U.S. patent application number 10/337993 was filed with the patent office on 2003-08-14 for bovine milk growth factor.
This patent application is currently assigned to GroPep Pty. Ltd.. Invention is credited to Belford, David Andrew, Dunbar, Andrew Jeremy, Goddard, Christopher.
Application Number | 20030152526 10/337993 |
Document ID | / |
Family ID | 3804598 |
Filed Date | 2003-08-14 |
United States Patent
Application |
20030152526 |
Kind Code |
A1 |
Dunbar, Andrew Jeremy ; et
al. |
August 14, 2003 |
Bovine milk growth factor
Abstract
The present invention relates to a novel growth factor from
bovine milk, milk products or milk product extracts. The present
invention also relates to the use of recombinant DNA technology to
isolate, clone and sequence nucleic acids encoding the mature and
precursor forms of the growth factor and, in addition, to the use
of these nucleic acids in the recombinant production of the growth
factor.
Inventors: |
Dunbar, Andrew Jeremy;
(Adelaide, AU) ; Goddard, Christopher; (Blackwood,
AU) ; Belford, David Andrew; (Seacliff, AU) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
GroPep Pty. Ltd.
|
Family ID: |
3804598 |
Appl. No.: |
10/337993 |
Filed: |
January 8, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10337993 |
Jan 8, 2003 |
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09554119 |
May 10, 2000 |
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6531134 |
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09554119 |
May 10, 2000 |
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PCT/AU98/00942 |
Nov 12, 1998 |
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Current U.S.
Class: |
424/50 ;
435/320.1; 435/325; 435/6.16; 435/69.1; 530/399; 536/23.2 |
Current CPC
Class: |
A61P 1/04 20180101; C07K
2319/00 20130101; A61K 38/00 20130101; A61P 17/02 20180101; A61P
1/02 20180101; A61P 1/00 20180101; C07K 14/475 20130101 |
Class at
Publication: |
424/50 ;
435/69.1; 435/320.1; 435/325; 530/399; 435/6; 536/23.2 |
International
Class: |
A61K 007/28; C12Q
001/68; C07H 021/04; C07K 014/475; C12P 021/02; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 12, 1997 |
AU |
PP-0318 |
Claims
1. A nucleic acid sequence encoding BMGF or a functionally active
fragment thereof, wherein said nucleic acid sequence comprises the
sequence shown in FIG. 5 or a sequence coding for a functionally
active fragment thereof.
2. A method for isolating a nucleic acid sequence encoding a BMGF
or a functionally active fragment thereof, comprising the steps of:
providing a source of cells having BMGF activity; treating the
cells to obtain mRNA therefrom; treating the mRNA thus obtained to
produce cDNA therefrom; and amplifying the cDNA thus obtained by
PCR using oligonucleotide primers to produce the nucleic acid.
3. The method of claim 2, wherein the oligonucleotide primers
comprise any one of the following sequences, mutants, analogues or
derivatives thereof: 5' ATC TAG GTT ACC ATG GAT GGG AAT TCA ACC AGA
3' (SEQ ID NO:10) 5' ATC TAG GTT ACC GGC GAT GGG AAT TCA ACC AGA 3'
(SEQ ID NO:11) 5'CTA GAT AAG GTT TCA TCA GTA AAA CAA GTC AAC TCT 3'
(SEQ ID NO:12) 5' GGG AAT TCA ACC AGA 3' (SEQ ID NO:13) 5' GTA AAA
CAA GTC AAC TCT 3' (SEQ ID NO:14) 5' GGG AAT TCA ACC AGA AGT CCT
GAA 3' (SEQ ID NO:15) 5' GTA AAA CAA GTC AAC TCT CTC ACA CCT 3'
(SEQ ID NO:16).
4. The nucleic acid sequence obtained by the method of claim 2.
5. An expression vector comprising a nucleic acid sequence
according to claim 1.
6. The expression vector of claim 5, further comprising a nucleic
acid sequence encoding a portion of porcine growth hormone (pGH)
linked to the 5' nucleotide sequence of BMGF.
7. The expression vector of claim 6, wherein the nucleic acid
sequence encoding a portion of pGH is linked through a nucleic acid
sequence encoding a cleavable amino acid sequence.
8. A method for enhancing wound healing and/or tissue repair,
comprising the step of: administering to a patient in need thereof
an effective amount of BMGF, or mutants, analogues and derivatives
of BMGF, precursor and fusion protein forms of BMGF and
functionally active fragments (peptides) of BMGF.
9. A method for preventing or treating periodontal disease,
comprising the step of: administering to a patient in need thereof
an effective amount of BMGF, or mutants, analogues and derivatives
of BMGF, precursor and fusion protein forms of BMGF and
functionally active fragments (peptides) of BMGF.
10. A method of providing a cosmetic delivery, comprising the step
of: administering to a subject in need thereof an effective amount
of BMGF, or mutants, analogues and derivatives of BMGF, precursor
and fusion protein forms of BMGF and functionally active fragments
(peptides) of BMGF.
11. An expression vector comprising a nucleic acid sequence
according to claim 4.
Description
[0001] The present invention relates to a novel growth factor from
bovine milk, milk products or milk product extracts. The present
invention also relates to the use of recombinant DNA technology to
isolate, clone and sequence nucleic acids encoding the mature and
precursor forms of the growth factor and, in addition, to the use
of these nucleic acids in the recombinant production of the growth
factor.
[0002] Growth factors are implicated in a wide range of
physiological and pathological processes such as cell
communication, growth and development, apoptosis, embryogenesis,
initiation of the immune response, cell survival and
differentiation, wound healing, and cancer. Justifiably, there is a
great deal of interest in isolating, characterising and defining
the functional mechanisms of growth factors, not only in
understanding the basic mechanisms behind normal growth control,
but also because of their potential therapeutic use.
[0003] Growth factors may also comprise an essential component of
defined media used in the growth of cells in culture by the
biotechnology industry. For many years animal sera have been used
to supplement culture media to provide essential components for
cell growth. Foetal bovine serum (FBS) is most commonly used,
however there are market and regulatory concerns about the safety
of animal and human sourced proteins in pharmaceutical
manufacturing processes.
[0004] Despite the considerable progress made using milk based
alternatives to serum, the safety concerns surrounding mammalian
body fluids as supplements for cell culture media (as a consequence
of the potential for infection with latent pathogens (eg. bovine
spongiform encephalopathy (BSE)) means that these are considered
unsafe. Therefore attention has focussed on the development of
completely defined cell culture media containing growth factors of
known origin and defined purity. This means that the growth factor
component of cell culture media is provided by purified native or
recombinant molecules.
[0005] Milk is an important nutrient required for the growth and
development of an infant. It is a source of nutrients such as
casein, lactoferrin, lactalbumin, lactose and various other
compounds such as vitamins, ions, enzymes etc. A number of growth
factors have been identified in human and bovine milk. These
include Insulin-like growth factor I and II, fibroblast growth
factors, transforming growth factor .beta., and platelet-derived
growth factor. Although epidermal growth factor is present in human
milk it has not been convincingly demonstrated in bovine milk
(Iacopetta et al, Acta Paediatr, 81, 287, (1992)). Several roles
have been proposed for milk-derived growth factors including
development and differentiation of the mammary gland (Collier et
al, Livestock Production Science, 35, 21, (1993)), regulation of
the developing neonatal immune system (Ishizaka et al. Cellular
Immunol, 159, 77, (1994)), gastrointestinal growth and maturation
(Read et al, Pediatric Research, 18, 133, (1984)), and possible
actions in other organs. Milk-derived growth factors have also been
shown to support the growth of a variety of cells in culture. In
addition, bovine milk whey, the by-product of cheese manufacture,
can also support the growth of mammalian cells in the short or long
term (Derouiche et al, Lait, 70, 313, (1990); Damerdji et al,
Biotechnology Tech, 2, 253, (1988)) and has been shown to possess
antitumor activity (Bounous et al, Clin. Invest. Med. 11, 312,
(1988)). The prior art also includes Australian patent 645589 to
the present applicant which describes the use of a bovine milk whey
extract, containing a plurality of cell growth stimulating factors,
as a replacement for serum in mammalian cell culture.
[0006] It is an object of the present invention to overcome, or at
least alleviate, one or more of the difficulties or deficiencies
associated with the prior art. Accordingly, in a first aspect of
the present invention there is provided a novel growth factor,
hereinafter referred to as Bovine Milk Growth Factor (BMGF)
obtainable from bovine milk, milk products or milk product
extracts. The term "milk" as used herein refers to lactational
secretions of human or animal origin.
[0007] The BMGF may be in a substantially pure or partially pure
form. The term "substantially pure" as used herein to describe the
purity of BMGF means at least 70% pure.
[0008] The term "partially pure" as used herein to describe the
purity of BMGF means a specific activity greater than 0.3 units per
milligram protein.
[0009] The term "one unit" as used herein as a measure of BMGF
activity is defined as the amount of BMGF required to compete for
50% of the binding of .sup.125I-labelled recombinant human
epidermal growth factor to AG2804 cells under the assay conditions
described in Example 1 of this specification.
[0010] The native BMGF in its glycosylated form may have a
molecular weight of approximately 21 to 25 kDa as determined by
SDS-PAGE. It has the ability to stimulate the proliferation of
fibroblasts and/or epithelial cells.
[0011] In a preferred form of this aspect of the invention the BMGF
has an amino acid sequence as follows:
[0012] DGNSTRSPEDDGLLCGDHAENCPATTTQPKRRGHFSRCPKQYKHYCI
KGRCRFWAEQTPSCVCDEGYAGARCERVDLFY (SEQ ID NO: 2)
[0013] The BMGF disclosed herein may be a member of the epidermal
growth factor family of growth factors. It contains eighty amino
acid residues and eight half cysteines, a pattern that has also
been described for an epidermal growth factor-like molecule
purified from the conditioned media of mouse pancreatic beta tumor
cells (Shing et al, Science, 259, 1604, (1993)). The amino acid
sequence of the human form of this factor, termed, betacellulin,
has also been deduced from a nucleotide sequence obtained from a
human adenocarcinoma cell line (Sasada et al, Biochem. Biophys.
Res. Commun. 190, 1173, (1993)). It is likely, based on the
sequence homologies between BMGF and mouse betacellulin (58
identical residues) and with human betacellulin (72 identical
residues), that BMGF is the bovine form of the betacellulin.
Clearly, it is not the same molecule. The BMGF isolated from bovine
cheese whey extract has a molecular mass of 21-25 kDa, which is
substantially smaller in size than the 32 kDa reported for the
natural mouse betacellulin.
[0014] The present invention also includes within its scope
precursor forms of BMGF and functionally active fragments
(peptides) of BMGF. Such fragments may have enhanced or diminished
growth stimulatory activity and/or may expand or limit the range of
cells responsive to BMGF's growth stimulatory activity. They may
find useful applications in areas such as, but not limited to, the
repair or prevention of gut damage or in wound healing.
[0015] They may be produced by methods known to those skilled in
the art. Procedures at the genetic level such as (but not limited
to) site-directed mutagenesis or at the protein level such as (but
not limited to) chemical modification are within the scope of the
invention.
[0016] In a preferred form of this aspect of the invention the
fragments of BMGF may include the following amino acid
sequences:
[0017] DGNSTRSPEDDGLLCGDHAENCPATTTQPKRRGHF (SEQ ID NO: 3); or
[0018] GYAGARCERVDLFY (SEQ ID NO: 4); or
[0019] DGNSTRSPEDDGLLCGDHAENCPATTTQPK (SEQ ID NO: 5); or
[0020] RRGHFSRCPK (SEQ ID NO: 6); or
[0021] QYK (SEQ ID NO: 7); or
[0022] HYCIK (SEQ ID NO: 8); or
[0023] GRCRFWAEQTPSCVCDEGYAGARCERVDLFY (SEQ ID NO: 9)
[0024] The present invention also provides BMGF that is
substantially non-glycosylated. This may be prepared, for example,
by subjecting glycosylated BMGF to an enzymatic de-glycosylation
step and recovering the de-glycosylated form. The BMGF in its
non-glycosylated or partially non-glycosylated form may have a
molecular weight of approximately 9-14 kDa as determined by
SDS-PAGE.
[0025] In a further preferred form of this aspect of the invention,
the BMGF is obtained from bovine milk, bovine milk products or
bovine milk product extracts. More preferably it is obtained from
cheese whey, most preferably from bovine cheese whey extract.
[0026] In a further aspect of the present invention, there is
provided a process for the isolation, in a substantially pure or
partially pure form, of BMGF from bovine milk or milk products.
More preferably it is isolated from cheese whey or a cheese whey
extract, most preferably from bovine cheese whey or bovine cheese
whey extract. The BMGF in its glycosylated form has a molecular
weight of approximately 21 to 25 kDa as determined by SDS-PAGE. It
may have the ability to stimulate the proliferation of fibroblasts
and/or epithelial cells, and/or osteoblast cells.
[0027] In a preferred form of this aspect of the invention the BMGF
has an amino acid sequence as follows:
[0028] DGNSTRSPEDDGLLCGDHAENCPATTTQPKRRGHFSRCPKQYKHYCI
KGRCRFWAEQTPSCVCDEGYAGARCERVDLFY (SEQ ID NO: 2)
[0029] The process of isolating substantially pure BMGF in this
aspect of the invention may include subjecting the milk or a milk
product or milk product extract to various purification steps such
as ion-exchange chromatography, size-exclusion chromatography,
affinity chromatography and reverse-phase high performance liquid
chromatography and/or if necessary further purification processes.
A "milk product" may include cheese whey, skim milk, acid (Casein)
whey and colostrum. Preferably, the milk or milk product is
subjected to a process outlined in AU645589 to provide a milk
product extract prior to the purification steps outlined above. The
contents of AU645589 are incorporated herein. A "milk product
extract" is defined herein as an extract prepared from milk or a
milk product by a process described in Australian Patent AU645589.
By way of clarification, the defining of a milk product extract
encompasses a cheese whey extract.
[0030] Sample fractions collected from each purification step, and
which show biological activity in a radioreceptor assay using
AG2804 cells are pooled and forwarded to the next purification
step. Sample material from each of the pooled fractions may be
subjected to a dose response analysis, thereby allowing for an
estimate of the amount of material required to elicit a 50%
response of the maximal activity in the said assay. This said
amount, in conjunction with the values for the amount of material
in each pool, may be used to calculate the yield of purification
recovery, and specific activity at each step of the process.
Homogeneity of substantially pure BMGF may be demonstrated by:
[0031] migration as a single band of approximately 21-25 kDa
following SDS-PAGE,
[0032] N-terminal sequence analysis,
[0033] mass spectroscopy and/or
[0034] specific binding to an EGF-receptor
[0035] In a preferred form of this aspect of the invention there is
provided a method of isolating substantially pure BMGF which method
includes
[0036] providing bovine milk, milk product or milk product
extract;
[0037] subjecting the bovine milk, milk product or milk product
extract to ultrafiltration to obtain a first fraction;
[0038] subjecting the first fraction to anion exchange
chromatography to obtain a second fraction;
[0039] subjecting the second fraction to gel filtration
chromatography to obtain a third fraction;
[0040] subjecting the third fraction to reverse phase high
performance liquid chromatography ((RP)HPLC) to obtain a fourth
fraction;
[0041] subjecting the fourth fraction to affinity chromatography to
obtain a fifth fraction;
[0042] subjecting the fifth fraction to (RP)HPLC to obtain a sixth
fraction;
[0043] subjecting the sixth fraction to (RP)HPLC to obtain the
substantially pure BMGF.
[0044] In a further preferred form of this aspect of the invention
there is provided a method of isolating substantially pure BMGF
which method includes
[0045] providing a milk product extract prepared according to
AU645589;
[0046] subjecting the milk product extract to acidification;
[0047] subjecting the acidified milk product extract to
ultrafiltration to obtain a first fraction;
[0048] subjecting the first fraction to anion exchange
chromatography to obtain a second fraction;
[0049] subjecting the second fraction to gel filtration
chromatography to obtain a third fraction;
[0050] subjecting the third fraction to reverse phase high
performance liquid chromatography ((RP)HPLC) to obtain a fourth
fraction;
[0051] subjecting the fourth fraction to affinity chromatography to
obtain a fifth fraction;
[0052] subjecting the fifth fraction to (RP)HPLC to obtain a sixth
fraction;
[0053] subjecting the sixth fraction to (RP)HPLC to obtain the
substantially pure BMGF.
[0054] Preferably, the milk, milk product or milk product extract
is subjected to an acidification step. Preferably the acidification
is conducted prior to ultrafiltration. More preferably the
acidification is conducted at a pH 2.5
[0055] Preferably the ultrafiltration is performed using a membrane
with an exclusion limit of approximately 50-150 kDa, more
preferably approximately 100 kDa.
[0056] Preferably the anion exchange chromatography is performed
using an agarose-based anion exchange column.
[0057] Preferably the gel filtration is performed using a column
which separates proteins having molecular weights in the range
approximately 3 kDa to 70 kDa.
[0058] Preferably the first (RP)HPLC is performed using a C4 or C18
matrix.
[0059] Preferably the affinity chromatography is performed using a
heparin/agarose-based affinity column.
[0060] Preferably the second (RP)HPLC is performed using a C4 or
C18 matrix.
[0061] Preferably the third (RP)HPLC is performed using a C4 or C18
matrix.
[0062] As an alternative to producing a milk product extract
prepared according to AU645589 as the starting material, a bovine
milk or milk product may be utilised. If this approach is adopted a
preliminary purification step may be used to remove the fat, solids
and acidic proteins before the anion exchange chromatography
step.
[0063] The substantially purified BMGF may be used in the
production of polyclonal and monoclonal antibodies which recognise
and/or bind to BMGF and this is considered within the scope of the
present invention.
[0064] Accordingly, in a further aspect of the present invention
there is provided a polyclonal or monoclonal antibody against
BMGF.
[0065] Various procedures are known in the art which may be used
for the production of antibodies to epitopes of BMGF. Various host
animals may be used in the production of these BMGF antibodies
following immunisation with BMGF protein including but not
restricted to rabbits, mice, goats etc. Adjuvants may be used to
increase the immunological response, depending on the host species,
and may include but are not restricted to Freunds (complete and
incomplete). BMGF monoclonal antibodies may be prepared by using
techniques which enable the continuous production of antibody
molecules by cell lines in vitro. These may include, but are not
limited to, the hybridoma technique (Kohler and Milstein, Nature
256, 495, (1975)). Antibodies to BMGF may find use in the detection
of mature and precursor forms of BMGF in various tissues, body
fluids and cell lines, for example in screening assays for the
growth factor, and in the affinity purification of BMGF
protein.
[0066] In a still further aspect of the present invention there is
provided a nucleic acid encoding BMGF or fragments thereof encoding
functionally active fragments of BMGF. The nucleic acid may encode
mature or precursor forms of BMGF.
[0067] Preferably the nucleic acid has the sequence shown in FIG.
5.
[0068] Due to the degeneracy of the genetic code, other nucleic
acid sequences which encode the same or functionally equivalent
amino acid sequence are included within the scope of the current
invention. Such alterations of the BMGF nucleotide sequence may
include substitutions of different nucleotides resulting in the
same or a functionally equivalent gene product. Also included
within the scope of this invention are nucleic acid sequences
having deletions and/or additions and which result in a
functionally equivalent gene product.
[0069] In a still further aspect of the present invention, there is
provided a method for isolating a nucleic acid encoding BMGF or
fragments thereof encoding functionally active fragments of BMGF.
The nucleic acid may encode mature or precursor forms of BMGF.
[0070] The nucleic acid encoding BMGF may be obtained from cell
sources that produce BMGF activity. For example, kidney cells may
be used as the source of the nucleic acid. The nucleic acid
encoding BMGF is preferably obtained by (but is not restricted to)
reverse transcription of BMGF mRNA into complementary cDNA and
subsequent Polymerase Chain reaction (PCR) amplification using
oligonucleotide primers containing nucleic acid sequences encoding
portions, preferably the extreme N and C terminal portions of the
mature or precursor protein. Preferably the oligonucleotide primers
have the following sequences or substantially homologous
sequences:
[0071] 5' ATC TAG GTT ACC ATG GAT GGG MT TCA ACC AGA 3' (SEQ ID NO:
10)
[0072] 5' ATC TAG GTT ACC GGC GAT GGG MT TCA ACC AGA 3' (SEQ ID NO:
1 1)
[0073] 5' CTA GAT MG CTT TCA TCA GTA AAA CM GTC MC TCT 3' (SEQ ID
NO: 12)
[0074] Preferably the oligonucleotide primers include restriction
enzyme sites to facilitate directional cloning of the nucleic
acid.
[0075] More preferably, the primers include the following
nucleotide sequence:
[0076] 5' GGG MT TCA ACC AGA 3' (SEQ ID NO: 13)
[0077] 5' GTA AAA CM GTC MC TCT 3' (SEQ ID NO: 14).
[0078] Most preferably, the primers are:
[0079] 5' GGG MT TCA ACC AGA AGT CCT GAA 3' (SEQ ID NO: 15)
[0080] 5' GTA AAA CM GTC MC TCT CTC ACA CCT 3' (SEQ ID NO: 16)
[0081] Thus, in a preferred form of this aspect of the invention
there is provided a method for isolating a nucleic acid encoding
BMGF or fragments thereof encoding functionally active fragments of
BMGF, said method including
[0082] providing a source of cells having BMGF activity;
[0083] treating the cells to obtain mRNA therefrom;
[0084] treating the mRNA thus obtained to produce cDNA therefrom;
and
[0085] amplifying the cDNA thus obtained by PCR using
oligonucleotide primers to produce the nucleic acid.
[0086] Other methods, well known to those in the art, for obtaining
nucleic acids encoding proteins exist, and the use of these methods
to obtain nucleic acids encoding mature or precursor forms of BMGF
is also included within the scope of the current invention. These
methods may include (but are not restricted to) either chemically
synthesising the nucleic acid from knowledge of the nucleic acid or
amino acid sequence of the mature and/or precursor forms of BMGF or
screening a cDNA and/or genomic library, preferably a bovine
library, with isotopically or non-isotopically labelled nucleic
acid sequences homologous to nucleotide sequence encoding part or
all of the mature and/or precursor forms of BMGF.
[0087] Using standard techniques of recombinant DNA technology,
well known to those skilled in the art, the nucleic acid encoding
the mature or precursor forms of BMGF may be cloned into an
appropriate vector, for example an expression vector.
[0088] Accordingly, in a further aspect of the present invention
there is provided a vector including a nucleic acid encoding BMGF
or fragments thereof encoding functionally active fragments of
BMGF.
[0089] The nucleic acid may encode mature or precursor forms of
BMGF.
[0090] Preferably the nucleic acid has the sequence shown in FIG.
5.
[0091] A large number of vector-host systems are available and
these include (but are not restricted to) plasmids such as pBR322
or pUC derivatives, or bacteriophage such as lambda
derivatives.
[0092] The vector of the present invention may be used to express
recombinant BMGF in host cells.
[0093] Accordingly in a still further aspect of the present
invention there is provided recombinant BMGF.
[0094] The recombinant BMGF may be in a substantially pure form.
Preferably it is at least about 70% pure, more preferably at least
about 90% pure, most preferably at least about 99% pure. The
recombinant BMGF may have a molecular weight of approximately 9 kDa
in the non-glycosylated form as determined by SDS-PAGE. It may have
the ability to stimulate the proliferation of fibroblasts and/or
epithelial cells and/or osteoblast cells.
[0095] In a preferred form of this aspect of the invention the
recombinant BMGF has an amino acid sequence as follows:
[0096] DGNSTRSPEDDGLLCGDHAENCPATTTQPKRRGHFSRCPKQYKHYCI
KGRCRFWAEQTPSCVCDEGYAGARCERVDLFY (SEQ ID NO: 2)
[0097] The present invention also includes within its scope
precursor forms of recombinant BMGF and functionally active
fragments (peptides) of recombinant BMGF.
[0098] In a preferred form of this aspect of the invention the
fragments of BMGF may include the following amino acid sequences or
substantially homologous sequences:
[0099] DGNSTRSPEDDGLLCGDHAENCPATTTQPKRRGHF (SEQ ID NO: 3); or
[0100] GYAGARCERVDLFY (SEQ ID NO: 4); or
[0101] DGNSTRSPEDDGLLCGDHAENCPATTTQPK (SEQ ID NO: 5); or
[0102] RRGHFSRCPK (SEQ ID NO: 6); or
[0103] QYK (SEQ ID NO: 7); or
[0104] HYCIK (SEQ ID NO: 8); or
[0105] GRCRFWAEQTPSCVCDEGYAGARCERVDLFY (SEQ ID NO: 9).
[0106] The present invention also provides a method for producing
recombinant BMGF, said method including
[0107] providing
[0108] a vector including a nucleic acid encoding BMGF or fragments
thereof encoding functionally active fragments of BMGF; and
[0109] a host cell;
[0110] introducing said vector into said host cell;
[0111] expressing said recombinant BMGF; and
[0112] isolating said recombinant BMGF.
[0113] The vector may be introduced into the host cell by methods
such as (but not restricted to) transformation, transfection,
electroporation and infection. Host cells may include but are not
restricted to bacteria such as E. coli cells.
[0114] The recombinant BMGF may be expressed as a fusion protein.
The fusion proteins formed according to this aspect of the present
invention, may be isolated as inclusion bodies within the host
cell.
[0115] It will be understood that the recombinant BMGF so formed
may be isolated, preferably following disruption of the host cell,
by conventional methods of polypeptide purification well known to
those skilled in the art, utilising techniques such as ion-exchange
chromatography, size-exclusion chromatography, affinity
chromatography, reverse-phase high performance liquid
chromatography and/or if necessary further purification processes.
The recombinant BMGF may be isolated as a fusion protein or cleaved
from its fusion partner using conventional methods well known to
those in the art.
[0116] In a preferred form of this aspect of the invention, the
recombinant BMGF may be prepared as a fusion protein according to
Australian Patent 633099 to the applicant, the entire disclosure of
which is incorporated herein by reference, wherein a portion of
porcine growth hormone is linked to the N-terminal sequence of
BMGF, optionally through a cleavable sequence.
[0117] The BMGF of the present invention, including the
naturally-derived mature or precursor forms of BMGF, and
recombinant BMGF protein isolated as either the mature or precursor
form with or without its fusion partner (and mutants, analogues,
fragments and derivatives thereof) may be utilised:
[0118] in the growth and/or proliferation of mammalian cells and
more organised structures such as skin in vitro either alone or in
combination with other factors (such as or not restricted to IGF-I
or IGF-I analogues) or, for example as a supplement to foetal
serum
[0119] in the enhancement of wound healing and/or tissue repair,
eg. in the treatment of surface wounds, either alone or in
combination with other factors
[0120] in the prevention, amelioration or treatment of conditions
associated with impaired gut barrier function, such as inflammatory
bowel disease and mucosal immunity, either alone or in combination
with other factors
[0121] as a supplement in infant milk formulae
[0122] in the prevention, treatment or amelioration of peridontal
disease
[0123] in cosmetic applications.
[0124] Accordingly, the present invention provides a composition
for promoting the growth and/or proliferation of mammalian cells
and tissues, said composition including an effective amount of
BMGF, or mutants, analogues and derivatives of BMGF, precursor and
fusion protein forms of BMGF and functionally active fragments
(peptides) of BMGF; together with a culture medium, preferably a
defined culture medium.
[0125] The present invention also provides a method for promoting
the growth and/or proliferation of mammalian cells and tissues,
said method including growing said cells or tissues in a culture
medium including an effective amount of BMGF, or mutants, analogues
and derivatives of BMGF, precursor and fusion protein forms of BMGF
and functionally active fragments (peptides) of BMGF.
[0126] The term "effective amount" as used herein in methods of use
for BMGF means an amount sufficient to elicit a statistically
significant response at a 95% confidence level (p<0.05 that the
effect is due to chance alone).
[0127] In a further aspect of the present invention there is
provided a method for promoting the growth and/or proliferation of
mammalian cells and tissues, said method including growing said
cells or tissues in a culture medium including an effective amount
of BMGF, or mutants, analogues and derivatives of BMGF, precursor
and fusion protein forms of BMGF and functionally active fragments
(peptides) of BMGF, together with an effective amount of an IGF
that produces a synergistic response.
[0128] The IGF or insulin-like growth factor that is included in
the culture medium of this further aspect of the present invention
may be IGF-I, IGF-II or a functionally effective mutant or analogue
of IGF such as LR.sup.3IGF-1 described in Australian Patent 633099
to the applicant.
[0129] In a further aspect of the present invention there is
provided a composition for the treatment of surface wounds, said
composition including an effective amount of BMGF, or mutants,
analogues and derivatives of BMGF, precursor and fusion protein
forms of BMGF and functionally active fragments (peptides) of BMGF;
together with a pharmaceutically acceptable excipient, diluent or
carrier
[0130] The present invention also provides a method for enhancing
wound healing and/or tissue repair, said method including
administering to a patient in need thereof an effective amount of
BMGF, or mutants, analogues and derivatives of BMGF, precursor and
fusion protein forms of BMGF and functionally active fragments
(peptides) of BMGF.
[0131] There are no limitations on the type of wound that may be
treated, and these may include (but are not restricted to): surface
ulcers (eg. pressure, venous stasis, diabetic and atherosclerotic
ulcers), burns or accidental wounds (such as lacerations and
incisions) and wounds to epithelial lined organs such as the
stomach and intestine (large and small) and corneal injury to the
eye. The application of BMGF to wound sites may be in the form of
(but is not restricted to) a powder, gel, ointment or bandages and
other wound dressings incorporating BMGF. The BMGF may be applied
to wounds either alone or in a mixture including other growth
factors, and may be in combination with other ingredients, such as
adjuvants, carriers and solubilising agents. The concentration of
BMGF in the composition is not critical but should be enough to
induce cell proliferation, particularly epithelial cell
proliferation.
[0132] The gastrointestinal tract (gut) is constantly challenged by
potentially harmful substances present in the intestinal lumen and
subsequently, maintenance of a normal mucosal barrier is crucial in
both adult and infant life. Barrier function may be compromised by
damage to the epithelial surface, for example as occurs during
high-dose chemotherapy, infection and trauma. In addition, a
pro-inflammatory response, generated after sensitisation to normal
and luminal antigens, may lead to epithelial damage and increased
permeability. This immune dysfunction is thought to play a
significant role in the pathogenesis of disorders including coeliac
and inflammatory bowel disease. Given the epithelial-cell
stimulatory activity of BMGF, a still further application of the
present invention is in the treatment, amelioration or prevention
of conditions associated with impaired gut function, such as
inflammatory bowel disease and mucosal immunity, either alone or in
combination with other growth factors, preferably as an enteral
formulation.
[0133] Accordingly, in a still further aspect of the present
invention there is provided a composition for the prevention,
amelioration or treatment of conditions associated with impaired
gut function, said composition including an effective amount of
BMGF, or mutants, analogues and derivatives of BMGF, precursor and
fusion protein forms of BMGF and functionally active fragments
(peptides) of BMGF; together with a pharmaceutically acceptable
excipient, diluent or carrier.
[0134] The present invention also provides a method for preventing,
ameliorating or treating conditions associated with impaired gut
function, said method including administering to a patient in need
thereof an effective amount of BMGF, or mutants, analogues and
derivatives of BMGF, precursor and fusion protein forms of BMGF and
functionally active fragments (peptides) of BMGF.
[0135] The present invention also provides a method for preventing
or treating periodontal disease said method including administering
to a patient in need thereof an effective amount of BMGF, or
mutants, analogues and derivatives of BMGF, precursor and fusion
protein forms of BMGF and functionally active fragments (peptides)
of BMGF.
[0136] There is also provided a use of BMGF in a cosmetic
application, said use including administering to a patient in need
thereof an effective amount of BMGF, or mutants, analogues and
derivatives of BMGF, precursor and fusion protein forms of BMGF and
functionally active fragments (peptides) of BMGF.
[0137] The present invention also provides an infant formula
including BMGF, or mutants, analogues and derivatives of BMGF,
precursor and fusion protein forms of BMGF and functionally active
fragments (peptides) of BMGF; together with nutrient components.
Preferably the infant formula is a milk formula.
[0138] Throughout the description and claims of this specification,
the word "comprise" and variations of the word, such as
"comprising" and "comprises", is not intended to exclude other
additives, components, integers or steps.
[0139] The present invention will now be more fully described with
reference to the accompanying Examples and drawings. It should be
understood, however, that the description following is illustrative
only and should not be taken in any way as a restriction on the
generality of the invention described above.
IN THE FIGURES
[0140] FIG. 1 bovine cheese whey extract (GFE-2) prepared according
to Australian Patent AU645589 was subjected to acidification and
ultrafiltration and a series of chromatographic steps. The figure
illustrates the elution profile of protein and activity in an
EGF-receptor binding assay following each chromatographic step.
[0141] FIG. 2 illustrates SDS-PAGE of BMGF. Purified preparation of
BMGF was analysed with a Pharmacia Phastsystem on a 8-25% pre-cast
phast gel under reducing (R) or non-reducing (N) conditions then
visualised with silver stain. The numbers on the left of each
figure represent the migration positions of size standards; 94 kDa,
phosphorylase b; 67 kDa, albumin; 43 kDa, ovalbumin; 30 kDa,
carbonic anhydrase; 20.1 kDa, trypsin inhibitor; 14.4 kDa,
.alpha.-lactalbumin.
[0142] FIG. 3 illustrates amino acid sequence determination of
BMGF. X indicates an amino acid that was not identified by
amino-terminal sequencing. Deduced Sequence--SEQ ID NO: 2,
N-terminal Sequence--SEQ ID NO: 18, Endoproteinase Lys-C Peptides:
SEQ ID NOS: 19, and 6-9.
[0143] FIG. 4 illustrates deglycosylation of BMGF. Purified BMGF
was incubated in the presence and absence of NANase II,
O-glycosidase DS and PNGaseF, and analysed by SDS-PAGE on a 10-20%
Tricine gel (see example 3 for details).
[0144] FIG. 5 illustrates nucleotide sequence (SEQ ID NO: 1) and
deduced amino acid sequence (SEQ ID NO: 2) of BMGF.
[0145] FIG. 6 illustrates construction of the pGH(1-46)-Met-BMGF
expression vector. The arrow indicates the site for cyanogen
bromide cleavage to produce authentic BMGF. (SEQ ID NOS: 20-21)
[0146] FIG. 7 illustrates C4 reverse-phase HPLC of purified
recombinant authentic BMGF (A) and recombinant pGH (1-46)-Met BMGF
fusion protein (B). Insets: SDS-PAGE analysis of the purified
preparations under reducing (R) or non-reducing conditions (N).
[0147] FIG. 8 illustrates effect of BMGF on the proliferation of a
variety of cultured cell lines. Cell proliferation is shown as the
response to increasing concentrations of BMGF and is expressed as
the percent increase above cells incubated in the absence of BMGF.
Balb/c 3T3 cells and IEC-6 cells were incubated with increasing
concentrations of BMGF in the presence or absence of an
insulin-like growth factor-I analogue (LR.sup.3IGF-1) at a
concentration of 50 ng.ml.sup.-1.
[0148] FIG. 9 illustrates weight of the gastrointestinal tract
relative to total body weight (g/kg) of Sprague-Dawley rats
following infusion of BMGF (500 .mu.g/kg/day) or vehicle (0.1 M
acetic acid) for 7 days.
EXAMPLES
Example 1
Isolation of BMGF from Bovine Cheese Whey Extract
[0149] Step 1: Ultrafiltration Size Exclusion:
[0150] 6 L of bovine cheese whey extract (GFE-2 prepared according
to that outlined in AU645589) (protein concentration=40
mg.ml.sup.-1) is acidified to pH 2.5 with HCl and then
microfiltered against a 100 kDa polysulfonate exclusion membrane
fitted to a Sartorius Sartocon II crossflow filtration unit
(Sartorius, Gottingen, Germany). The permeate obtained from this
step is then diafiltered using an Amicon DC-10 ultrafiltration unit
(Amicon, Danvers, Mass.) equipped with a 0.1 .mu.m hollow fibre
cartridge and then concentrated to approximately 1.8 L against a 3
kDa cellulose triacetate membrane using the same unit.
[0151] Step 2: Anion Exchange Chromatography
[0152] The desalted and concentrated permeate is made 20 mM
Tris-HCl (pH 7.5) with solid Tris Base and pH adjustment to 7.5
with 5 M HCl, filtered through a 1 .mu.m membrane, and applied to a
Q-Sepharose column (5.times.15 cm; Pharmacia, Sweden) attached to
an FPLC system (Pharmacia, Sweden) at a flow rate of 5
ml.min.sup.-1. The column is then washed with 20 mM Tris-HCl and
the proteins that remain bound to the column (which include BMGF)
are eluted with a 2.1 L linear salt gradient of 0-0.6 M NaCl in 20
mM Tris-HCl at a flow rate of 5 ml min.sup.-1. Fractions of 30 ml
are collected and analysed for BMGF activity (FIG. 1). Activity was
measured as Epidermal Growth Factor (EGF) Receptor Binding
Activity. Fractions containing BMGF are pooled and then dialysed
for 16 h against H.sub.2O (2.times.20 L) followed by
freeze-drying.
[0153] EGF-Receptor Binding Assay.
[0154] BMGF was measured by competition for [.sup.125I]-rhEGF
binding to specific EGF receptors present on AG2804 fibroblasts.
Briefly, AG2804 cells were grown to 70-80% confluence in DMEM
supplemented with 10% FBS in 24-well plates. The cells were washed
twice with binding buffer (100 mM Hepes pH 7.6, 120 mM NaCl, 5 mM
KCl, 1.2 mM MgSO.sub.4.7H.sub.2O, 8 mM glucose, and 0.1% BSA) and
then incubated with column fractions and [.sup.125I]-rhEGF (10 000
cpm) in binding buffer for 18h at 40.degree. C. At the end of this
period, cells were washed three times with Hanks buffered salt
solution (HBSS) and lysed with 1 ml of 0.5 M NaOH, 0.1% Triton
X-100 for 30 min at room temperature. Radioactivities of cell
lysates were determined with a gamma counter. Total binding was
determined by adding binding buffer in place of column fractions.
Non-specific binding was determined by adding an excess (100 ng) of
unlabelled rhEGF in place of sample and was usually about 5% of
total binding. Standard curves for EGF competition were obtained by
using increasing amounts of unlabelled rhEGF (0.2-100 ng) in the
assay.
[0155] Step 3: Gel/Filtration Chromatography
[0156] The freeze-dried BMGF pool from the above step is
reconstituted in 15 ml 150 mM NaCl, 1 M glacial acetic acid, and
10% (v/v) CH.sub.3CN, filtered through a 0.22 .mu.m membrane and
applied to a Superdex-75 35/600 (3.5.times.60 cm, Amersham
Pharmacia Biotech, Sydney, Australia) column attached to the FPLC
at a flow rate of 3.5 ml.min.sup.-1. Fractions of 17.5 ml are
collected and analysed for BMGF activity (FIG. 1).
[0157] Step 4: C4 RP-HPLC
[0158] Fractions containing BMGF activity are pooled, diluted 1:4
with 0.1% TFA and applied to a Delta-Pack C4 RP-HPLC column (15
.mu.m, 300 .ANG., 25.times.100 mm, Millipore-Waters, Lane Cove, New
South Wales, Australia) equilibrated with 0.1% TFA. The column is
washed extensively with 0.1% TFA and bound protein then eluted with
a linear gradient of 0-80% CH.sub.3CN and 0.08% TFA over 80 min at
a flow rate of 5 ml.min.sup.-1. Fractions of 10 ml are collected
and those containing BMGF activity (FIG. 1) pooled and
freeze-dried.
[0159] Step 5: Affinity Chromatography
[0160] The freeze-dried pool from the above step is reconstituted
in 20 ml of 20 mM Tris-HCl (pH 7.5) and applied to a 5 ml HiTrap
Heparin-Sepharose affinity column (Amersham Pharmacia Biotech,
Sydney, Australia) attached to the FPLC at a flow rate of 0.5
ml.min.sup.-1. The column is washed with 20 mM Tris-HCl (pH 7.5)
until the O.D..sub.280nm returns to baseline and bound proteins are
then eluted with 75 ml of a linear salt gradient of 0-1M NaCl in 20
mM Tris-HCl (pH 7.5) (FIG. 1). Fractions of 1 ml are collected and
those containing BMGF activity pooled.
[0161] Step 6: C18 RP-HPLC #1
[0162] The Heparin-Sepharose pool is diluted 1:4 with 10% propanol,
0.13% HFBA and applied to a Nova-Pack C18 RP-HPLC column (4 .mu.m,
60 .ANG., 8.times.100 mm, Millipore-Waters, Lane Cove, New South
Wales, Australia) at a flow rate of 1 ml.min.sup.-1. The column is
washed with 10% propanol, 0.13% HFBA and bound protein eluted with
a two-step gradient of 10-24% propanol, 0.13% HFBA over 10 min and
then 24-40% propanol, 0.13% HFBA over 160 min. 1 ml fractions are
collected and analysed for BMGF activity (FIG. 1).
[0163] Step 7: C18 RP-HPLC #2
[0164] Fractions containing BMGF activity from the above step are
pooled, diluted 1:3 with 0.1% TFA and applied to the same column as
above at a flow rate of 1 ml.min.sup.-1. The column is washed with
0.1% TFA and bound protein eluted with a two step gradient of 0-10%
CH.sub.3CN over 10 min and then 10-35% CH.sub.3CN over 250 min at a
flow rate of 1 ml.min.sup.-1 (FIG. 1). 2 ml fractions are collected
and analysed for BMGF activity. The fractions containing BMGF
activity are pooled and stored at -20.degree. C. until
required.
[0165] A summary of the result of purification is shown in Table 1.
The BMGF is present in the active fractions from the final C18
RP-HPLC step in a form which is up to, and including, 99% pure and
has a molecular weight of approximately 21-25 kDa. These criteria
being estimated from silver stained 8-25% gradient SDS-PAGE gels
run on the Pharmacia Phast system (see FIG. 2) and by N-terminal
sequence analysis.
1TABLE 1 Summary of the purification of BMGF Total Specific Purifi-
Purification Protein Activity.sup.a Activity cation- Yield Step mg
Units Units/mg fold % GFE-2 209559.sup.b 5365.7 0.025 2 100.0
Permeate 16326.sup.b 2808.0 0.172 7 52.3 Q-Sepharose 2820.sup.b
924.9 0.328 13 17.2 Superdex-75 228.sup.b 372.2 1.630 63 6.9 C4
RP-HPLC 16.5.sup.b 157.6 9.502 371 2.9 Heparin 0.372.sup.c 138.7
376.0 14687 2.5 Sepharose C18 RP-HPLC#1 0.152.sup.c 48.0 320.0
12500 0.9 C18 RP-HPLC#2 0.029.sup.d 27.7 957.6 37406 0.5 .sup.aOne
unit of activity is defined as the amount of factor required to
compete for 50% of the binding of [.sup.125I]-rhEGF to AG2804
cells. .sup.bEstimated by BCA Protein assay kit (Pierce).
.sup.cEstimated by micro BCA Protein assay kit (Pierce).
.sup.dEstimated by N-terminal sequence analysis.
Example 2
N-Terminal and Peptide Sequence Analysis of BMGF
[0166] The amino acid sequence of bovine BMGF and of peptide
fragments generated by Endoproteinase Lys-C digestion was
determined using a Hewlett-Packard G1000A protein sequencer. Twenty
.mu.g of purified BMGF was reduced with 100 .mu.l 4 mM DTT in 400
.mu.l of denaturation buffer (6 M guanidine-HCl, 100 mM Tris-Cl pH
8.5, and 5 mM EDTA) for 30 min at room temperature in the dark. The
denatured and reduced BMGF was S-carboxymethylated by adding 1
.mu.mol of iodoacetic acid containing 200 .mu.Ci of
iodo[2-.sup.3H]acetic acid in denaturation buffer and incubated as
above. Subsequently 16 .mu.mol of iodoacetic acid dissolved in
denaturation buffer was added, and the incubation continued for a
further 15 min. The reaction was stopped by the addition of 5 .mu.l
TFA and the [.sup.3H]carboxymethylated BMGF recovered by RP-HPLC.
The [.sup.3H]carboxymethylated BMGF was dried under vacuum and
sequenced from the N-terminus or digested with endoproteinase Lys-C
(0.5 .mu.g, Promega) in 100 .mu.l Tris-HCl (pH 8.5) at 37.degree.
C. for 16 h. The reaction was stopped by the addition of 5 .mu.l
TFA and the resulting peptide fragments separated on a C18 RP-HPLC
column (2.1.times.30 mm, Brownlee Lab, Santa Clara, Calif.) using a
linear gradient of 0-50% CH.sub.3CN and 0.08% TFA over 70 min at a
flow rate of 0.25 ml.min.sup.-1. Peptide-containing fractions were
dried under vacuum and sequenced (FIG. 3).
Example 3
De-Glycosylation of BMGF
[0167] 10 .mu.g pure BMGF was dried in a 1.5 ml polypropylene
microfuge tube, resuspended in 16 .mu.l 50 mM sodium phosphate (pH
6.0), and 0.2 units .alpha.2-3,6-Neuraminidase (NANase 11) and
0.002 units Endo-.alpha.-acetylgalactosaminidase (O-glycosidase DS)
(BioRad, Richmond, Calif.) added and the reaction incubated at
37.degree. C. for 1 h. The reaction volume was then increased to 40
.mu.l with 500 mM sodium phosphate (dibasic) and 2.5 .mu.l 2% SDS,
1 M .beta.-mercaptoethanol added. The reaction was then heated to
95.degree. C. for 5 min. placed on ice and then 2.5 .mu.l
Nonidet-P40 and 0.005 units
Peptide-N.sup.4(acetyl-.beta.-glucosaminyl)-asparagine aminidase
(PNGase F) (BioRad, Richmond, Calif.) added and incubated at
37.degree. C. for 3 h. The reaction was then dried under vacuum,
resuspended in SDS-PAGE reducing buffer and run on a 10-20% Tricine
gel (Novex) (FIG. 4).
Example 4
Cloning of the Mature Form of BMGF
[0168] Two oligonucleotide primers (see below) corresponding to the
N- and C-terminal amino acid sequences of BMGF (see example 2) were
chemically synthesised and used to amplify by PCR (Polymerase Chain
Reaction) the nucleotide sequence encoding the complete mature form
of bovine BMGF.
[0169] Primer 1: 5' GAT GGG MT TCA ACC AGA AGT CCT GAA 3' (SEQ ID
NO: 17)
[0170] Primer 2: 5' GTA AAA CM GTC MC TCT CTC ACA CCT 3' (SEQ ID
NO: 16)
[0171] Total RNA was isolated from 80-90% confluent Madin-Darby
bovine kidney cells (MDBK, ATCC CCL 22) using a RNeasy Mini kit
(Qiagen, Clifton Hill, Victoria, Australia). cDNA was synthesised
from 1 .mu.g total MDBK RNA using oligo dT primer and Superscript
II (Life Technologies, Melbourne, Australia). The subsequent cDNA
was used as a template for PCR with the above primers. PCR was
carried out in 50 .mu.l of 60 mM Tris-SO.sub.4, 18 mM
(NH.sub.4).sub.2SO.sub.4, 1.5 mM MgSO.sub.4 (pH 9.1), 0.2 mM dNTPs,
200 ng each primer, 1 U eLONGase (Life Technologies, Melbourne,
Australia) and 1 ul cDNA. Following an initial incubation at
94.degree. C. for 3 min. 30 cycles of amplification were carried
out as follows: 94.degree. C. for 1 min. 50.degree. C. for 1 min.
and 68.degree. C. for 1 min. followed by a final 3 min extension at
68.degree. C.
[0172] The PCR reaction was analysed by electrophoresis through a
2% agarose gel and a 240 bp product excised from the gel and
purified using a Promega PCR Preps Purification kit. The purified
PCR product was blunt-end ligated into the vector pCR-Blunt
(Invitrogen) according to the manufacturer's instructions. BMGF
vector constructs were transformed into E. coli TOP10 (Invitrogen)
cells and selected on LB agar plates containing 50 .mu.g ml.sup.-1
kanamycin. The complete nucleotide sequence of mature BMGF (see
FIG. 5) was determined by the dideoxy chain termination method
(Sanger et al. Proc. Natl. Acad. Sci. U.S.A. 74, 5463, (1977))
using an Amplicycle sequencing kit (Perkin-Elmer) and universal M13
forward (-40) and reverse primers.
Example 5
Construction of BMGF cDNA Expression Plasmid of E. coli
[0173] To obtain the DNA encoding the complete 80 amino acids of
mature BMGF [Asp.sup.1-Tyr.sup.80] for expression vector
construction, total RNA was isolated from 80-90% confluent
Madin-Darby bovine kidney cells (MDBK, ATCC CCL 22) using an RNeasy
Mini kit (Qiagen, Clifton Hill, Victoria, Australia). cDNA was
synthesised from 1 .mu.g total MDBK RNA using oligo dT primer and
Superscript II (Life Technologies, Melbourne, Australia). The
subsequent cDNA was used as a template for PCR with the primers
shown below. The PCR conditions were the same as described
above.
[0174] Primer 1: 5' ATC TAG GTT ACC ATG GAT GGG MT TCA ACC AGA 3'
(SEQ ID NO: 11)
[0175] Underlined: Hpa I restriction enzyme site
[0176] Double underlined: Methionine site for cleavage with
cyanogen bromide
[0177] Bold: nucleotide sequence of amino acids 1-6 of BMGF
[0178] Primer 2: 5' CTA GAT MG CTT TCA TCA GTA MA CM GTC MC TCT 3'
(SEQ ID NO: 12)
[0179] Underlined: Hind III restriction enzyme site
[0180] Double underlined: two stop codons
[0181] Bold: nucleotide sequence of amino acids 75-80 of BMGF
[0182] The PCR reaction was analysed by electrophoresis through a
2% agarose gel and a 273 bp product excised from the gel and
purified using a Promega PCR Preps Purification kit. The purified
PCR product and the plasmid p[Met.sup.1]-pGH(1-46) were digested
with the restriction enzymes Hpa I and Hind lIl (New England
Biolabs) and the digested PCR product ligated into linearised
p[Met.sup.1]-pGH(1-46) with T4 DNA ligase (Promega) (FIG. 6).
p[Met.sup.1]-pGH(1-46) is an expression vector (owned and patented
by GroPep Pty Ltd) which contains the nucleotide sequence encoding
the first 46 amino acids of methionyl porcine growth hormone
downstream of the strong tee promoter. Vector constructs were
transformed into E. coli JM101 (lac.sup.q) and selected on LB agar
plates containing 50 .mu.G ml.sup.-1 ampicillin.
Example 6
Purification of Authentic BMGF Produced by an E. coli
Transformant
[0183] The E. coli JM101 strain harbouring the plasmid
[Met.sup.1]-pGH(1-46)-Met-BMGF was selected as a single colony and
used to inoculate a 20 ml starter culture consisting of 60 mM
K.sub.2HPO.sub.4, 33 mM KH.sub.2PO4, 7.5 mM
(NH.sub.4).sub.2SO.sub.4, 1.7 mM sodium citrate, 10PM
MgSO.sub.4.7H.sub.2O, 0.2% D-glucose, 0.0005% thiamine and 50 .mu.g
ml.sup.-1 ampicillin. The culture was grown at 37.degree. C. for 16
h. The starter culture was then in turn used to inoculate two 5 L
fermenters (Applicon) containing in each 3 L of growth medium (30
mM NH.sub.4Cl, 7 mM K.sub.2SO.sub.4, 12 mM KH.sub.2PO.sub.4, 19 mM
Na.sub.2HPO.sub.4, 139 mM D-glucose, 2.4 mM MgSO.sub.4.7H.sub.2O,
0.0004% thiamine, 0.035 mM Fe(II)SO.sub.4.7H.sub.2O, 0.0074 mM
MnSO.sub.4.7H.sub.2O, 0.0008 mM CuSO.sub.4.7H.sub.2O, 0.074 mM
tri-sodium citrate and 50 ug.ml.sup.-1 ampicillin pH 6.9). Bacteria
were grown at 37.degree. C. until the absorbance at 600 nm reached
an O.D. of 4.0 and then induced with 0.33 mM IPTG and the
cultivation was continued until glucose became limiting indicated
by a sharp rise in pH. Regulation of temperature, pH and oxygen was
under automatic control (FC4 Data system, Real Time Engineering).
Cells were disrupted at 5000 p.s.i. following five passes through a
Rannie homogeniser and inclusion bodies collected by centrifugation
(10 000 rpm, 25 min. 4.degree. C.). The inclusion bodies were
washed twice with 30 mM NaCl, 10 mM KH.sub.2PO.sub.4, 0.5 mM
ZnCI.sub.2 by centrifugation at 6000 rpm and stored at -80.degree.
C.
[0184] Washed inclusion bodies, in 20 g batches, were thawed,
suspended at 10% (w/v) in 8 M urea, 0.1 M Tris-HCl, 40 mM glycine,
40 mM dithiothreitol and 0.5 mM ZnCI.sub.2 (pH 9.0) and stirred for
30 min at room temperature. The solubilised inclusion bodies were
centrifuged at 14 000 rpm for 20 min and the resultant supernatant
desalted on a Pharmacia-LKB XK column packed with Cellufine
GCL-1000 and equilibrated with 8 M urea, 0.1 M Tris-HCl, 40 mM
glycine, 1.6 mM dithiothreitol and 0.5 mM ZnCI.sub.2 (pH 9.0) at a
flow rate of 2 ml.min.sup.-1. 30 ml fractions were collected and
those containing recombinant [Met.sup.-1]-pGH(146)-Met-BMGF fusion
protein were pooled and subject to oxidative refolding by diluting
the pool to a final protein concentration of 0.1 mg.m.sup.-1 in 4 M
urea, 40 mM glycine, 0.1 M Tris-HCl, 5 mM EDTA, 0.4 mM DTT and 1 mM
2-hydroxethyl disulphide, pH 9.0. After stirring for 3 h at room
temperature, the reaction was stopped by pH adjustment to 6.45 with
HCl.
[0185] The refolded fusion protein was next loaded onto a
Pharmacia-LKB XK50 column packed with 100 ml of Sepharose Fast Flow
S (Amersham Pharmacia Biotech, Sydney, Australia) equilibrated with
8 M urea, 50 mM ammonium acetate (pH 6.45) at a flow rate of 15
ml.min.sup.-1. The column was washed with the above buffer until
OD.sub.280nm returned to baseline. The column was then eluted with
a linear salt gradient of 0-0.7 M NaCl in the same buffer at a flow
rate of 15 ml.min.sup.-1. Fractions of 30 ml were collected and
those containing fusion protein pooled.
[0186] The fusion protein pool was desalted and further purified by
reverse-phase HPLC chromatography on a C4 Prep-Pak column (40
mm.times.100 mm; 300 A, 15 .mu.m; Millipore-Waters, Lane Cove, New
South Wales, Australia). The protein pool was adjusted to 0.1% TFA
and loaded onto the C4 column at 50 ml.min.sup.-1. The column was
washed extensively with 0.1% TFA and protein eluted with a gradient
of 18-50% (v/v) acetonitrile over 90 min in the presence of 0.08%
TFA at a flow rate of 20 ml.min.sup.-1. Fractions of 30 ml were
collected and those containing fusion protein pooled and
lyophilised.
[0187] Analysis of the fusion protein pool by microbore C4
reverse-phase HPLC (2.1 mm.times.100 mm, Brownlee Lab, Santa Clara,
Calif.) identified a single protein peak (FIG. 7B). The purity of
the BMGF preparation was further confirmed by N-terminal sequence
analysis which gave the expected N-terminal sequence for BMGF with
an approximate purity of >99%. A single protein of the expected
14.5 kDa was also detected following SDS-PAGE under reducing or
non-reducing conditions (FIG. 7B, inset).
[0188] To produce authentic BMGF the fusion protein was cleaved by
solubilising the lyophilised protein in 0.13 M HCl containing a
100-fold molar excess of cyanogen bromide at a protein
concentration of 10 mg.ml.sup.-1. The cleavage reaction was
performed at room temperature in the dark for 25 h. Following
cleavage, cyanogen bromide was removed from the reaction by
ion-exchange chromatography as described above, except that the
protein was eluted from the column batchwise with 1 M NaCl.
[0189] Authentic BMGF was separated from its fusion partner by
reverse phase HPLC. The S-Sepharose protein pool was diluted 1:4
(v/v) with 0.1% TFA and applied to a C4 Prep-Pak column (25
mm.times.100 mm; 300 A, 15 .mu.m; Millipore-Waters, Lane Cove, New
South Wales, Australia) at a flow rate of 50 ml.min.sup.-1. The
column was washed with 0.1% TFA until OD.sub.280nm returned to
baseline and the column then eluted with a gradient of 16-32% (v/v)
acetonitrile over 150 min in the presence of 0.08% TFA at a flow
rate of 20 ml.min.sup.-1. Fractions of 26 ml were collected and
those containing pure BMGF pooled.
[0190] Analysis of the final BMGF protein pool by microbore C4
reverse-phase HPLC (2.1 mm.times.100 mm) identified a single
protein peak (FIG. 7A). The purity of the BMGF preparation was
further confirmed by N-terminal sequence analysis which gave the
expected N-terminal sequence for BMGF with an approximate purity of
>99%. The molecular mass of recombinant BMGF determined by
electrospray ionization mass spectrometry was 8995.1.+-.0.83. This
is consistent with the theoretical mass of 8995.02 calculated from
the BMGF amino acid sequence. Following SDS-PAGE a single protein
peak of approximately 9 kDa was detected under reducing or
non-reducing conditions (FIG. 7A, inset).
Example 7
Mitogenic Activity of BMGF
[0191] The mitogenic activity of authentic BMGF and recombinant
BMGF on a range of cell lines was determined by methylene blue cell
proliferation assay. Cell lines were sub-cultured in 96 well plates
at a density of 10-20.times.10.sup.4 cells.ml.sup.-1 and incubated
overnight at 37.degree. C., 5% CO.sub.2. Plates were then washed
extensively with DMEM to remove any residual medium after which
either native or recombinant BMGF was added at various
concentrations (see FIG. 8). In some cell lines the effect of
co-incubation of BMGF with 50 ng.ml.sup.-1 LR.sup.3IGF-1 was
tested. After incubation for 48 h, the plates were washed twice
with 0.15 M NaCl, the monolayers of cells fixed, and the cell mass
quantified by addition of 1% methylene blue and measuring
Absorbance at 600 nm. Both the BMGF isolated from bovine cheese
whey extract and BMGF produced recombinantly were equipotent in
Balb/c 3T3 cells. BMGF stimulated a wide range of cell lines
covering both epithelial cells, fibroblastic cells and osteoblast
cells. These include Balb/c 3T3, HaCaT, IEC-6, SF3169, Mv1Lu and
CaIOst cells. In some cases, for example in the gut epithelial cell
line IEC-6, BMGF acted synergistically with LR.sup.3IGF-1.
Example 8
Effect of BMGF on Gut Growth
[0192] Osmotic mini-pumps containing vehicle (0.1 M acetic acid) or
BMGF (500 .mu.g/kg/day) were implanted subcutaneously in the
suprascapular region of male Sprague Dawley rats (8 animals per
treatment group) and the rats kept in Tecniplast metabolism cages
for 7 days in an environment maintained at 25.degree. C. with a 12
h light/dark cycle. Animals had a continual access to water and a
high carbohydrate diet. After 7 days the rats were sacrificed by
CO.sub.2 overdose and total gut weight measured. Total gut weight
per kilogram body weight was 25% higher after 7 days in rats
treated with BMGF (FIG. 9).
Example 9
Cream (O/W-Type)
[0193] Ingredients: %
[0194] Sorbitan monostearate 2.0
[0195] Polyoxyethylene sorbitanmonostearate 3.0
[0196] Cetyl alcohol 5.0
[0197] Light liquid paraffin 8.0
[0198] Isopropyl myristate 2.0
[0199] Active substance BMGF 1.0-10.sup.-5
[0200] Propylene glycol 2.0
[0201] Glycerin 2.0
[0202] Deionised water 76
[0203] Preservatives and other q.s. stabilisers
[0204] Heat the aqueous phase to 55-60.degree. C., dissolve the
active substance in it, and disperse the melted lipid phase in it
by vigorous stirring. Cool to room 5 temperature and homogenise. In
a similar manner a cream comprising 0.4, 4, or 20 .mu.g.ml.sup.-1,
respectively, can be produced. Of this cream 100 ul.cm.sup.-2 of
wound is applied.
Example 10
Ointment (W/O-Type)
[0205] Ingredients: %
[0206] Sorbitan trioleate 5.0
[0207] Wax, microcrystalline 3.0
[0208] Light liquid paraffin 9.0
[0209] Isopropyl myristate 10.0
[0210] Lanolin alcohols 3.0
[0211] Active substance BMGF 1.0-0-5
[0212] Propylene glycol 2.0
[0213] Glycerin 2.0
[0214] Magnesium sulphate, hydrous 0.7
[0215] Deionised water 65.3
[0216] Preservatives and other q.s.
[0217] Dissolve the active substance in the aqueous phase with
gentle heating, and disperse the solution in the melted lipid
phase. Cool to room temperature and 5 homogenise. In a similar
manner an ointment 100 .mu.l.cm.sup.-2 of wound is applied.
Example 11
Mouthwash
[0218] Ingredients: %
[0219] Active substance BMGF 1.0-10.sup.-3
[0220] Polyethylene glycol(7)-glycerol cocoate 2.0
[0221] Deionised water 13
[0222] Glycerin (86%) 18
[0223] Peppermint oil 10
[0224] Ethanol 55
[0225] Preservatives and other q.s.
[0226] Dissolve the active substance in deionised water. Add and
dissolve PEG(7)-glyceryl cocoate and glycerin n the solution.
Dissolve peppermint oil in ethanol and mix the two solutions with
stirring. The solution is to be diluted up to 1:10 before use.
Example 12
Parenteral Solution Ingredients
[0227] Active substance BMGF 1 mg.ml.sup.-1
[0228] .+-.Human Serum Albumin 1 mg.ml.sup.-1
[0229] Arginine or Glycine 20 mg.ml.sup.-1
[0230] .+-.Carbohydrate 5-20 mg.ml.sup.-1
[0231] pH 7
[0232] The carbohydrate is glucose, mannose, dextran, hydroxyethyl
starch or a mixture thereof. The pH is adjusted with phosphate,
succinate, amino acids or a mixture thereof.
[0233] Vials with 0.5 mg BMGF/0.5 ml are made and lyophilised.
Example 13
Toothpaste
[0234] Active substance BMGF 1.0-10.sup.-5 g
[0235] Methyl cellulose 0.8 g
[0236] Calcium carbonate 30 g
[0237] Colloidal silica 3 g
[0238] Light liquid paraffin 2 g
[0239] Glycerin 20 g
[0240] Sweetening agent
[0241] Flavouring agent
[0242] Preservatives
[0243] Deionised water to 100 g
[0244] The powders are wetted with the mixture of the active
substance and methyl cellulose in a part of deionised water,
paraffin and glycerin. The additives are added in solution. After
making up with the remaining water the paste is homogenised.
[0245] Finally, it is to be understood that various alterations,
modifications and/or additions may be made without departing from
the spirit of the present invention as outlined herein.
* * * * *