Note: Descriptions are shown in the official language in which they were submitted.
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W O 97/12963 PCTAEP96/04268
P~F-ACEllnJrYDROLASE AND USEIN THE~UUFY
New Use
The present invention relates to the use of a novel lipase in therapy.
Lipoprotein Associated Phospholipase A2 (Lp-PLA2) [also known as Platelet
Activating Factor Acetyl Hydrolase (PAF acetyl hydrolase)] is responsible, during the
conversion of LDL to its oxidised form, for hydrolysing the sn-2 ester of oxidatively
modified phosphatidylcholine to give Iyso-phosphatidylcholine and an oxidativelymodified fatty acid. Both of these products of Lp-PLA2 action are potent
chemoattractants for circulating monocytes. As such, this enzyme is thought to be
responsible for the accumulation of cells loaded with cholesterol ester in the arteries,
10 causing the characteristic 'fatty streak' associated with the early stages ofatherosclerosis. The arnino acid and DNA sequence of the enzyme lipoprotein
associated Lp-PLA2 are disclosed in WO95/00649 (SmithKline Beecharn plc).
WO 95/09921 (Icos Corporation) suggests a range of possible therapeutic use
for platelet activating factor acetylhydrolase, in regulating pathological infi:~mm~tory
15 events such as asthma, anaphylaxis, reperfusion injury and central nervous system
ischemia, antigen-induced arthritis, atherogenesis, Crohn's Disease, icçh~mic bowel
necrosis/necrotising enterocolitis, ulcerative colitis, ischemic stroke, icch~mic brain
injury, systemic lupus eryth~m~tosus, acute pancreatitis, septi~emi~l~ acute post
streptococcal glomerulonephritis, pulmonary edema resulting from IL-2 therapy,
20 allergic infl~mm~tion, ischemic renal failure, preterm labour and adult respiratory
distress syndrome. It is being developed by Icos for the treatment of ~ ces
associated with PAF, including acute respiratory distress syndrome, asthma, acute
pancreatitis, infl~mm~tQry bowel disease, solid organ transplant and necrotizingentercolitis.
More recently, International application number PCT/GB95/02320
(SmithKline Beecham plc) has disclosed a novel polypeptide of human origin having
the amino acid sequence given in SEQ ID NO 1, and fragments. analogs or derivative
thereof. In addition, it discloses polynucleotides (DNA or RNA) which encode such
polypeptide sequences, in particular a polynucleotide having the DNA sequence given
30 in SEQ ID NO 2. cDNA molecules showing extended identity sections with the
cDNA of SEQ ID NO 2 have been identified in cDNA libraries from foetal heart,
pineal gland, activated T cells, microvascular endothelial cells and secondary breast
tumour tissues. The polynucleotide of SEQ ID NO 2 was discovered in a cDNA
library derived from prostate (benign possible hyperplasia). It is structurally related
35 to the lipase family. It contains an open reading frame encoding a protein of about
393 amino acid residues. The protein exhibits the highest degree of homology to Lp-
PLA2 (WO95/00649, WO 95/09912) with 40% identi~y and 60% similarity over a
390 amino acid stretch. Although the overall identity is only 405~, the residues
- 1 -
CA 02233300 l998-03-27
WO 97/12963 PCT/EP96/04268
identifird for the catalytic triad in Lp-PLA2 (WO 95/09912) are conserved between
the two polypeptides implying that they are likely to have a similar biochemicalfilnrtion The positions of the Ser, Asp and His, are underlined below. SEQ ID NO 1
is the lower seqence and Lp-PLA2 the upper sequence. Vertical lines inllir~
S i-lentirnl residuec,
238 DIDHGKPVKNAIRLKFDMEQLKDSIDREKIAVIGHSFGGATVIQTLSEDQ 287
::. 1..1 1 :. :1: 11:.11..::11:11111111.1 .1..:
202 EVTAGQTVFNIFPGGLDLMTLKGNIDMSRVAVMGH~FGGATAILALAKET 251
288 RFRcGIAL~!AwMFpLGDEvysRIpQpLFFINsEyFQypANIIKMKKcysp 337
.111::111111111: :.1.: ..1:1111.1 11 ..: 111.:..
252 QFRcAvAL~2AwMFpLERDFypKARGpvFFI~ K~,y~ IEsvNLMKKIcAQ 301
338 DKERKMITIRGSV~QNFADFTFATGKIIGHML..KLKGDIDSNAAIDLSN 385
..:.::11: 1111.. . I 1.1.11.:11.:: . :1.:1. .: ::
302 HEQSRIITVLGSVE~RSQTDFAFVTGNLIGKFFSTETRGSLDPYEGQEVMV 351
The aforementioned patent application (International application number
PCT/GB95/02320) discloses that inhibitors of the polypeptide may be of use in
various therapeutic applications such as atherosclerosis, myocardial infarction,reperfusion injury, acute and chronic innnmmntion~ rhrl-m~toid arthritis, stroke,
diabetes and neuropsychiatric illnes~s but fails to suggest a possible therapeutic role
for the polypetide per se.
We have now established that this novel polypetide, already implicated as
probable lipase by the sequence companson shown above, shows PAF-
acetylhydrolase activity in vitro.
Accordingly, the present invention provides for the use of a polypeptide
having the amino acid sequence given in SEQ ID NO 1, and fr~gmentc. analogs or
derivative thereof in therapy, in particular for the trentment of diseases associated
with PAF.
Within the limits imposed by the application of an active protein therapeutic
agent, the clinical indications are already, or will be, indicated by the progression of
PAF antagonists in revealing the pathological mechanism of disorders involving PAF.
These are detailed in a review by Kotai et al. (Platelet-activating factor antagonists:
scientific background and possible clinical applications, Koltai M, Guinot P, Hosford
D & Braquet G, Advances in Pharamacology, Vol 28, 1994, Ar~drmic Press, NY).
Such diseases include those hereinbefore mentioned for PAF-acetyl hydrolase,
in particular, acute respiratory distress syndrome, asthma, acute pancreatitis,
intlnmmntory bowel disease, solid organ transplant and necrotizing entercolitis, as
well as AIDS tPlatlet activating factor: a candidate HIV- 1 induced nerotoxin, Gelbard
et al. J.Virol, 68, 4828-4635 (1994)~.
- 2 --
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W O 97/12963 PCTAEP~6/01~6
As used herein, the term 'polypeptide(s)' refers to a polypeptide having the
amino acid sequence given in SEQ ID NO 1 and fragments, analogs and derivatives
thereof.
The polypeptide of the present invention may be a recombinant polypeptide, a
5 natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
,~ The fragment, derivative or analog of the polypeptide of SEQ ID NO 1 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 acidresidue) and such substituted amino acid residue may or may not be one encoded by
10 the genetic code, or (ii) one in which one or more of the amino acid residues includes
a sut)stitl~ent 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
15 which is employed for purification of the mature polypeptide or a proprotein
sequence, (v) one in which amino acid substitutions have been made to induce a
glycosylation pattern to increase half-life and/or bioavalability, or (vi) one in which
amino acid substitutions, addtions or truncations have been made to e~h~n~e the
capability of the enzyme to utilise PAF as a substrate. Such fr~gments, derivatives
20 and analogs are deemed to be within the scope of those skilled in the art from the
teachings herein.
The terms "fragment," "derivative" and "analog" when referring to the
polypeptide of SEQ ID NO 1, means a polypeptide which retains essenti~lly the same
biological function or activity as such polypeptide. Thus, an analog includes a
25 proprotein which can be activated by cleavage of the proprotein portion to produce an
active mature polypeptide.
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
30 environment (e.g., the natural environment if it is naturally occurring). For example,
a naturally-occurring 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
35 composition, and still be isolated in that such vector or composition is not part of its
natural environment. The polypeptide is preferably in purified form. By purifiedform is meant at least 80%, more preferably 90%, still more preferably 95% and most
preferably 99% pure with respect to other protein cont~min:~nts.
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W O 97/12963 PCTAEP96/04268
The polypeptides of the present invention may be employed in combination
with a suitable pharn~euti~l carrier. Such compositions comprise a therapeutically
effective amount of the active agent, and a pharmaceutically acceptable carrier or
excipient. Such a carrier includes but is not limited to saline, buffered saline,
S dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should
suit the mode of :~ministration ,~
The pharmaceutical compositions may be ;l-lminictPred in a convenient
manner such as by the oral, topical, intravenous, intraperitoneal, intr~mllscul~r,
subcutaneous, intranasal or intradermal routes. The polypeptides or polynucleotides
10 of the present invention is ~lmini~stered in an amount which is effective for tre~tment
and/or prophylaxis of the specific in-1ic~tion The amounts and dosage regimens of
active agent a~minictpred to a subject will depend on a number of factors such as the
mode of ~llminictration~ the nature of the condition being treated and the judgme~t of
the prescAbing physician.
The invention also provides a pharmaceutical pack or kit comprising one or
more cnntzlinPrs filled with one or more of the ingredients of the pharm~t~eutical
compositions of the invention. Associated with such cnnt~inPr(s) can be a notice in
the form prescribed by a governm~nt~l agency regulating the manufacture, use or sale
of phslrm~-~eutir~lc or biological products, which notice reflects approval by the
20 agency of m~mlfartllre, use or sale for human ~lminictration In addition, thepolypeptides of the present invention may be employed in conjunction with other
therapeutic compounds.
Polypeptide for use in the present invention may be obtained from genetically
modif1ed host cells using gentic en,inPering techniques well known to those skilled in
25 the art.
The polynucleotide for use in 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
30 sequence which encodes the polypeptide may be identical to the coding sequence
shown in SEQ ID NO 1 or may be a different coding sequence which, as a result ofthe rednnd~ncy or degener~cy of the genetic code, encodes the same polypeptide as
the DNA of SEQ ~ NO 1. Also included are variants of the hereinabove described
polynucleotides which encode fr~gm.ontc, analogs and derivatives of the polypeptide
35 having the arnino acid sequence of SEQ ID NO 1. The variant of the polynucleotide
may be a naturally occurring allelic variant of the polynucleotide or a non-naturally
occurring variant of the polynucleotide.
The polynucleotide may have a coding sequence which is a naturally
occurring allelic variant of the coding sequence of SEQ ID NO 2. As known in the-- 4 --
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W O 97/12963 PCT~EP96/04268
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 polynucleotide which encodes for the polypeptide of SEQ ID NO 1 may
5 include: only the coding sequence for the polypeptide; the coding sequence for the
polypeptide and ~1ition~l coding sequence such as a leader or secretory sequence or
a proprotein sequence; the coding sequence for the polypeptide (and optionally
additional coding sequence) and non-coding sequence, such as introns or non-coding
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 therefore includes polynucleotides, wherein the coding
sequence for the polypeptide may be fused in the same reading frame to a
15 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
20 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
25 both a prosequence and a presequence (leader sequence).
The polynucleotides 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 hexa-histidine tag supplied by a pQE-9
vector to provide for purification of the mature polypeptide fused to the marker in the
30 case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin
(HA) tag when a m~mm~ n host, e.g. COS-7 cells, is used. The HA tag corresponds
to an epitope derived from the infl~en7~ hemagglutinin protein (Wilson, I., et al.,
Cell, 37:767 (1984)).
Suitable polynucleotides also include those which hybridize to the
35 hereinabove-described sequences if there is at least 50% and preferably 70% identity
between the sequences, in particular 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
CA 02233300 1998-03-27
W O 97/12963 . PCTAEP96/04268
hybridize to the hereinabove described polynucleotides in a preferred embodimentencode polypeptides which retain subst~n~i~lly the same biological function or
activity as the polypeptide of SEQ ID NO 1.
Suitable host cells are oen~ti~ ~lly engineered (transduced or transformed er
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 aplasmid, a cosmid, a phage, etc. The engineered host cells can be cultured in
conventional nutrient media modified as appropriate for activating promoters,
sel.octing transfo~ ntc or amplifying the genes. The culture conditions, such astemperature, pH and the like, are those previously used with the host cell selected for
e,~ ession, and will be apparent to the ordinarily skilled artisan.
Suitable expression 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 ~. coli. Iac or rrp, the phage lambda PL promoter and other promoters
known to control expression of genes in prokaryotic or eukaryotic cells or theirviruses. 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 recict:-nc e in E. coli.
The gene can be placed under the control of a promoter, ribosome binding site
(for bacterial expression) and, optionally, an operator (collectively referred to herein
as "control" elements), so that the DNA sequence encoding the desired protein istranscribed into RNA in the host cell transformed by a vector containing this
expression construction. The coding sequence may or may not contain a signal
peptide or leader sequence. The protein sequences of the present invention can be
-- 6 -
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W O 97/12963 PCT~EP96/04268
~p.l;ssed using, for example, the E. coli tac promoter br the protein A gene (spa)
promoter and signal sequence. Leader sequences can be removed by the bacterial
host in post-translational procec~ing Promoter regions can be selected from any
desired gene using CAT (chlor~mphPnirol transferase) vectors or other vectors with
5 sPhPct~ble markers. Two appropriate vectors are PKK232-8 and PCM7. Particular
named bacterial promoters include lacI, lacZ. T3, T.7, gpt, lambda PR, PL and trp.
Eukaryotic promoters include CMV imme~ te 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 addition to control sequences, it may be desirable to add regulatory
sequences which allow for regulation of the expression of the protein sequences
relative to the growth of the host cell. Regulatory sequences are known to those of
skill in the art, and examples include those which cause the ~p-ession of a gene to be
turned on or off in response to a chemical or physical stimulus, including the presence
15 of a regulatory compound. Other types of regulatory elements may also be present in
the vector, for example, enh~nrer sequences.
An expression vector is constructed so that the particular coding sequence is
~ located in the vector with the appropAate regulatory sequences, the positioning and
oAentation of the coding sequence with respect to the control sequences being such
20 that the coding sequence is transcAbed under the "control" of the control sequences
(i.e., RNA polymerase which binds to the DNA molecule at the control sequences
transcAbes the coding sequence). Modification of the coding sequences may be
desirable to achieve this end. For example, in some cases it may be necessary tomodify the sequence so that it may be attached to the control sequences with the25 appropAate orientation; i.e., to m~int~in the reading frame. The control sequences
and other regulatory sequences may be ligated to the coding sequence pAor to
insertion into a vector, such as the cloning vectors described above. Alternatively, the
coding sequence can be cloned directly into an expression vector which already
contains the control sequences and an appropAate restAction site. Modification of the
30 coding sequences may also be performed to alter codon usage to suit the chosen host
cell, for enh~nred expression.
Generally, recombinant expression vectors will include oAgins of replication
and selectable markers permitting transformation of the host cell, e.g., the ampicillin
resi~t~nre gene of ~. coli and S. cerevisiae TRPl gene, and a promoter deAved from a
35 highly-expressed gene to direct transcription of a downstream structural sequence.
The heterologous structural sequence is assembled in appropAate phase with
translation initiation and termination sequences, and preferably, a leader sequence
capable of directing secretion of tr~nsl~terl protein into the peAplasmic space or
extracellular medium. Optionally, the heterologous sequence can encode a fusion
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W O 97/12963 PCTrEP96/04268
protein including an N-terrninal idl~ntifirati~n peptide impar~ing desired character-
istics, e.g., stabilization or simplified purification of expressed recombinant product.
The vector CC!nt~ining 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.
Examples of recombinant DNA vectors for cloning and host cells which they v
can transform include the bacteriophage ~ (E. coli), pBR322 (E. coli), pACYC177
(E. coli), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria),
pLAFR1 (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria),
pHV14 (E. coli and Rncj~r~ su~tilis), pBD9 (Bactllus), pIJ61 (Streptomyces), pUC6
(Streptomyces), YIpS (Saccharomyces), a baculovirus insect cell system,, YCpl9
(Saccharomyces). See, generally, "DNA Cloning": Vols. I & II, Glover et al. ed.
IRL Press Oxford (1985) (1987) and; T. Maniatis et al. ("Molecular Cloning" ColdSpring Harbor Laboratory (1982).
In some cases, it may be desirable to add sequences which cause the secretion
of the polypeptide from the host organism, with subsequent cleavage of the secretory
signal.
Yeast expression vectors are also known in the art. See, e.g., U.S. Patent Nos.
4,446,235; 4,443,539; 4,430,428; see also European Patent Applications 103,409;
100,561; 96,491. pSV2neo (as described in J. Mol. Appl. Genet. 1:327-341) which
uses the SV40 late promoter to drive expression in m~mm~ n cells or pCDNAlneo,
a vector derived from pCDNAl(Mol. Cell Biol. 7:4125-29) which uses the CMV
promoter to drive expression. Both these latter two vectors can be employed for
transient or stable(using G418 resi~t~nce) expression in m~mm~ n cells. Insect cell
expression systems. e.g., Drosophila. are also useful, see for example, PCT
applications WO 90/06358 and WO 92/06212 as well as EP 290,261-Bl.
Polypeptides can be expressed in host 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 including the SV40 enhancer on
the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter
CA 02233300 1998-03-27
W O 97/12963 PCT~EP96/0426S
enhancer, the polyoma enh~nrer on the late side of the replication origin, and
adenovirus enh:~nrPrs
Suitable hosts include prokaryotes for example bacterial cells, such as E. coli,,, Streptomyces, Salmonella tvphimurium; and eukaryotes for example fungal cells, such
S as yeast, insect cells such as Drosophila and Spodoptera frugiperda, m~mm~ n cells
such as CHO, COS or Bowes melanoma, plant cells, etc.
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)).10Following transformation of a suitable host strain and growth of the host strain
to an appropriate cell density, the sPlPc~ed promoter is ind~lce~ by applopiiate 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
15 chemical means, and the res-llting c;ude 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 know to those skilled in the art.
Various m~mm~ n cell culture systems can also be employed to express
20 recombinant protein. Examples of m~mm~ n expression systems include the COS-7lines of monkey kidney fibroblasts, described by Gl~ 7.m~n, 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. ~mm~ n expression vectors will comprise
an origin of replication, a suitable promoter and enhancer, and also any necessary
25 ribosome binding sites, polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and S' flanking nontranscribed sequences.DNA sequences derived from the SV40 splice, and polyadenylation sites may be used
to provide the required nontranscribed genetic elements.
Depending on the expression system and host selected. the polypeptide of the
30 present invention may be produced by growing host cells transformed by an
expression vector described above under conditions whereby the polypeptide of
interest is expressed. The polypeptide is then isolated from the host cells and
purified. If the expression system secretes the polypeptide into growth media, the
polypeptide can be purified directly from the media. If the polypeptide is not
35 secreted, it is isolated from cell Iysates or recovered from the cell membrane fraction.
Where the polypeptide is localized to the cell surface, whole cells or isolated
membranes can be used as ~n assayable source of the desired gene product.
Polypeptide expressed in bacterial hosts such as E. coli may require isolation from
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W O 97/12963 PCT~EP96/04268
inrlllsion bodies and refolding. The selection of the appropriate growth conditions
and recovery methods are within the skill of the art.
The polypeptide can be recovered and purified from recombinant cell cultures
by methods inrlurling ammonium sulfate or ethanol precipitation, acid extraction,
5 anion or cation exch~nge chromatography, phosphocellulose chromatography,
hydrophobic interaction chromatography, affinity chromatography hydroxylapatite
chromatography and lectin chromatography. Protein refolding steps can be used, as
nPce~s~ry, in completing configuration of the mature protein. Finally, high
perfo~ nre liquid chromatography (HPLC) can be employed for final purifir~tion
10 steps.
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.
Cells may be enginçered in vivo for expression of a polypeptide in vivo by, for
exarnple, procedures known in the art. As known in the art. a producer cell for
producing a retroviral particle containing RNA encoding the polypeptide of the
present invention may be ~(lminictPred to a patient for enginrering cells in vivo and
expression of the polypeptide in vivo. These and other methods for ~ln~inictering 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. For example, theexpression vehicle for engineering cells may be other than a retrovirus, for example,
an adenovirus which may be used to enginrer cells in vivo after combination with a
suitable delivery vehicle.
"Recombinant" polypeptides refer to polypeptides produced by recombinant
DNA techniques; i.e., produced from cells transforrned by an exogenous DNA
construct encoding the desired polypeptide. "Synthetic" polypeptides are those
prepared by chemical synthesis.
A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that
functions as an autonomous unit of DNA replication in vivo; i.e., capable of
replication under its own control.
A "vector" is a replicon, such as a plasmid, phage, or cosmid, to which
another DNA segment may be ~tt~rh~d so as to bring about the replication of the
7~ttz~hed segment.
A "double-stranded DNA molecule" refers to the polymeric form of
deoxyribonucleotides (bases a-lenine, guanine, thymine, or cytosine) in a double-
stranded helix, both relaxed and supercoiled. This term refers only to the primary and
secondary structure of the molecule, and does not limit it to any particular tertiary
forms. Thus, this term includes double-sLranded DNA found, inter alia, in linear
- 10-
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W O 97/12963 PCTAE~G~ 58
DNA molecules (e.g., restriction fragmt-ntc), viruses, pl~cmi~1c, and chromosomes. In
~liccuccing the structure of particular double-ctr~n~led DNA molecules, sequences may
be described herein according to the normal convention of giving only the sequence
in the 5' to 3' direction along the sense strand of DNA.
A DNA "coding sequence of" or a "nucleotide sequence encoding" a particular
protein, is a DNA sequence which is transcribed and tr~ncl~t-o~l into a polypeptide
when placed under the control of appropriate regulatory sequences.
A "promoter sequence" is a DNA regulatory region capable of binding RNA
polymerase in a cell and initi~ting transcription of a downstream (3' direction) coding
sequence. Within the promoter sequence will be found a transcription initiation site
(conveniently defined by mapping with nu~!e~ce Sl), as well as protein binding
dom~inc (ConcenClls sequences) responsible for the binding of RNA polymerase.
Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT"
boxes.
DNA "control sequences" refers collectively to promoter sequences, ribosome
binding sites, polyadenylation signals, transcription termination sequences, upstream
regulatory domains, enh~n~ers, and the like, which collectively provide for the
expression (i.e., the transcription and translation) of a coding sequence in a host cell.
A control sequence "directs the expression" of a coding sequence in a cell
when RNA polymerase will bind the promoter sequence and transcribe the coding
sequence into mRNA, which is then translated into the polypeptide encoded by thecoding sequence.
A "host cell" is a cell which has been transformed or transfected, or is capableof transformation or transfection by an exogenous DNA sequence.
A cell has been "transformed" by exogenous DNA when such exogenous
DNA has been introduced inside the cell membrane. Exogenous DNA may or may
not be integrated (covalently linlced) into chromosomal DNA making up the genomeof the cell. In prokaryotes and yeasts, for example, the exogenous DNA may be
m~int~ined on an episomal element, such as a plasmid. With respect to eukaryoticcells, a stably transformed or transfected cell is one in which the exogenous DNA has
become integrated into the chromosome so that it is inherited by daughter cells
through chromosome replication. This stability is demonstrated by the ability of the
eukaryotic cell to establish cell lines or clones comprised of a population of ~l~ugh~er
cell containing the exogenous DNA.
A "clone" is a population of cells derived from a single cell or common
~ncestor by mitosic A "cell line" is a clone of a primary cell that is capable of stable
growth in virro for many generations.
Two DNA or polypeptide sequences are "substantially homologous" or
"substantially the same" when at least about 85~o (preferably at least about 90%, and
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most preferably at least about 95%) of the nucleotides or amino acids match over a
defined length of the molecule and includes allelic variations. As used herein,
subst~nti~lly homologous also refers to sequences showing identity to the specified
DNA or polypeptide sequence. DNA sequences that are substantially homologous
can be identified in a Southern hybridization experiment under, for example, stringent
conditions, as defined for that particular system. Defining appropriate hybricii7~ti~
conditions is within the skill of the art. See, e.g., "Current Protocols in Mol. Biol."
Vol. I & II, Wiley Interscience. Ausbel e~ al. (ed.) (1992). Protein sequences that are
substantially the same can be i~entified by proteolytic digestion, gel electrophoresis
and microsequencing.
The term "functionally equivalent" intends that the amino acid sequence of the
subject protein is one that will exhibit enzymatic activity of the same kind as that of
the lipase.
A "heterologous" region of a DNA construct is an i~i~ntifi~ble segment of
DNA within or attached to another DNA molecule that is not found in association
with the other molecule in nature.
The present invention will be further described with reference to the following
examples; however, it is to be llndprstood that the present invention is not limited to
such examples. All parts or amounts, unless otherwise specified, are by weight.
In order to facilitate lln-lerst~lnding of the following examples certain
frequently occurring methods and/or terms will be described.
"Plasmids" are ~IP~ign~t~d 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.
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or
two complementary polydeoxynucleotide strands which may be chemically
synth~i7p~l 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 (M~ni~ti~, 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 ~lg of approximately
equimolar amounts of the DNA fragments to be ligated.
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EXAMPLE
Gene Cloning
cDNA Li~rar~ construction
Poly A+ (mRNA) was isolated from human prostate (benign possible hyperplasia)
5 using standard methods (ref Maniatis et al). First strand cDNA was primed using an
oligo dT primer. The cDNA library was constructed with the Stratagene ZAP-cDNA
synthesis kit, packaged with Gigpack 11 gold p~k~ing extract and amplified in XLI-
blue MRF bacterial cells. The cDNA inserts were cloned unidirectionally into thevector.
DNA Sequencing
The phage clone cont~ining the EST was excised from the ~ Unizap XR bacteriophage
vector into the Bluescript phagemid (according to the Stratagene manual) for
characterisation. The insert of 1823bp was m~nu~lly sequenced on both strands (using
the Amersham -USB Sequenase 2.0 DNA sequencing kit) by primer walking (SEQ ID 2).
15 The cDNA has an open reading frarne with the potential to code for a polypeptide of 393
amino acids (SEQ ID 1). The predicted MW for the full reading frame is 44143Da.
Producffon of an NSDL* recombinant baculovirus and expression of the
functional protein.
The NSDL cDNA was isolated from its host plasmid pBluescript 11 SK+/- as an
approximate 1800bp EcoRl-Xhol fragment, the enzyme activity inactivated for 10
minutPs at 80~ C followed by blunt ending with the Klenow fragment of DNA
polymerase 1, in the presence of excess dNTPs. The blunt end fragment was ligated
25 into the Sma 1 site of the baculovirus expression vector, pBacPAK9 (Clontech) with
the resulting plasmid transformed into E.Coli (high efficiency JM109 -Promega) and
plated on LB/1.5% w/v agar plates cont~inin~, lOO,uglml ampicillin. Recombinant
clones were predicted by DNA restriction enzyme analysis and confirmed by DNA
sequencing using the USB Sequenase 2.0 DNA sequencing kit.
* NSDL (novel serine dependent lipase~ is name given to the novel polypeptide of SEQ ID NO 1.
Co-transfection of plasmid DNA into baculovirus DNA
DNA from selected clones was diluted to lOOng/~ll with water and co-transfected with
BakPAK6 DNA into BSU 36I digested baculovirus DNA (Clontech), using lipid
mediated transfer (Lipofectin-Life Technologies). The viral DNA was used to infect
1.5X106 Spodoptera frugiperda cells (sf-9; ATCC CRL 1711) seeded in 2ml of IPL-
41 medium plus lipid and yeast supplements (complete medium). 72 hours post-
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transfection, the virus particles released into the medium were harvested and used to
create a plaque assay (Summers, M.D. and Smith G.E.; Texas Agricultural
Experiment Station Bulletin No 1555, 1987). Ten plaques were chosen at random
and each used to infect 5xlO5 fresh sf-9 cells in 2ml complete me~ m Four days
S post-infection 1.8ml meflillm from each plaque was used to infect a further 5xlO5
fresh sf-9 cells. Four days post infection the sf-9 cells were lysed in 200~L1 50mM
HEPES buffer, lOmM CHAPS pH 7.4, plus 10 ~LgJml each of the protease inhibitors
pepstatin A, antipain. Leupeptin, aprotinin), the debris removed by centrifugation at
3000xg for 4 minutes and 1~L1 of the supçrn~t~nt assayed in a colourimetric assay.
10 Virus titre was improved by further rounds of infection in sf-9 cells using a moi of
0.01 and protein expression optimised at 2 days post-infection. Purification of NSDL
was carried out from a 30 litre culture of infected cells.
Purification of Recombinant NSDL
Sfg cells were lysed by freeze thawing 3 times in liquid nitrogen after the addition of
40ml/L of lysis buffer (SOmM Tris (pH 8.5), SmM Chaps, lmM EDTA, lmM DTT,
20% glycerol,50 ~lg/ml Ben7~mil1inl~, plus lOIlg/ml protease inhibitors. The sample
was centrifuged at 50,000xg for 20 minutPc to pellet the cell debris, following the
20 addition of DNase to reduce the viscosity.
The supernatant was loaded onto a Q sepharose column (Pharmacia) which had been
equilibrated with lysis buffer minus protease inhibitors (buffer A). The column was
washed with buffer A and the protein was eluted using a linear gradient of NaCI. (O-
lM) The enzyme eluted at 300mM NaCI.
25 Active fractions were concentrated using a YM30 membrane in an Amicon
ultr~filtration cell, followed by dialysis against 50mM Mes,( pH 6.0) ,SmM Chaps,
lmM DTT, lmM EDTA, 20% Glycerol (buffer B). The sample was loaded onto a
blue sepharose 6 fast flow column (Pharmacia), equilibrated with buffer B. The
column was washed with buffer B, followed by 50mM Mops, (pH 7.0), 5mM Chaps,
30 lmM DTT, lmM EDTA, 20% glycerol, 0.3M NaCl (buffer C) and enzyme activity
was eluted using 50mM Tris (pH 8.0), 5mM Chaps, lmM DTT, lmM EDTA, 20%
glycerol, lM NaCI (buffer D).
Active fractions were concentrated over a YM30 membrane and dialysed against
lOmM Tris (pH 7.4), 5mM Chaps, lmM DTT, 20% Glycerol (buffer E).
35 The concentrated protein was applied to a hydroxyapatite column (Biorad),
equilibrated with buffer E and the enzyme was eluted with a linear gradient frombuffer E alone, to buffer E containing 150mM KH2PO~. The activity eluted at around
60mM KH2PO~.
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The protein was concentrated over a YM30 membrane followed by a 10 fold dilutionwith buffer A and was fhen applied to a Resource Q column (Pharmacia) pre-
equilibrated with buffer A.The enzyme does not bind to fhe column and is collected
by washing with buffer A, cont~min~nt~ are eluted using buffer A plus lM NaCl.
s
Activity of Recomhin~nt NSDL
~,
Activity assays were carried out as described in Tew D G, Southan C, Rice S Q. J.,
Rice G., Lawrence M P, Li H, Gloger I S, Saul H F, Moores K & MacPhee C H.,
10 Purifi~tion, properties, sequencing and cloning of a lipoprotein associated, serine
dependent phospholipase which is involved in the oxidative modification of low
density lipoprofeins. Arteriosclerosis, 16, 591-599, 1996. The Km values for PAF as a
substrate with purified NSDL was determined to be 42 mM and the tum over rate for
PAF clef~-rminPd as 35 mmol/min/mg.
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Sequence Data:
SEQ ID NO 1
MGVNQSVGFPPVTGPHLVGCGDVMEGQNLQGSFFRLFYPCQKAEETMEQPLWIPRYEYCTGLAEYLQFN
KRCAGACCSTWRWDLVACLLAGMAPFKTKDSGYPLIIFSHGLGAFRTLYSAFCMELASRGFVVAVPEPQ
DRSAATTYFCKQAPEENQPTNESLQEEWIPFRR~ :K~:~RNPQVHQRVSECLRVLKILQEVTAGQ
TVFNIFPGGLDLMTLKGNIDMSRVAVMGHSFGGATAILALAKETQFRCAVALDAWMFPLERDFYPKARG
PVFFI~ K~'Q'l'MESVNLMKKICAQHEQSRIITVLGSVHRSQTDFAFVTGNLIGKFFSTETRGSLDPYE
GQEVMVRAMLAFLQKHLDLKEDYNQWNNLIEGIGPSLTPGAPHHLSSL
SEQIDNO2
GGCACGAGCT TCTGAGGAAT CAGCTTGACT GGCCAGCAAG TTCAGCTCCG
GCAAGTCATT TGATTCACCC GGTGATGAAA TGGGGGTCAA CCAGTCTGTG
GGCTTTCCAC CTGTCACAGG ACCCCACCTC GTAGGCTGTG GGGATGATGA
TGGAGGGGTC AGAATCTCCA GGGGAGCTTC TTTCGACTCT TCTACCCCTG
CCAAAAGGCA GAGGAGACCA TGGAGCAGCC CCTGTGGATT CCCCGCTATG
AGTACTGCAC TGGCCTGGCC GAGTACCTGC AGTTTAATAA GACGACTGCG
GGGGCTTGCT GTTCAACCTG GCGGTGGGAT CTTGTCGCCT GCCTGTTAGC
TGGAATGGCC CCCTTTAAGC ACMAAAGGAC TCTGGATAAC CCCATTGATC
20 A~l~ll~l~CC CATGGCCTAG GAGCCTTCAG GACTTTGTAT TCAGCCTTCT
GCATGGAGCT GGCCTACACG TG~l~l~l~lG GTTGCTGTGC CAGAGCCACA
GGACCGGTCA GCGGCAACCA CCTATTTCTG CAAGCAGGCC CCAGAAGAGA
ACCAGCCCAC CAATGAATCG CTGCAGGAGG AATGGATCCC ~l~llCC~lCGA
GTTGAGGAAG GGGAGAAGGA ATTTCATGTT CGGAATCCCC AGGTGCATCA
25 GCCGGGTAAG CGAGTGTTTA CGG~l~l~lGA AGATCCTGCA AGAGGTCACT
GCTGGGCAGA CTGTCTTCAA CATCTTTCCT GGTGGCTTGG ATCTGATGAC
TTTGAAGGGC AACATTGACA TGAGCCGTGT GGCTGTGATG GGACATTCAT
TTGGAGGGGC CACAGCTATT CTGGCTTTGG GCCAAGGAAG ACCCAATTTC
TCGTGTGCGG TGGCTCTGGA TGCTTGGATG TTTCCTCTGG AACGTGACTT
TTACCCCAAG GCCCGAGGAC CTGTGTTCTT TATCAATACT GAGAAATTCC
AGACAATGGA GAGTGTCAAT TTGATGAAGA AGATATGTGC CCAGCATGAA
CAGTCTAGGA TCATAACCGT TCTTGGTTCT GTTCATCGGA GTCAAACTGA
CTTTGCTTTT GTGACTGGCA ACTTGATTGG TAAATTCTTC TCCACTGAAA
CCCGTGGGAG CCTGGACCCC TATGAAGGGC AGGAGGTTAT GGTACGGGCC
ATGTTGGCCT TCCTGCAGAA GCACCTCGAC CTGAAAGAAG ACTATAATCA
ATGGAACAAC CTTATTGAAG GCATTGGACC GTCGCTCACC CCAGGGGCCC
CCCACCCATC TGTCCAGCCT GTAGGCGACA ACTGGCTCAT TTGTAAAGTC
ACTTCAGCCA AGCTTTTCAT TTGGGAGCTA CCCAAGGGCA CCCATGAGCT
CCTATCAAGA AGTGATCAAC GTGACCCCTT TTCACAGATT GAAAGGTGTA
ATCACACTGC TGCTTGGATA ACTGGGTACT TTGATCTTAG ATTTGATCTT
AAAATCACTT TGGGACTGGG ATCCCTTGCT GATTGACAAA CAGACTTTCT
GGGACCTTGA TGGAGTGGGG AACAAGCAGT AGAGTGGGAC TGGGGGAGAC
CCAGGCCCCG GGCTGAGCAC TGTGAGGCCT GGATGTGAAG ACTCAMCCCA
CGAACGCTCA TTCCCTTACC CCCGGCCAGT GCTGCTGCTT CAGTGGAAGA
GATGAAGCCA AAGGTAACAG AATGAAAAAT CCCTACCTTC AGAGACTCTA
GCCCAGCCCA ACACCATCTC TTCCTACCTC TCAGCCTTCT CCCTCCCCAG
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GGCCACTTGT TGAGAAGTCT GAGCACTTTA TGTAAATTTC TAGGTGTGAG
CCGTGAAAAA AAAAAAAAAA AAAA
M is defined as either A or C, where the actual base is unclear from either DNA
strand.
