Note: Descriptions are shown in the official language in which they were submitted.
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MATRIX BINDING FACTOR
The present invention relates to the design,
manufacture and use of novel polypeptide bioactive factors.
More particularly, the invention relates to the targeting
of polypeptide bioactive factors to biological or chemical
substrates via engineered amino acid motifs, while
maintaining or extending the biological activities
attributed to that polypeptide :bioactive factor.
l0
BACKGROUND OF THE INVENTION
Polypeptide bioactive factors, particularly
growth factors, are crucial for many cellular processes.
Consequently, much research is undertaken in relation to
the presence and mechanism of action of such factors in
cellular processes, with the objective of identifying novel
polypeptide bioactive factors, identifying growth factor
synergies, and understanding the mechanisms of processes
such as cellular maintenance, growth, development, and
apoptosis. The potential usefulness of polypeptide
bioactive factors, particularly growth factors, in
diagnostics, pharmaceuticals and therapeutics is well
recognised.
Considerable interest has been directed to a
family of polypeptide bioactive factors, the insulin-like
growth factors (IGFs). It is well established in the art
that these proteins are structurally related, and that
their expression patterns, localisation and bioactivity are
important for the differentiation and proliferation of
various cellular types. This has led to the suggestion
that IGFs are pivotal for cellular migration and
proliferation, for connective tissue production, and for
turnover processes associated with tissue remodelling,
repair and wound healing (Clemmons, D.R., British Medical
Bulletin, 1989 45 465-480; Gartner et a1, J. Surg. Res.,
1989 52 389-394).
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Despite the demonstrated effects of IGFs in
cultured vertebrate cells and tissues, as yet there have
been no observations in vivo which support any unequivocal
role in tissue remodelling or wound healing for
exogenously-administered IGFs, whether given systemically,
locally or topically. It is therefore accepted in the art
that there must be other moieties which influence the
bioavailability of IGFs.
The bioavailability of a polypeptide bioactive
l0 factor is a measure of that factor's ability to remain
active at a site where it can effect a desired cellular
response. Bioavailability is modulated by the stability,
protease susceptibility and rate of clearance of a factor
from the site where it interacts with its cellular
receptors.
The bioavailability of IGFs can be modulated by
one or more of the six insulin-like growth factor binding
proteins (IGFBPs) which are so far known. Furthermore, the
bioavailability of an IGF may also be modified by
structural changes to the amino acid sequence of the
polypeptide. As a hypothetical example, introducing an
affinity for one or more biological or chemical substrates
may serve to slow or prevent IGF clearance from the local
environment, or may even localise an IGF in a biologically
active and accessible form. Moreover, this general concept
may be extended to polypeptide bioactive factors other than
IGFs.
The nature of many biological substrates, such as
basement membrane, extracellular matrix (ECM), bone matrix
3o and other connective tissue components, is such that
localised and more general patterns of negative charge are
created. For example, both heparan sulphate proteoglycans
present in the ECM and hydroxyapatite in bone have net
negative charges. These charged moieties provide sites of
attachment for factors with the appropriate affinities, as
determined by their amino acid sequences. The potential of
factors able to elicit cell growth effects at the site of
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such localisation offers opportunities to create a
significant improvement in the delivery of therapeutic
agents to their preferred site of action. Similarly,
therapeutic agents that are retained at the preferred site
of action when administered locally may prove useful for
the treatment of many conditions, such as bone or ligament
grafts and tendonitis, or they may improve the post-
operative recovery associated with tissue manipulations,
cartilage repair and reconstructive surgery.
Gastrointestinal tract impairment associated with
either iatrogenic disorders such as chemotherapy-induced
epithelial damage or pathologies such as inflammatory bowel
disease are significant causes of morbidity. Accordingly,
therapeutic agents directed to the healing of the
epithelial lining of the gastrointestinal tract are useful
in the prevention and/or treatment of conditions associated
with impaired gut function.
The ability of negatively-charged substrates,
such as heparan sulphate proteoglycans, to protect bound
polypeptides from degradation due to physical stresses such
as temperature, chemical stresses such as low pH, and/or
biological stresses such as protease degradation, as
described in the prior art, provides potential mechanisms
for the prevention and treatment of pathological states
associated with impaired gut function.
Accordingly, there is a need in the medical and
veterinary fields for novel delivery and targeting
technologies which create local concentrations of bioactive
factors .
SUN~IARY OF THE INVENTION
In a first aspect, the present invention provides
a polypeptide bioactive factor, hereinafter referred to as
a matrix binding factor (MBF), comprising a polypeptide
bioactive factor in which the naturally-occurring amino
acid sequence of the factor has been modified to introduce
one or more amino acid substitutions, deletions and/or
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additions which increase the affinity of the polypeptide
bioactive factor for a negatively-charged site or surface.
The term "polypeptide bioactive factor" means a
polypeptide or small protein which either modulates
cellular responses directly, as is the case with growth
factors, or acts indirectly by its association with other
components, including the extracellular matrix, such that
cellular responses are potentiated. This term is also to be
understood to encompass mutants, fragments and analogues of
such a factor which retain at least one of the biological
activities of the factor.
The term "negatively-charged surface" means any
surface which displays an array of negative charge that
provides association sites for positively-charged
polypeptide motifs. Such negatively-charged surfaces may
be of natural or artificial origin, and may be in an animal
body. They include, but are not limited to, extracellular
matrix, dextran sulphate, chondroitin sulphate, dermatan
sulphate, collagen, fibronectin, vitronectin, laminin,
heparan sulphates, heparin, hydroxyapatite, anionic
plastics, silicates, and physiologically-compatible metals
and other materials used in surgical implants or
prostheses, such as stainless steel, titanium, metal
alloys, ceramics, polymers and plastic coated metals.
Metals and ceramics are widely used in
orthopaedic applications.
As an integral part of their biological activity,
a number of polypeptide bioactive factors have affinities
for sites other than their high affinity cellular
receptors. For example, they may bind to cell attachment
factors, basement membrane moieties, extracellular matrix
components, or soluble circulating proteins.
Accordingly, the amino acid modifications include
introduction of sequences which are deduced from motifs in
the amino acid sequences of polypeptide bioactive factors
which have been identified by their particular affinity for
specific substrates. Such motifs include, but are not
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limited to, the heparin-binding amino acid motif
characteristic of fibroblast growth factors (FGFs),
heparin-binding epidermal growth factor, vitronectin,
fibronectin, histidine-rich glycoprotein or purpurin.
Preferably the polypeptide bioactive factor into
which changes are introduced in order to create an MBF is a
polypeptide bioactive factor which does not already include
similar sequence motifs which confer an ability to bind to
negatively-charged surfaces.
More preferably the polypeptide bioactive factor
into which changes are introduced in order to create an MBF
is a growth factor which stimulates proliferation,
differentiation, migration or cellular activity, including,
but not limited to, members of the insulin-like growth
factor (IGF), transforming growth factor-~3, platelet-
derived growth factor, vascular endothelial growth factor
or epidermal growth factor families of growth factors.
The present invention also includes within its
scope biologically-active mutants, analogues and
derivatives of the MBF. Preferably such modified MBFs are
in a biologically pure form.
The term "biologicall.y pure" as used herein means
a product essentially devoid of unavoidable biologically
active impurities or contaminants.
According to a second aspect, the invention
provides a nucleic acid molecule whose sequence encodes a
polypeptide bioactive factor of the invention. The nucleic
acid molecule may be a cDNA, a genomic DNA, or an RNA, and
may be in the sense or anti-sense orientation. Preferably
the nucleic acid molecule is a cDNA, more preferably a
sense cDNA.
In a third aspect, the invention provides a
method for producing a recombinant MBF, comprising the
steps of subjecting a cloning vector comprising a nucleic
acid sequence encoding the polypeptide growth factor to
mutagenesis to generate a nucleotide sequence encoding an
MBF.
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The nucleic acid sequence of the polypeptide
bioactive factor must be subcloned into an appropriate
cloning vector or plasmid. This permits mutagenesis of the
DNA encoding a polypeptide growth factor to generate a
sequence encoding a MBF, by means well known to those in
the art.
Preferably the mutagenesis is achieved using
site-directed mutagenesis, more preferably using
oligonucleotide primers which are based on binding sites
able to bind to negatively-charged surfaces, which binding
sites are present in other polypeptide bioactive factors,
or sequences substantially homologous thereto. Examples of
polypeptide bioactive factors comprising such binding sites
include vitronectin, 10 kilodalton gamma-interferon
inducible protein, histidine-rich glycoprotein, purpurin,
beta-thromboglobulin, antithrombin III, heparin
cofactor II, FGF-1, FGF-2, heparin-binding epidermal growth
factor, lipocortin, protein C inhibitor, fibronectin,
thrombospondin, lipoprotein lipase, hepatic triglyceride
lipase, vascular endothelial cell growth factor, thrombin,
neural cell adhesion molecule and glial-derived nexin.
The altered nucleic acid sequence encoding an MBF
may be subcloned into a suitable expression vector, which
may be introduced into host cells by conventional means
familiar to those skilled in the art.
Thus the method of the invention preferably also
comprises the steps of
subcloning the nucleic acid sequence encoding the
MBF into a suitable expression vector;
transforming the expression vector into a
suitable bacterial, yeast or tissue culture host cell;
cultivating the host cell under conditions
suitable to express the MBF; and
isolating the MBF.
It will be appreciated that host cells comprising
selected constructs so formed may express the MBF as a
fusion protein within inclusion bodies (IB). By the term
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"MBF fusion protein" we mean a polypeptide consisting of
two linked protein components, one of which is selected so
as to be expressed in the host cell under the control of a
suitable promoter, and the other of which comprises the
polypeptide bioactive factor incorporating the motif that
confers MBF activity. The fusion protein is produced in
order to facilitate the expression and/or processing of the
amino acid sequence of the MBF activity. Preferably the
MBF fusion protein is produced by an appropriate host cell
in a fermenter by conventional means understood by those
skilled in the art.
Preferably, the MBF is isolated from the host
cell following disruption of the host cell by
homogenisation, and processed to its biologically pure form
using conventional methods of protein purification well
recognised by those skilled in the art. These include
oxidative refolding to achieve correct disulphide bonding,
chemical cleavage of the fusion partner (if used) from the
MBF, and various chromatographic steps. The MBF may be
isolated as a biologically pure form of the fusion protein,
and may then be cleaved from its fusion partner, yielding a
peptide that is not extended.
In a preferred embodiment an MBF is prepared as a
fusion protein using the methods described in our
Australian Patent No. 633099, the entire disclosure of
which is incorporated herein by reference. In this method a
fragment of porcine growth hormone is linked to the
N-terminal sequence of an MBF, optionally via a cleavable
sequence.
In another preferred embodiment of the invention
there is provided a process for the production of a
cleavable MBF fusion protein, comprising the step of
transforming a susceptible bacterial, yeast or tissue
culture cell hosts with one or more recombinant DNA
plasmids which include DNA sequences capable of
facilitating the expression of an MBF fusion protein.
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In a further preferred embodiment, the MBF fusion
protein is expressed as an insoluble aggregate or inclusion
body within the host cell, and isolated by cell disruption
and centrifugation. The conventional methodologies which
may be employed in the isolation of the MBF include fusion
protein dissolution, oxidative refolding, hydroxylamine or
proteolytic cleavage, and various chromatographic
processes, including size exclusion chromatography, ion
exchange chromatography, reversed-phase high performance
liquid chromatography and affinity chromatography. Sample
fractions may be collected from each purification step,
with those exhibiting biological activity in an appropriate
assay being pooled and carried forward to the next
purification step. This may result in the isolation of
biologically pure MBF with or without its fusion partner.
Preferably, the presence of biologically pure MBF
is detected by one or more of
a) migration as a single band of the appropriate
size on SDS-PAGE gel chromatography;
b) N-terminal sequence analysis, or
c) mass spectroscopy.
The biologically pure MBF of the present
invention is useful for a wide variety of purposes,
including but not limited to
maintenance, growth or differentiation of animal
or human cells in culture;
maintenance, growth or differentiation of more
organised cellular structures, for example, skin,
cartilage, tendon, ligament or bone;
coating a negatively-charged surface to promote
cell adhesion, growth, migration, or activity, for example
culture vessels for use in keratinocyte expansion to
provide partial thickness skin grafts for burns patients,
or coating of a surgical implant or a prosthesis;
enhancement of tissue remodelling and repair
associated with trauma or manipulation, for example in the
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treatment of wounds of all types, including burns,
traumatic injuries, or surgical wounds;
as an orally active product for the prevention
and/or treatment of impaired gut function;
facilitating tissue targeting following systemic
administration, for example by localisation of the factor
to bone matrix following intravenous injection; and
maintaining higher bioactive factor
concentrations at the site of administration in order to
effect a prolonged pharmacological action.
The MBF may be used in conjunction with or in
combination with one or more other growth factors or
therapeutic agents.
Accordingly, in a fourth aspect the present
invention provides a composition for promoting adhesion,
growth, migration, the prevention of apoptosis, or activity
of cells in culture, comprising an effective amount of an
MBF together with a pharmaceutically- or veterinarily-
acceptable carrier.
In a preferred embodiment, this aspect of the
invention provides a composition for the coating of a
negatively-charged surface with an effective amount of MBF,
thereby to promote adhesion, growth, migration or activity
of cells.
In a fifth aspect the invention provides a method
for promoting adhesion, growth, migration or activity of
cells on a negatively-charged surface coated with an MBF,
comprising the step of growing vertebrate cells in a
culture medium over or on an appropriate surface pretreated
with an MBF.
In both the fourth and fifth aspect of the
invention the cells may be of vertebrate, preferably
mammalian, or of insect origin.
In a sixth aspect, the invention provides a
composition for the enhancement of tissue remodelling or
tissue repair associated with tissue trauma or wound
healing, comprising an effective amount of an MBF
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formulated with a carrier such as an injectible, excipient,
carrier, lotion, medicated body wash, dressing, liniment,
toothpaste, mouthwash or powder.
In an alternative embodiment, this aspect of the
invention provides a composition for alleviation of skin
damage associated with ageing or with exposure to
ultraviolet or ionizing radiation, comprising an effective
amount of an MBF together with a cosmetically-acceptable
carrier.
In a seventh aspect, the invention provides a
composition for the prevention or treatment of a condition
associated with impaired gut function, comprising an
effective amount of an MBF formulated with a carrier
suitable to produce an orally stable, bioactive enteral
formulation.
In an eighth aspect, the invention provides a
composition for the targeting or localisation of an MBF to
cells or tissues, thereby to promote cell adhesion, growth,
migration or activity in vivo, comprising an effective
amount of MBF formulated in a pharmaceutically-acceptable
carrier.
In a ninth aspect, the invention provides a
method for the enhancement of tissue remodelling or tissue
repair associated with tissue trauma or wound healing,
comprising the step of administering an effective amount of
an MBF to a subject in need of such treatment.
In a preferred embodiment, this aspect of the
invention provides a method for the prevention or treatment
of impaired gut function, comprising the step of
administering an effective amount of an MBF to a subject in
need of such treatment.
In a second preferred embodiment there is
provided a method for the targeting and localisation of an
MBF to cells or tissues, thereby to promote cell adhesion,
growth, migration or activity in vivo, comprising the step
of systemic or local administration of an MBF to a subject
in need of such treatment:
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It will be appreciated that in the therapeutic
methods of the invention, the subject to be treated may be
a human, or may be a domestic, companion or zoo animal.
The carrier to be used and the dose and route of
administration will depend on the nature of the condition
to be treated and the age and general health of the
subject, and will be at the discretion of the attending
physician or veterinarian. Suitable carriers and
formulations are known in the art, for example by reference
to Remington's Pharmaceutical Sciences, 19th Edition, Mack
Publishing Company, Easton, Pennsylvania (1995). Suitable
dosing regimens are established using methods standard in
the art.
For the purposes of this specification it will be
clearly understood that the word "comprising" means
"including but not limited to", and that the word
"comprises" has a corresponding meaning.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the results of SDS/PAGE analysis
of the biologically pure MBF, as visualised by Novex
Tricine gel chromatography.
Figure 2a shows dose-response curves of the
extended forms of MBFs, L-MBF-1, L-MBF-2, L-MBF-3, L-MBF-4
and the extended form of IGF-I (L-IGF-I) for the
competitive displacement of iodinated IGF-I from the IGF-I
receptor isolated from human placental membranes.
Figure 2b shows dose-response curves of the
cleaved forms of MBFs, MBF-1, MBF-2, MBF-3, MBF-4 and IGF-I
for the competitive displacement of iodinated IGF-I from
the IGF-I receptor isolated from human placental membranes.
Figure 3a shows dose-response curves of four
extended forms of MBFs, L-MBF-1, L-MBF-2, L-MBF-3, L-MBF-4,
and L-IGF-I in a protein synthesis assay using a myoblast
cell line.
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Figure 3b shows dose-response curves of four
cleaved forms of MBFs, MBF-1, MBF-2, MBF-3, MBF-4 and IGF-I
in a protein synthesis assay using a myoblast cell line.
DETAILED DESCRIPTION OF THE INVENTION
The present invention will now be more fully
described with reference to the accompanying non-limiting
examples. It should be understood that the following
description is illustrative only, and should not be taken
in any way as a restriction on the generality of the
invention. In particular, while the invention is
specifically exemplified with reference to MBFs derived
from IGF-I, it will be clearly understood that the methods
described herein are applicable generally to the
modification of polypeptide bioactive factors to generate
MBFs, and to evaluation of the MBFs thus produced for
biological activity. In particular, once the MBF is
produced its testing for suitability for the purposes of
the invention is a matter of routine.
Representative MBFs according to the invention
were produced by introduction of heparin-binding motifs of
other polypeptide bioactive factors into the sequence of
human IGF-1. The methods used are described in detail
below. It will be clearly understood that while the
examples utilize the heparin-binding motif from bovine
FGF-1, FGF-1 sequences from the human protein or from other
species may also be used.
Sequence MBF-1 involves the deletion of the IGF-1
D-domain post Pro63 and its substitution with the heparin-
binding motif Lys127 to G1n142 from bovine FGF-1,
represented by the single letter code for amino acids as
shown below. The introduced heparin-binding motif is shown
in bold.
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEM
YCAPKKNGRSKLGPRTHFGQ
(SEQ ID NO. 1)
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Sequence MBF-2 contains all of the amino acids of
native IGF-1, and Lys127 to G1n142 of FGF-1, through the
insertion of the FGF-1 fragment Lys128 to G1y135 in between
the residues Lys65 and Pro66 of IGF-1. This is followed by
a second insertion of the FGF-1 segment Arg137 to G1n142 in
between residues Pro66 and A1a67 of IGF-1. Overall, this
results in the insertion of the entire FGF-1 fragment
Lys128 to G1y142 which includes by Pro66 of IGF-land is
represented below by the single letter code for amino
acids.
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEM
YCAPLKKNGRSKLGPRTHFGQAKSA
(SEQ ID NO. 2)
Sequence MBF-3 was constructed using a helical
wheel optimised sequence element of FGF-1 (Lys127 to
G1n142) that maximised the polarity of a theoretical
helical model of the proposed IGF-1 variant. The
optimisation analysis resulted in one glycine spacer amino
acid being inserted in front of the FGF-1 sequence element
and the substitution of glutamine for Lys133 and lysine for
Leu134. The IGF-1 D-domain post Pro63 was deleted and this
new FGF-1 segment was added and is represented below by the
single letter code for amino acids.
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEM
YCAPGKKNGRSQKGPRTHFGQ
(SEQ ID N0. 4)
Sequence MBF-4 most closely represents the native
structure of the parent IGF-1 peptide. This variant has
been designed utilising substitutions of amino acids and
creates two recognised heparin-binding sequences; the
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octapeptide XBBBXXBX (Asp-Lys-Arg-Gln-Leu-Glu-Lys-Tyr) and
the hexapeptide XBBXB (Gly-Lys-Arg-Gly-Arg-Ser). These two
structures are positioned either side of Cys61 and
collectively constitute 7 changes in the A- and D-domains.
This sequence represents an attempt to mimic heparin-
binding structures seen in insulin-like growth factor
binding proteins (IGFBPs) and heparin-binding EGF where the
sequences surround a cysteine residue. The changes
described are represented below by the single letter code
for amino acids.
GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDKRQLEK
YCAPGKRGRSA
(SEQ ID NO. 5)
IS
In every case, MBF cDNA constructs encode a
fusion protein that results in a polypeptide containing the
first 11 amino acids of porcine growth hormone, a linker of
valine and asparagine (MFPAMPLSSLFVN) and the mutagenised
hIGF-I sequence (MBF) and results in the expression of an
extended form of MBF or Long MBF (L-MBF). These components
permit the restriction digestion of the hIGF-I mutagenised
sequence, the bacterial expression of the fusion protein
and the hydroxylamine chemical cleavage of the leader
sequence from the MBF sequence.
Example 1 Mutagenesis of a Nucleotide Sequence Encoding
a Polypeptide Bioactive Factor (IGF-I) to
Generate a Nucleotide Sequence Encoding an
MBF
In order to perform site-directed mutagenesis on
a nucleotide sequence encoding a polypeptide bioactive
factor, a suitable plasmid cloning vector PTZ18 was
obtained. The cDNA nucleotide sequence pMpGH(11)VN/IGF-I
according to Example 5 of Australian Patent No. 633099,
encoding pGH(1-11) joined via a potential hydroxylamine-
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cleavable linkage N-terminal to IGF-I, was optimised for
codon usage in bacteria, and subcloned into the PTZ18
plasmid cloning vector EcoR1/HindIII restriction site in
5'-3' orientation. The construct is hereinafter referred
S to as PTZ18/pGH(11)/hIGF-I. The optimisation involved
generation, extraction and precipitation of PTZ18 plasmid
cloning vector DNA and pMpGH(11)VN/IGF-I DNA, restriction
enzyme digestions and legations using conventional methods,
for example as described in Molecular Cloning: A Laboratory
Manual, Eds Sambrook, Fritsch and Maniatis (second
edition), 1989; Pages 1.23-1.24, 1.62-1.68 respectively.
The correct nucleotide sequence was confirmed
using the dideoxy-mediated chain termination (Sanger)
method as described in Molecular Cloning, Pages 13.3-13.6.
1S Transformation of 200 x..1.1 of competent MV1190
bacterial cell suspension with 5 ~tl of PTZ18/pGH(11)/hIGF-I
legation reaction for the generation of quantities of
PTZ18/pGH(11)/hIGF-I double stranded (ds) DNA to be used
for restriction digests was carried out as described in
Molecular Cloning, Pages 1.74-1.84. The remaining 5ul of
legation reaction was used to transform 200 ~1 of CJ236
bacterial cell suspension for the production of single
stranded (ss) uracil containing DNA to be used for the
production of replicative mutagenised ds DNA. Replicative
2S ds mutagenised DNA was generated using site-directed
mutagenesis. In each case two oligonucleotide primers were
employed to insert the changes necessary to create a
nucleotide sequence encoding an MBF. The complete
mutagenesis in each case employed the prime (')
oligonucleotide for the first reaction, while the second
reaction employed the double prime(") oligonucleotide and
used the methods described in Molecular Cloning,
Pages 15.74-15.79 and 15.63-15.65. For example, to complete
the mutagenesis of MBF-2, IGFS-2' was employed to achieve
3S the first round of mutagenic changes and IGFS-2 " was
employed to achieve the second round of mutagenic changes.
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Oligonucleotide IGFS-2' (54 mer)
5'-TGCGCTCCGCTGF~i~AAAAAACGGTCGTTCTAAACTGGGCCCGGCTAAATCTGCT-3'
(SEQ ID NO. 6)
Oligonucleotide primer IGFS-2 " (48 mer)
5'-TCTAAACTGGGTCCGCGTACCCACTTCGGCCAGGCTAAATCTGCTTGA-3'
(SEQ ID N0. 7)
Transformants carrying the correct ds MBF-2 DNA,
hereinafter referred to as PTZ18/pGH(11)/MBF-2, were
determined by the dideoxy-mediated chain termination
(Sanger) method, and used to generate quantities of
PTZ18/pGH(11)/MBF-2 DNA.
A number of other PTZ18/pGH(11)/MBF analogue
constructs were generated using different oligonucleotide
primers and identical molecular biology techniques (see
examples).
Oligonucleotide primers IGFS-1' and IGFS-1" encoding MBF-1:
Oligonucleotide primer IGFS-1' (54 mer)
5'-ATGTACTGCGCTCCGF~~AAAAAACGGTCGTTCTAAACTGCTGAAACCGGCTAAA-3'
(SEQ ID NO. 8)
Oligonucleotide primer IGFS-1 " (54 mer)
5'-GGTCGTTCTAAACTGGGCCCGCGTACCCACTTCGGTCAGTGATGATGC AAGCTT-3'
(SEQ ID NO. 9)
Oligonucleotide primers IGFS-3' and IGFS-3 " encoding MBF-3:
Oligonucleotide primer IGFS-3' (57 mer)
5'-ATGTACTGCGCTCCGGGTP~~AAAAAACGGCCGTTCTCAGAAACTGAAACCGGCTAAA-3'
(SEQ ID N0. 10)
Oligonucleotide primer IGFS-3 " (54 mer)
5'-GGTCGTTCTCAGAAAGGCCCGCGTACCCACTTCGGTCAGTGATGATGCAAGCTT-3'
(SEQ ID N0. 11)
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Oligonucleotide primers IGFS-4' and IGFS-4 " encoding MBF-4:
Oligonucleotide primer IGFS-4' (48 mer)
5'-TTCCGTTCTTGCGACAAACGTCAGCTGGAAAAATACTGCGCTCCGCTG-3'
(SEQ ID NO. 12)
Oligonucleotide primer IGFS-4 " (45 mer)
5'-AAATACTGCGCTCCGGGTAAACGTGGCCGTTCTGCTTGATGATGC-3'
1~> ( SEQ ID NO . 13 )
Example 2 Subcloning the Nucleotide Sequence Encoding
an MBF from PTZ18/pGH(11)/MBF into Expression
Vector pGHXSC.4
pGHXSC.4 already contains within its DNA the
nucleotide sequence encoding pGH(11). The nucleotide
sequence encoding only the MBF was excised from
PTZ18/pGH(11)/MBF DNA using restriction enzymes
HpaI/HindIII. This MBF nucleotide sequence was subcloned
into expression vector pGHXSC.4, using the techniques
described in Example 1. The thus-formed expression vector
construct is hereinafter referred to as pGHXSC.4/MBF.
ds DNA sequencing as described in Example 1 was
again employed to confirm the expected nucleotide sequence
of the MBF fusion protein in the expression vector.
Example 3 Transformation of JM101 Bacterial Cells with
the Expression Vector pGHXSC.4/MBF Containing
the Nucleotide Sequence Encoding an MBF
To facilitate the expression of the MBF fusion
protein, pGHXSC.4/MBF (Example 2) was transformed by
methods outlined in Example 2 into a suitable host cell, in
this case IacIq JM101 cells.
Transformants containing pGHXSC.4/MBF
successfully grew on agarose plates containing ampicillin.
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Example 4 Induction of ,IM101 Bacterial Cells
Transformed with pGHXSC.4/MBF to Determine
Cell Clones Expressing MBF Fusion Proteins as
IBs
To confirm that the cells were expressing the
fusion protein as inclusion bodies (IBs}, inductions of
single clones were undertaken. Single pGHXSC.4/MBF
transformed colonies (Example 3) were inoculated into Luria
Bertani (LB) medium and cultured overnight. An aliquot
from each of these cultures was transferred to a fresh
sample of LB medium and incubated at 37°C until an
absorbance at 600 nm (A6o~) of 0.8-2.0 was reached. At this
time an aliquot was taken from each culture and reserved.
To the remainder of the culture was added isopropyl
~3-D-thiogalactopyranoside (IPTG) to a final concentration
of 0.2 mM, to induce the cells into the production of the
MBF fusion protein encoded within pGHXSC.4/MBF. Following
further incubation, both the reserved culture and the
induced culture were centrifuged to pellet the cell, and
following removal of the supernatant the cells were treated
with 2% ~3-mercaptoethanol/10o SDS to lyse the cells and
denature the protein.
Pre-induced cultures were compared directly with
post-induced cultures, using SDS/PAGE gel chromatography,
8-25o gradient Phast gel (Pharmacia) to determine MBF
fusion protein expressing clones and to confirm the
estimated size of the MBF.
Example 5 Production of MBF Fusion Protein Using Cells
Expressing MBF Fusion Protein as Inclusion
Bodies (IBs)
Clones identified as expressing the fusion
protein in Example 4 were used in a scale-up process to
produce appropriate quantities of the fusion protein for in
vi tro and in vivo experiments .
Four 2 litre Applicon fermenters were employed,
each containing 1L of minimal medium and inoculated with an
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aliquot of a culture, established with clones expressing
MBF fusion proteins, growing in log phase. Inoculated
fermentation cultures were incubated at 37°C overnight. At
an absorbance at 600 nm (A6oo) of between 4-6, IPTG was
added to the cultures to a final concentration of 0.2 mM
and the cells further incubated until an A6oo of
approximately 15-20 was reached, after which the
fermentation suspension was subjected to a number of passes
through a homogeniser. This process disrupted the cells,
facilitating the further isolation and purification of IBs
by three centrifugation and washing steps, using 30 mM
NaCl/10 mM KHZP04 washing buffer.
For pGHXSC.4/MBF-2 this process yielded a wet IB
pellet of 8.2 grams, which was stored at -20°C.
Example 6 Dissolution, Refolding, and Cleavage of MBF
Fusion Protein Produced in Inclusion Bodies,
Purification of Biologically Pure MBF
The wet IB pellet containing the MBF-2 fusion
protein from Example 5 was solubilised, desalted and
refolded by conventional methods. The MBF fusion protein
was then isolated and cleaved, followed by chromatographic
steps to yield a biologically pure MBF, employing known
methods. These processes included, in sequence:
1) dissolution of IBs in buffer (8 M urea,
0.1 M Tris, 40 mM glycine, 0.5 mM ZnCl2 and 40 mM
dithiolthreitol (DTT) pH 9.1), centrifugation and
filtration (1 ~.m gradient Whatman filter) to remove
particulate contaminants and desalting into 8 M urea,
0.1 M Tris, 40 mM glycine, 0.5 mM ZnCl2 and
1.6 mM dithiothreitol pH 9.1 by size exclusion
chromatography on Cellufine GCL-1000m;
2) a 330 ml pool of buffer containing 104.1 mg
of fusion protein was reconstituted to 1.320 L in
2.5 M urea, 40 mM glycine, 0.1 M Tris, 0.4 mM DTT and
10 mM ethylenediaminetetra-acetic acid (EDTA) pH 9.0;
refolded over 120 minutes with the addition of 0.12m1.L-1
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of oxidised (3-mercaptoethanol and re-acidified to pH 2.5
with concentrated HC1;
3) cation exchange on a SP Sepharose Fast Flow
(FFS) matrix, eluting the protein with 8 M urea, 50 mM
Ammonium acetate and 1M NaCl pH 4.8;
4) a 160 ml (FFS) pool of protein containing
97.3 mg was divided with 20% reserved, after which 80% was
reconstituted to 2 M urea, 0.1 M Tris, 1 M NH?OH and 1 mM
EDTA pH 8.65 followed by cleavage (see below) for 24 hrs at
to 40°C;
5) buffer exchange of both the reserved
material and cleaved material was achieved by C18 matrix
fast performance liquid chromatography (FPLC) using an
XK50/20 column (Pharmacia), washing with
0.1% trifluroacetic acid (TFA) and eluting with
80% acetonitrile/0.08% TFA, followed by
6) final desalting and purification by high
performance liquid chromatography (HPLC) on a C4 matrix
PrepPak column (Waters) washing with 0.1% TFA and eluting
with an 80% acetonitrile/0.08% TFA gradient at 0.1% per
minute.
Cleavage of the MBF fusion protein yields a
fusion partner and MBF, and may be employed to further
potentiate the bioactivity of the MBF if necessary. Thus
two MBFs may be derived from the one MBF fusion protein, an
extended form of MBF having the first 11 amino acids from
porcine growth hormone (pGH(1-11)) N-terminally linked to
the MBF amino acid sequence (L-MBF), and a cleaved form not
having pGH(1-11).
Dissolution, refolding and cleavage (if used) of
MBF fusion protein derived from the pGHXSC.4/MBF expression
vector construct in the foregoing manner yielded material
that ran as a single band following SDS/PAGE Novex Tricine
gel chromatography, as shown in Figure 1. This material
was represented as the major species following electrospray
mass analysis, and was observed at the calculated
theoretical mass.
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Example 7 The Affinity of Biologically Pure MBF for
Heparin as Measured by Heparin Affinity
Chromatography
10 ~,g aliquots of biologically pure MBF-2 and
L-MBF-2 from Example 6 were reconstituted in 10 ~,l of
mM HCl, taken up into a final volume of 100 ~l and
loaded in 10 mM Tris pH 7.0 on to a Pharmacia heparin-
Sepharose CL6B affinity column connected to a FPLC and
10 eluted with a linear gradient (0 M NaCl-1M NaCl) of
10 mM Tris/1 M NaCl pH 7.0 over 50 minutes. A
10 ~g aliquot of authentic IGF-I and pGH(1-11) IGF-I
(L-IGF-I) was also loaded and eluted using the same
conditions.
The affinity of cleaved MBFs was such that they
required salt concentrations of between 0.26 M and 0.33 M
to elute them from the heparin matrix. L-MBFs required
salt concentrations of between 0.24 M and 0.32 M, whereas
IGF-I and L-IGF-I eluted at salt concentrations of 0.12 M
and 0.11 M respectively.
Similarly, 10 ~g aliquots of MBFs, L-MBFs, IGF-I
and L-IGF-I were prepared as described and loaded in
10 mM Tris pH 7.0 on to a Progel TSK Heparin affinity
column connected to a HPLC. Proteins were again eluted
with the previously described salt gradient.
MBFs required salt concentrations of between
0.53 M and 0.59 M, while L-MBFs required salt concentration
of between 0.56 M and 0.89 M to elute them from this
column. IGF-I and L-IGF-I eluted at salt concentrations of
0.28 M and 0.29 M respectively.
This example shows that MBFs do indeed bind more
avidly to negatively charged materials as exemplified by
two types of heparin-affinity columns.
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Example 8 In vitro Binding Affinity of Biologically
Pure MBF for the IGF-I Receptor Isolated from
Human Placental Membranes
The biologically pure MBFs derived in Example 6
had affinities for the IGF-I receptor which ranged from
equipotent to three fold lower than authentic IGF-I or
uncleaved long (L-IGF-I}. IGF type I receptors isolated
from human placental membranes (Cuatrecasas, P., J. Biol.
Chem., 1972 247 1980-1991) were incubated with iodinated
hIGF-I or L-hIGF-I in the presence of increasing
concentrations of MBFs or L-MBFs (0.01 pmol to 100 pmol).
The affinity of the MBFs or L-MBFs was measured by the
competitive displacement of iodinated hIGF-I or L-hIGF-I
from the receptors and the results expressed as percentages
IS of iodinated IGF-I remaining bound to the IGF-I receptor.
The results are shown in Figures 2a and 2b.
Example 9 In vitro Stimulation of Protein Synthesis by
Biologically Pure MBF in a Myoblast Cell Line
The biologically pure MBFs from Example 6
stimulated the production of proteins by rat L6 myoblasts
in serum-free medium (Francis et a1, Biochem. J., 1985 233
207). The ability of increasing concentrations of MBFs,
ranging from 1 ng/ml to 1 Eig/ml, to stimulate protein
synthesis in rat L6 myoblasts was measured, and compared
with the ability of both commercially-derived IGF-I and
L-IGF-I (GroPep) to stimulate protein synthesis in rat L5
myoblasts. Results are expressed as the percentage
stimulation of protein synthesis above that observed in
growth factor-free or serum free medium, and shown in
Figures 3a and 3b.
Example 10 Characteristics of Binding of Iodinated
Biologically Pure MBF to Negatively-Charged
~mrfa~a~
The biologically pure MBFs from Example 6 were
iodinated by conventional methods, and shown to have the
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ability to bind to two negatively-charged surfaces.
Iodinated L-MBFs and reference peptides (10,000 counts per
minute cpm/well), when incubated overnight at 4°C in
24-well polyanionic tissue culture plastic plates in the
presence of 1 ml of l.Oo bovine serum albumin (BSA)
dissolved in phosphate-buffered saline (PBS) and then
washed twice using 1 ml of l.Oo BSA/PBS, demonstrated an
increase of approximately 6 to 15-fold in their ability to
remain bound to the substrate, compared to that of
t0 iodinated L-IGF-I (10,000 cpm/well) incubated under the
same conditions.
The results, shown in Table 1, are expressed as
the number of counts per minute (cpm) retained by the
iodinated polypeptide bioactive factor following the
washing steps.
Table 1
Radioactive counts per minute {cpm)
retained on tissue culture plastic
Iodinated polypeptide Counts per minute retained
bioactive factor (means sem)
FGF-2 1911.6 16.7
IGF-II 967.6 29.3
L-IGF-I 291.3 34.6
L-MBF-1 4758.3 107.4
L-MBF-2 4523.1 89.6
L-MBF-3 4976.3 122.5
L-MBF-4 1970.2 77.4
Similarly, biologically pure, iodinated L-MBFs
(10,000 cpm/well) when incubated overnight at 4°C in
l.Oo BSA/PBS on HaCat epithelial cell-derived matrix in
24-well plates (Jones et a1, J. Cell Biol., 1993 121 679)
and then washed twice with l.Oo BSA/PBS exhibited increases
of approximately up to 5-fold in their ability to remain
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bound to the matrix when compared with iodinated L-IGF-I
(10,000 cpm/ml), as shown in Table 2.
Table 2
Radioactive counts per minute (cpm) retained on
HaCaT cell derived matrix
Iodinated polypeptide Counts per minute retained
bioactive factor
(means sem)
FGF-2 1697.4 73.6
IGF-II 2168.6 201.3
L-IGF-I gp_g 4,7
L-MBF-1 370.6 29.7
L-MBF-2 406.2 16.8
L-MBF-3 398.7 14.1
L-MBF-4 78.8 7.9
Example 11 In vitro Stimulation of Protein Synthesis in
an Epithelial Cell Line by Biologically Pure
MBF Bound to a Negatively-Charged Surface
The four extended forms of biologically pure MBFs
from Example 6, which were shown to stimulate protein
synthesis in a myoblast cell line (Example 9) and to have
the ability to bind to negatively-charged surfaces
(Example 10), were compared with authentic IGF-II, IGF-I
and L-IGF-I for their ability to stimulate protein
synthesis in an epithelial cell line following pre-
incubation and retention on two negatively-charged
surfaces. MBFs, IGF-II, IGF-I and L-IGF-I were incubated
overnight at 4°C in 0.5 ml of 1.0% BSA/PBS at
concentrations of 2 ng/ml, 20 ng/ml and 200 ng/ml in
24-well tissue culture plates which were either untreated
or coated with HaCat epithelial cell-derived matrix, and
then washed twice using 1 ml of 1.0°s BSA/PBS, as described
in Example 10.
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HaCat epithelial cells were serum starved for
2 hours, harvested and resuspended in serum-free medium
containing 1 ~Ci/ml H3 leucine, after which they were
seeded on to MBF, IGF-II, IGF-I or L-IGF-I pre-incubated
and washed wells at a density of 2.85 x 105 cells/well, and
incubated for a further 18 hrs at 37°C. Wells were washed
twice with 1 ml of cold Hanks balanced salt solution,
followed by a single wash in 0.5 ml of cold 5%
trichloroacetic acid , after which wells were washed with
0.5 ml of cold reverse osmosis quality water. Finally
0.25 ml of 0.1o Triton X-100/0.5 M NaOH was added to each
well and shaken for 30 mins. The Triton X-100/NaOH
solution from each well was then assayed for beta-emitting
radiation, indicative of 3H-leucine which had been
incorporated into proteins produced by the cells during the
18 hr incubation at 37°C. The results are shown in
Tables 3 and 4.
Bound MBFs showed dose-dependent stimulation of
protein synthesis in HaCat epithelial cells, between 1.5
and 2-fold greater than that exhibited by IGF-II, IGF-I or
L-IGF-I in the untreated negatively-charged plastic tissue
culture vessel. For matrix-bound MBFs, stimulation of
protein synthesis in HaCat epithelial cells was
approximately 30o above that induced by IGF-I and L-IGF-I,
and was observed only at the highest concentration of
200 ng/ml (Table 3 and 4).
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Table 3
Stimulation of protein synthesis in HaCaT cells
seeded onto polypeptide bioactive factor pre-treated
tissue culture plastic.
Polypeptide 2 ng/ml 20 200
bioactive factor ng/ml ng/ml
FGF-2 112.3 7.5 102.6 4.7 130.4 7.0
IGF-II 117 13 109.8 5.1 134.1 2.7
L-IGF-I 114 6.3 109.8 13.6 132.3 6.3
L-MBF-1 131.7 3.7 175.4 9.4 213.0 10.6
L-MBF-2 131.2 5.2 159.9 10.2 220.5 9.6
L-MBF-3 133.2 10.2 153.7 13.2 226.8 5.6
~L-MBF-4 143.3 5.6 165.6 6.6 214.6 5.2
Table 4
Stimulation of protein synthesis in HaCaT cells seeded
onto polypeptide bioactive factor pre-treated HaCaT cell
derived matrix
Polypeptide 2 ng/ml 20 200
bioactive ng/ml ng/ml
factor
FGF-2 82.4 3.3 110.5 13.0 120.9 8.3
IGF-II 96.1 6.1 98.6 10.0 107.3 2.2
L-IGF-I 102,3 3.8 103.3 8.7 113.1 7.3
L-MBF-1 101.3 4.9 100.4 7.3 130.9 6.4
L-MBF-2 98.3 4.2 102 10.5 133.2 3.6
L-MBF-3 103.8 7.9 102.5 9.9 135.2 7.2
L-MBF-4 103.7 5.9 114.1 17.4 124.1 11.8
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Example 12 In vitro Binding of Pure MBF-2 and L-MBF-2 to
Titanium Screws
Biologically pure MBF-2 and L-MBF-2 from Example
6 were iodinated by conventional methods and shown to have
an increased ability to bind to titanium screws, compared
to iodinated IGF-I. Iodinated MBF-2 and L-MBF-2 were
diluted into Dulbecco's modified minimal medium (DMEM)
(10,000 counts per minute/ml).Titanium screws were
incubated in the presence of 1ml of iodinated MBF or IGF-I
solution (10,000 cpm/ml) overnight at 4°-C in 24-well tissue
culture plastic plates. The medium was removed, and the
screws were each washed twice with 1 ml of cold DMEM. The
washing medium and the screws were analysed for the
presence of the iodinated MBF or IGF-I species.
The MBFs demonstrated between 2.5 and 4.5-fold
increases in their ability to remain bound to the titanium
screws when compared to iodinated IGF-I. The results,
shown in Table 5, are expressed as the number of counts per
minute (cpm) retained on the screws following the washing
steps .
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Table 5
Radioactive Counts
Remaining Associated with Titanium Screws
Following Two DMEM Washes (n=3)
Treatment cpm/SCREW cpm/SCREW
(mean sem)
IGF-I 97.9
IGF-I 87.3 83.8 11.4
IGF-I 66.1
MBF-2 337.4
MBF-2 416.9 373.4 28.5
MBF-2 365.8
L-MBF-2 211.5
IL-MBF-2 189.7 209.3 13.1
L-MBF-2 226.7
Example 13 In vitro Retention of MBF-2, L-MBF-2 and
IGF-I in Fibrin Gels
Biologically pure MBF-2 and L-MBF-2 from
Example 6 were iodinated by conventional methods, and shown
to have an increased ability to remain bound within fibrin
gels or clots, compared to iodinated IGF-I. 50 ~1 of
0.4o fibrinogen containing 10,000 cpm of iodinated MBF or
IGF-I was combined with 5 ~.l of 0.020 thrombin in 24-well
tissue culture plastic plates to form a fibrin clot or gel.
1 ml of DMEM was added to each well to cover the clots and
incubated at 4°C for 24 hours, after which the medium was
collected and replaced with fresh DMEM. This process of
medium collection and replacement continued for 48 hours.
Collected medium was analysed for the presence of iodinated
MBF or IGF-I.
The MBFs demonstrated up to 50o increases in
retention within the fibrin gels as compared to IGF-1 at
all time points, as shown in Table 6.
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Table 6
Retention of Radioactive Counts (Iodinated Peptide)
Within Fibrin Gels over 48 hours
Sample o Counts Retained
24 hours 48 hours
IGF-I 19.4 14.9
MBF-2 24.5 19.4
L-MBF-2 26.2 20.9
Example 14 Adsorption of MBF-2, L-MBF-2 and IGF-I on to
Polyanionic Tissue Culture Plastic
Solutions of MBF-2, L-MBF-2 and IGF-I were
prepared to a final concentration of 100 ng/ml in DMEM.
1 ml of each solution was applied to the first well of
separate 24-well tissue culture plastic plates, and
incubated at room temperature for 15 minutes. Following
this incubation period the 1 ml solutions were transferred
to the second well of each 24-well plate, and incubated at
room temperature for another 15 minutes. This process was
repeated for a total of 18 out of the total 24-wells in
each plate.
Following the sequential coating of 18 wells by
either a 1 ml solution of MBF-2, L-MBF-2 or IGF-I, the
wells were washed twice with 1 ml of DMEM, and air dried
within a laminar flow cabinet. 1 m1 of DMEM containing
2.5 x 105 HaCat epithelial cells and 1 ~Ci of tritiated
leucine was added to each well and incubated at 37°C for
18 hours. Wells were washed twice with 1 ml each of Hank's
balanced salt solution, twice with 1 ml each of 5~
trichloroacetic acid (TCA), and once with 2 ml of Milli-Q
water. 1 ml of 0.5M sodium hydroxide/0.1o triton X-100 was
added to each well and incubated at room temperature for at
least 30 minutes, with shaking. 100 ~,1 samples from each
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well were transferred to scintillation vials, 2 ml of
scintillation fluid was added to each vial, and mixed well
with shaking. Samples were analysed for the presence of
~3-emitting tritiated leucine incorporated into newly-
synthesized protein in response to the MBF or IGF-I bound
to the plastic surface.
Results are expressed as a percentage of protein
synthesis compared to a growth factor-free control, and are
shown in Table 7. 1 ml solutions of biologically pure MBF-2
and L-MBF-2 (100 ng/ml) from Example 6 were able to be used
repeatedly (at least 18 applications) to coat tissue
culture plastic surfaces, with 5-8 fold stimulation of
protein synthesis in HaCat cells grown on these surfaces.
In contrast, the repeated coating of tissue culture plastic
surfaces with IGF-1 solution resulted in a lower
stimulation of protein synthesis by the HaCat cells, which
returned to baseline after 13 applications.
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Table 7
Stimulation of Protein Synthesis in HaCat Cells Grown
on to 24-well Tissue Culture Plates Treated with
MBF-2, L-MBF-2 or IGF-I, Expressed as a Percentage
of a Growth Factor-Free Control
Diluted Series IGF-i NSF-2 L-NSF-2
1 334.4 _ 876.8
611.9
2 442.3 578.7 751.3
3 400.'7 560 705.5
4 267 608.2 708.9
293.3 592.6 742
6 243.7 660.1 814.3
7 234.4 643.1 758.7
8 203.5 642.6 830.1
192.9 613.4 706.9
202.3 603.5 712.8
11 230.1 549.1 655.9
12 178.1 539.3 706.2
13 86.4 550.9 632.4
14 128.1 533.7 676.2
184.7 566.5 686
16 122.2 572.7 675.1
17 106 615.3 774
18 97.9 628.2 717.3
Example 15 In vivo Tissue Distribution of Systemically
10 Administered Iodinated MBF-2, L-MBF-2
Compared to IGF-I
Biologically pure MBF-2 and L-MBF-2 from Example
6 iodinated by conventional methods and injected via
jugular catheter into male rats appeared to localise
15 preferentially to a variety of tissues, when compared to
similarly iodinated and administered IGF-I.
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Male Sprague Dawley rats (118-130 grams) were
administered 1 x 10' cpm of either MBF-2, L-MBF-2 or IGF-I,
and decapitated at either 1 minute or 15 minutes, after
which samples of blood and the gut, left hind limb, pelt,
heart, liver, spleen, lungs, adrenals, kidneys and thymus
were frozen in liquid nitrogen. Samples were thawed before
being homogenised in 5 volumes (w/v) of 10% TCA and
analysed for the presence of TCA precipitable iodinated
MBF-2, L-MBF-2 or IGF-I.
Results for each tissue were expressed as
cpm/gram of tissue and normalised compared to cpm in the
plasma of each respective rat, to produce a ratio of
between 0 and 1 correlating to cpm/gram of tissue. Table 8
shows the tissues where preferential localisation of MBFs
occurs compared to IGF-I.
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SUBSTITUTE SHEET (Rule 26) (RO/AU)
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Example 16 Effect of L-MBF-2 on Growth and Cellular
Activity of CHO Cells Grown on Polypropylene
Discs
30 mls of 0.9o saline, 2 grams of polypropylene
discs were placed in four 100 ml spinner flasks and
autoclaved. The saline in two of the spinner flasks was
removed, replaced with 30 mls of DMEM containing 100 ng/ml
L-MBF-2, and all four flasks stored overnight at 4°-C. The
polypropylene discs in all four-spinner flasks were washed
twice with 50 mls of DMEM and seeded with 1 x 10' CHO cells
expressing marmoset chorionic gonadotrophin in 50 mls of
PF-CHO protein-free medium. The spinner flasks were
allowed to stand for 1 hour, after which they were
incubated at 5o COZ, 37°C on a multiple magnetic stirrer.
1 ml sub-samples of medium were collected from each spinner
flask at 2 hrs, 4 hrs, 16 hrs, 20 hrs, 24 hrs, 48 hrs,
72 hrs and frozen immediately. The sub-samples were
analysed for glucose concentration and marmoset chorionic
gonadotrophin (ELISA) as indicators of cellular growth
and/or activity.
Results at each sub-sample time are expressed as
glucose concentrations (mM), as shown in Table 9, and as
protein (chorionic gonadotrophin) production, as measured
by colorimetric ELISA assay at 650 nm, shown in Table 10.
These results showed that CHO cells grown on polypropylene
discs treated with biologically pure L-MBF-2 from example 6
contained within 200 ml spinner flasks were more active, as
indicated by glucose consumption and marginally increased
protein production, than identical cells grown on untreated
polypropylene discs.
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Tahla ~
Glucose Consumption over the 72 hour Period
Sample Glucose
Concentration
(mM)
Treated Untreated
Discs Discs
2 hours 21.8 21.8 21.8 21.8
4 hours 20.8 20.7 21.6 21.6
16 hours 19.3 19.2 21.1 21.0
20 hours 18.7 18.9 20.9 20.7
24 hours 15.9 15.7 20.7 20.3
48 hours 13.9 13.6 16.6 16.5
72 hours 9.8 8.5 11.5 12.1
Ta~-,~ ~~ ~ n
Marmoset Chorionic Gonadotrophin in
Conditioned Medium as Measured by ELISA
Sample Absorbance
(650
nm)
Treated Untreated
Discs Discs
2 hours 0.43 0.4 0.36 0.4
(1:4 dilution}
4 hours 0.44 0.4 0.37 0.4
(1:4 dilution)
16 hours 0.41 0.41 0.38 0.37
(1:8 dilution)
hours 0.46 0.44 0.39 0.39
(1:8 dilution)
'24 hours 0.48 0.51 0.45 0.47
(1:8 dilution)
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Example 17 The Protective Effects of MBF Coated Tissue
Culture Plastic Against Apoptosis induced by
Serum Deprivation or Camptothecin
The tissue culture wells were each incubated with
1 ml of DMEM containing 100 ng/ml of MBF-2 or L-MBF-2 for
18 hours at 4°C. Following the 18 hour incubation all
wells were washed twice with 1 ml of DMEM, and allowed to
air dry in a laminar flow cabinet. An identical number
(0.2 x 10') of MCF7 mammary tumour cells was seeded on to
i0 and grown on the MBF-treated and untreated 24-well tissue
culture plastic plates. The number of cells on the
respective plates induced into apoptosis by either serum
deprivation or the addition of camptothecin was indicated
by the detection of propidium iodide staining.
Results are expressed as the number of cells per
field staining positive for propidium iodide. Four fields
in each of two wells were assessed for each treatment
(n=8), and the results are shown in Table 11. This
experiment showed that biologically pure MBF-2 and L-MBF-2
from Example 6 coated on 24-well tissue culture plastic
plates had a protective effect against serum deprivation or
drug-induced apoptosis in a mammary tumour cell-line.
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r-i,..
I
m
3 a
N ro
1 ~ w M
w ~
m +i +~
a ~ --~ M
.~,
0
0
m ~
s~
0
I
~ m +I
~
o ~ o ~r
a ~- ~ co
U
ro
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v
w M d~
W ~ m +~ +~
r
l
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3 n
.-,
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'I-a m 11
0 3 ~
00
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+~
+~
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b
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s~
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!~ N
O
b
m ~ O
U
SUBSTITUTE SHEET (Rule 26) (RO/AU)
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Example 18 The Stimulation of Protein Synthesis in HaCat
Epithelial Cells Seeded on to Various
Biological Substrates Pre-Treated with MBF-2,
L-MBF-2 or IGF-I
Preparation of substrates
24-well tissue culture plastic plates were coated
with 1 ml of poly-L-lysine solution (O. lo) for 10 minutes,
after which each well was washed twice with sterile Milli-Q
water and allowed to air dry for at least 2 hours.
to Heparin, dextran sulphate and chondroitin sulphate A were
dissolved in sterile water (100 ~,g/ml), 1 ml of each
solution added to respective wells and incubated for
18 hours at 4°C. The wells were washed twice with 1 ml of
sterile Milli-Q water, and allowed to air dry inside a
t5 laminar flow cabinet for at least 1 hour. These plates were
stored at 4°C until pre-treatment with MBFs or IGF-I.
Rat tail collagen was prepared according to a
conventional method and 250 x..1,1 of the stock collagen
solution added to respective poly-L-lysine coated wells.
20 After a 5 minute incubation inside a laminar flow cabinet
the collagen solution was aspirated, leaving only a thin
film of collagen remaining, and the collagen-coated wells
air dried for at least 1 hour, after which they were stored
at 4°C until pre-treatment with MBFs or IGF-I.
25 Fibronectin and laminin coated plates were
purchased from Falcon, and pre-treated with MBFs or IGF-I
as supplied.
Pre-treatment of substrates
30 Biologically pure MBF-2 and L-MBF-2 from
Example 6 and receptor grade IGF-I were dissolved in DMEM
at 100 ng/m1. 1 ml of these solutions was added to the
respective pre-prepared wells and incubated for 4 hours at
4°-C. All wells were washed twice with 1 ml of cold DMEM
35 and stored at 4°C prior to inoculation with cells.
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Seeding with cells
HaCat cells were grown in the absence of serum
for 4 hours and harvested. Cells were counted and
resuspended into DMEM containing 1 ~Ci/ml of tritiated
leucine at 2 x 105 cells/ml. 1 ml of cell suspension
containing radioactive tracer was added to each of the pre-
prepared and pre-treated wells, and incubated at 37°C for
18 hours.
Harvesting
The wells were washed twice with 2 ml of cold
Hank's balanced salt solution, twice with 1 ml of cold 50
trichloroacetic acid, and once with 2 ml of cold Milli-Q
water. 1 ml of 0.1o Triton X-100/0.5M NaOH was added to
each well and incubated at room temperature for 30 minutes
with shaking. 100 ~l sub-samples from each well were
transferred to scintillation vials, 2 ml of scintillation
fluid added to each vial and mixed well with shaking. Sub-
samples were assayed for the presence of newly synthesized
tritiated-leucine containing protein.
Results are expressed as the percentage of
protein synthesis stimulation compared to a growth factor-
free control, and are shown in Table 12. This study showed
that HaCat cells grown on 24-well tissue culture plastic
plates coated with heparin, dextran sulphate, chondroitin
sulphate A, fibronectin, laminin and rat-tail collagen
respectively that had been pre-treated with MBF-2 or L-MBF-
2 all exhibited increased stimulations of protein
synthesis, compared with the same substrates pre-treated
with IGF-I. No effect was found with poly-L-lysine coated
plates. Similar results were obtained in a second
experiment.
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M
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I
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SUBSTITUTE SHEET (Rule 26) (RO/AU)
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Example 19 The Stimulation of Tenocyte Cell Migration
from Tendon Biopsies by MBF-2
The migration of fibroblast type cells (tenocytes)
from circular tendon biopsies into fibrin clots was
stimulated by biologically pure MBF-2 from Example 6. 2 mm
diameter tendon biopsies were taken from chicken toe flexor
tendons and embedded into fibrin clots. The fibrin clots
containing tendon biopsies were incubated in medium
containing MBF-2 (500 ng/ml) + 5% fetal bovine serum (FBS),
IGF-I (500 ng/ml) + 5o FBS or in 5% FBS alone and incubated
at 5o COz, 37QC for 4 days. The migration distances of
cells were measured four times each day at three-hourly
intervals using phase-contrast light microscopy
(4X magnification) and the mean calculated for each
treatment.
Results were expressed as the migration distance
in millimetres at each of the four days, and are shown in
Table 13. This experiment demonstrated greater migration
distances with MBF-2 and IGF-I above control (5oFBS)
cultures. MBF-2 was slightly more active than IGF-I at the
earlier time points.
m ~ ~.,, ~ , ~
Migration Distances ~(mm) of Tenocytes
from their Biopsy Interface
IGF-1 I~F-2 (500 5% Fetal bovine
(500 ng/ml) ser~un
ng/ml) + 5% FBS
+ 5 %
FBS
Day 1 0.8 0.1 1.0 0.1 0.4 0.1
Day 2 2.7 0.4 3.4 0.3 1.5 0.2
Day 3 5.3 0.6 5.7 0.5 3.5 0.4
Day 4 7.8 0.8 7.8 0.4 5.7 0.6
It will be apparent to the person skilled in the
art that while the invention has been described in some
detail for the purposes of clarity and understanding,
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various modifications and alterations to the embodiments
and methods described herein may be made without departing
from the scope of the inventive concept disclosed in this
specification.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: GROPEP PTY LTD
(B) STREET: GATE 11, VICTORIA DRIVE
(C) CITY: ADELAIDE
(D) STATE: SOUTH AUSTRALIA
(E) COUNTRY: AUSTRALIA
(F) POSTAL CODE (ZIP): 5000
(ii) TITLE OF INVENTION: MATRIX BINDING FACTOR
(iii) NUMBER OF SEQUENCES: 12
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version
#1.30 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: AU PP 2984
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
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(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
G1y Pro G1u Thr Leu Cys Gly Ala Glu Leu Val Asp Ala
Leu Gln Phe
1 5 10
15
Val Cys Gly Asp Arg G1y Phe Tyr Phe Asn Pro Thr
Lys
Gly Tyr Gly
20 25 30
Ser Ser Ser Arg Arg A1a Pro Gln Thr Gly Val Asp
Ile
Glu Cys Cys
35 40 45
Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Tyr Cys
Met
Ala Pro Lys
50 55 60
Lys Asn Gly Arg Ser Lys Leu Gly Pro Arg His Phe
Thr
Gly Gln
65 70 75
(2) INFO RMATION FOR SEQ ID NO:
2:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 84 amino
acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE
TYPE:
peptide
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(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Gly Pro Glu Thr Leu Cys G1y Ala Glu Leu Val Asp Ala
Leu Gln Phe
1 5 10
l0 15
Val Cys Gly Asp Arg G1y Phe Tyr Phe Asn Lys Pro Thr
Gly Tyr Gly
20 25 30
Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp
Glu Cys Cys
35 40 45
Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys
Ala Pro Leu
50 55 60
Lys Lys Asn Gly Arg Ser Lys Leu Gly Pro Arg Thr His
Phe Gly Gln
65 70 75
80
Ala Lys Ser Ala
(2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 80 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
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4
(D) TOPOLOGY: linear
(ii)MOLECULE TYPE: peptide
(iii)HYPOTHETICAL: NO
(iv)ANTI-SENSE: NO
(xi)SEQUENCE DESCRIPTION: NO: 3:
SEQ ID
t0
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala
Leu Gln Phe
1 5 10
15
Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr
Gly Tyr Gly
20 25 30
Ser Ser Ser Arg Arg Ala Pro G1n Thr Gly Ile Va1 Asp
Glu Cys Cys
35 40 45
Phe Arg Ser Cys Asp Leu Arg Arg Leu Glu Met Tyr Cys
Ala Pro Gly
50 55 60
Lys Lys Asn Gly Arg Ser G1n Lys Gly Pro Arg Thr His
Phe Gly Gln
65 70 75
80
(2) INFORMATION FOR SEQ ID NO: 4:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 amino acids
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(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Gly Pro Glu Thr Leu Cys Gly Ala Glu Leu Val Asp Ala
Leu Gln Phe
1 5 10
Val Cys Gly Asp Arg Gly Phe Tyr Phe Asn Lys Pro Thr
Gly Tyr Gly
20 25 30
Ser Ser Ser Arg Arg Ala Pro Gln Thr Gly Ile Val Asp
Glu Cys Cys
35 40 45
Phe Arg Ser Cys Asp Lys Arg Gln Leu Glu Lys Tyr Cys
Ala Pro Gly
50 55 60
Lys Arg Gly Arg Ser Ala
65 70
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(B) TYPE: nucleic acid
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6
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii)HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
TGC GCTCCGC
TGP.,~~~1AAAAA
CGGTCGTTCT
AAACTGGGCC
CGGCTAAATC
TGCT
54
15(2) INFORMATION
FOR
SEQ
ID
NO:
6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii)HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
TCT AAACTGG
GTCCGCGTAC
CCACTTCGGC
CAGGCTAAAT
CTGCTTGA
48
(2) INFORMATION
FOR
SEQ
ID
NO:
7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
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7
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETTCAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
ATGTACTGCG
CTCCGAA.AAA
AAACGGTCGT
TCTAAACTGC
TGAAACCGGC
TAAA
54
(2) INFORMATION
FOR SEQ
ID NO:
8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GGTCGTTCTA
AACTGGGCCC
GCGTACCCAC
TTCGGTCAGT
GATGATGCAA
GCTT
54
(2) INFORMATION
FOR SEQ
ID NO:
9:
(i) SEQUENCE CHARACTERISTICS:
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8
(A) LENGTH: 57 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
ATGTACTGCG CTCCGGGTAA AAAAAACGGC CGTTCTCAGA AACTGAAACC
GGCTAAA 57
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 54 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
GGTCGTTCTC AGAAAGGCCC GCGTACCCAC TTCGGTCAGT GATGATGCAA GCTT
54
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_ g _
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
IS (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
TTCCGTTCTT GCGACAA.ACG TCAGCTGGAA AAATACTGCG CTCCGCTG
48
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
AAATACTGCG CTCCGGGTAA ACGTGGCCGT TCTGCTTGAT GATGC
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