behind the promoter are single cleavage sites for 
the restriction endonucleases HindIII, BamHI, Pvull and Asp 718. After the 
Asp 718 cleavage site there is situated the polyadenylation site of the 
preproinsulin gene of the rat (Lomedico et al., Cell, 18:545-558 (1979)). 
The plasmid contains in addition the replication origin of the SV40 virus 
and a fragment from pBR322 which confers E.coli bacteria ampicillin 
resistance and the replication in E.coli. Plasmid pCMV11 was digested with 
BamHI and Asp718 and then dephosphorylated using calf intestinal 
phosphatase as described in Example 1. The dephosphorylated vector was 
thereafter isolated from an agarose gel as described in Example 1. 
The vector fragment V3 was ligated with fragment F2, E.coli HB101 bacteria 
were transfonned and the plasmids of the transformed cells isolated by 
procedures known in the art. By means of restriction analysis and DNA 
sequencing according to known methods, transformants were identified which 
contained the plasmid with the insert in the correct orientation. This 
vector received the designation pCMV-FLAP II. 
Transfections of the COS1 (ATCC CRL 1650) Raji-(ATCC CRL 8163) and 
Jurkart-(ATCC CCL 86) cells with the plasmid pCMV-FLAP II were carried out 
either according to the lipofection method published by Felgner et al. 
(Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987)) or by the well known 
technique using DEAE Dextran (Pharmacia). The expression vector pCMV11 
without the FLAP II gene served as a control. 72 hours after the 
transfections were carried out the cells were harvested and analyzed for 
the activation of 5-lipoxygenase. 
EXAMPLE 4 
Expression via Gene Therapy 
Fibroblasts are obtained from a subject by skin biopsy. The resulting 
tissue is placed in tissue-culture medium and separated into small pieces. 
Small chunks of the tissue are placed on a wet surface of a tissue culture 
flask, approximately ten pieces are placed in each flask. The flask is 
turned upside down, closed tight and left at room temperature over night. 
After 24 hours at room temperature, the flask is inverted and the chunks 
of tissue remain fixed to the bottom of the flask and fresh media (e.g., 
Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added. This 
is then incubated at 37.degree. C. for approximately one week. At this 
time, fresh media is added and subsequently changed every several days. 
After an additional two weeks in culture, a monolayer of fibroblasts 
emerge. The monolayer is trypsinized and scaled into larger flasks. 
pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by the long 
terminal repeats of the Moloney murine sarcoma virus, is digested with 
EcoRI and HindIII and subsequently treated with calf intestinal 
phosphatase. The linear vector is fractionated on agarose gel and 
purified, using glass beads. 
The cDNA encoding a polypeptide of the present invention is amplified using 
PCR primers which correspond to the 5' and 3' end sequences respectively. 
The 5' primer contains an EcoRI site and the 3' primer includes a HindIII 
site. Equal quantities of the Moloney murine sarcoma virus linear backbone 
and the amplified EcoRI and HindIII fragment are added together in the 
presence of T4 DNA ligase. The resulting mixture is maintained under 
conditions appropriate for ligation of the two fragments. The ligation 
mixture is used to transform bacteria HB101, which are then plated onto 
agar-containing kanamycin for the purpose of confirming that the vector 
had the gene of interest properly inserted. 
The amphotropic pA317 or GP+am12 packaging cells are grown in tissue 
culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) 
with 10% calf serum (CS), penicillin and streptomycin. The MSV vector 
containing the gene is then added to the media and the packaging cells are 
transduced with the vector. The packaging cells now produce infectious 
viral particles containing the gene (the packaging cells are now referred 
to as producer cells). 
Fresh media is added to the transduced producer cells, and subsequently, 
the media is harvested from a 10 cm plate of confluent producer cells. The 
spent media, containing the infectious viral particles, is filtered 
through a millipore filter to remove detached producer cells and this 
media is then used to infect fibroblast cells. Media is removed from a 
sub-confluent plate of fibroblasts and quickly replaced with the media 
from the producer cells. This media is removed and replaced with fresh 
media. If the titer of virus is high, then virtually all fibroblasts will 
be infected and no selection is required. If the titer is very low, then 
it is necessary to use a retroviral vector that has a selectable marker, 
such as neo or his. 
The engineered fibroblasts are then injected into the host, either alone or 
after having been grown to confluence on cytodex 3 microcarrier beads. The 
fibroblasts now produce the protein product. 
Numerous modifications and variations of the present invention are possible 
in light of the above teachings and, therefore, within the scope of the 
appended claims, the invention may be practiced otherwise than as 
particularly described. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- (1) GENERAL INFORMATION: 
- (iii) NUMBER OF SEQUENCES: 7 
- (2) INFORMATION FOR SEQ ID NO:1: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 444 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 1..441 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- ATG GCC GGG AAC TCG ATC CTG CTG GCT GCT GT - #C TCT ATT CTC TCG GCC 
48 
Met Ala Gly Asn Ser Ile Leu Leu Ala Ala Va - #l Ser Ile Leu Ser Ala 
# 15 
- TGT CAG CAA AGT TAT TTT GCT TTG CAA GTT GG - #A AAG GCA AGA TTA AAA 
96 
Cys Gln Gln Ser Tyr Phe Ala Leu Gln Val Gl - #y Lys Ala Arg Leu Lys 
# 30 
- TAC AAA GTT ACG CCC CCA GCA GTC ACT GGG TC - #A CCA GAG TTT GAG AGA 
144 
Tyr Lys Val Thr Pro Pro Ala Val Thr Gly Se - #r Pro Glu Phe Glu Arg 
# 45 
- GTA TTT CGG GCA CAA CAA AAC TGT GTG GAG TT - #T TAT CCT ATA TTC ATA 
192 
Val Phe Arg Ala Gln Gln Asn Cys Val Glu Ph - #e Tyr Pro Ile Phe Ile 
# 60 
- ATT ACA TTG TGG ATG GCT GGG TGG TAT TTC AA - #C CAA GTT TTT GCT ACT 
240 
Ile Thr Leu Trp Met Ala Gly Trp Tyr Phe As - #n Gln Val Phe Ala Thr 
# 80 
- TGT CTG GGT CTG GTG TAC ATA TAT GGC CGT CA - #C CTA TAC TTC TGG GGA 
288 
Cys Leu Gly Leu Val Tyr Ile Tyr Gly Arg Hi - #s Leu Tyr Phe Trp Gly 
# 95 
- TAT TCA GAA GCT GCT AAA AAA CGG ATC ACC GG - #T TTC CGA CTG AGT CTG 
336 
Tyr Ser Glu Ala Ala Lys Lys Arg Ile Thr Gl - #y Phe Arg Leu Ser Leu 
# 110 
- GGG ATT TTG GCC TTG TTG ACC CTC CTA GGT GC - #C CTG GGA ATT GCA AAC 
384 
Gly Ile Leu Ala Leu Leu Thr Leu Leu Gly Al - #a Leu Gly Ile Ala Asn 
# 125 
- AGC TTT CTG GAT GAA TAT CTG GAC CTC AAT AT - #T GCC AAG AAA CTG AGG 
432 
Ser Phe Leu Asp Glu Tyr Leu Asp Leu Asn Il - #e Ala Lys Lys Leu Arg 
# 140 
# 444 
Arg Gln Phe 
145 
- (2) INFORMATION FOR SEQ ID NO:2: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 147 amino 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- Met Ala Gly Asn Ser Ile Leu Leu Ala Ala Va - #l Ser Ile Leu Ser Ala 
# 15 
- Cys Gln Gln Ser Tyr Phe Ala Leu Gln Val Gl - #y Lys Ala Arg Leu Lys 
# 30 
- Tyr Lys Val Thr Pro Pro Ala Val Thr Gly Se - #r Pro Glu Phe Glu Arg 
# 45 
- Val Phe Arg Ala Gln Gln Asn Cys Val Glu Ph - #e Tyr Pro Ile Phe Ile 
# 60 
- Ile Thr Leu Trp Met Ala Gly Trp Tyr Phe As - #n Gln Val Phe Ala Thr 
# 80 
- Cys Leu Gly Leu Val Tyr Ile Tyr Gly Arg Hi - #s Leu Tyr Phe Trp Gly 
# 95 
- Tyr Ser Glu Ala Ala Lys Lys Arg Ile Thr Gl - #y Phe Arg Leu Ser Leu 
# 110 
- Gly Ile Leu Ala Leu Leu Thr Leu Leu Gly Al - #a Leu Gly Ile Ala Asn 
# 125 
- Ser Phe Leu Asp Glu Tyr Leu Asp Leu Asn Il - #e Ala Lys Lys Leu Arg 
# 140 
- Arg Gln Phe 
145 
- (2) INFORMATION FOR SEQ ID NO:3: 
- (i) SEQUENCE CHARACTERISTICS: 
#acids (A) LENGTH: 160 amino 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: protein 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- Met Asp Gln Glu Thr Val Gly Asn Val Val Le - #u Leu Ala Ile Val Thr 
# 15 
- Leu Ile Ser Val Val Gln Asn Gly Phe Phe Al - #a His Lys Val Glu His 
# 30 
- Glu Ser Arg Thr Gln Asn Gly Arg Ser Phe Gl - #n Arg Thr Gly Thr Leu 
# 45 
- Ala Phe Glu Arg Val Tyr Thr Ala Asn Gln As - #n Cys Val Asp Ala Tyr 
# 60 
- Pro Thr Phe Leu Ala Val Leu Trp Ser Ala Gl - #y Leu Leu Cys Ser Gln 
#80 
- Val Pro Ala Ala Phe Ala Gly Leu Met Tyr Le - #u Phe Val Arg Gln Lys 
# 95 
- Tyr Phe Val Gly Tyr Leu Gly Glu Arg Thr Gl - #n Ser Thr Pro Gly Tyr 
# 110 
- Ile Phe Gly Lys Arg Ile Ile Leu Phe Leu Ph - #e Leu Met Ser Val Ala 
# 125 
- Gly Ile Phe Asn Tyr Tyr Leu Ile Phe Phe Gl - #y Ser Asp Phe Glu Asn 
# 140 
- Tyr Ile Lys Thr Ile Ser Thr Thr Ile Ser Pr - #o Leu Leu Leu Ile Pro 
145 1 - #50 1 - #55 1 - 
#60 
- (2) INFORMATION FOR SEQ ID NO:4: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 40 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
# 40 AACT CGATCCTGCT GCTGGCTGCT 
- (2) INFORMATION FOR SEQ ID NO:5: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 35 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
# 35 GCCG CCTCAGTTTC TTGGC 
- (2) INFORMATION FOR SEQ ID NO:6: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 34 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
# 34 GGCC GGGAACTCGA TCCT 
- (2) INFORMATION FOR SEQ ID NO:7: 
- (i) SEQUENCE CHARACTERISTICS: 
#pairs (A) LENGTH: 30 base 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- (ii) MOLECULE TYPE: DNA (genomic) 
- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
# 30 TGCA AGCATTAAAG 
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