U.S. patent application number 09/902460 was filed with the patent office on 2003-02-27 for recombinant fibroblast growth factors.
Invention is credited to Abraham, Judith A., Fiddes, John C., Protter, Andrew.
Application Number | 20030040042 09/902460 |
Document ID | / |
Family ID | 23825960 |
Filed Date | 2003-02-27 |
United States Patent
Application |
20030040042 |
Kind Code |
A1 |
Fiddes, John C. ; et
al. |
February 27, 2003 |
Recombinant fibroblast growth factors
Abstract
The DNA sequences encoding analogs of human acidic and basic
fibroblast growth factors (FGF) can be recombinantly expressed to
obtain practical amounts of proteins useful in effecting both
pathologies related to persistent angiogenesis and wound healing
and related tissue repair.
Inventors: |
Fiddes, John C.; (Palo Alto,
CA) ; Abraham, Judith A.; (Sunnyvale, CA) ;
Protter, Andrew; (Palo Alto, CA) |
Correspondence
Address: |
BURNS, DOANE, SWECKER & MATHIS, L.L.P.
P.O. Box 1404
Alexandria
VA
22313-1404
US
|
Family ID: |
23825960 |
Appl. No.: |
09/902460 |
Filed: |
July 9, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09902460 |
Jul 9, 2001 |
|
|
|
09098628 |
Jun 16, 1998 |
|
|
|
6294359 |
|
|
|
|
09098628 |
Jun 16, 1998 |
|
|
|
07459739 |
Feb 12, 1990 |
|
|
|
5859208 |
|
|
|
|
07459739 |
Feb 12, 1990 |
|
|
|
07070797 |
Jul 7, 1987 |
|
|
|
07070797 |
Jul 7, 1987 |
|
|
|
07050706 |
May 15, 1987 |
|
|
|
07050706 |
May 15, 1987 |
|
|
|
06869382 |
May 30, 1986 |
|
|
|
06869382 |
May 30, 1986 |
|
|
|
06809163 |
Dec 16, 1985 |
|
|
|
5439818 |
|
|
|
|
06809163 |
Dec 16, 1985 |
|
|
|
06775521 |
Sep 12, 1985 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/503 20130101;
C12N 15/67 20130101; A61K 38/1825 20130101; C07K 14/50 20130101;
C12N 2830/00 20130101; C12N 2830/55 20130101; C07H 21/04 20130101;
C07K 14/501 20130101; C12N 2830/15 20130101; C12N 15/85
20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 530/350; 536/23.5 |
International
Class: |
C07K 014/475; C07H
021/04; C12P 021/02; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 9, 1986 |
CA |
518137 |
Claims
1. A recombinant DNA sequence encoding an analog of mammalian
FGF.
2. The DNA sequence of claim 1 which encodes a human FGF protein
analog.
3. The DNA sequence of claim 2 which encodes a human basic FGF
protein analog.
4. The DNA sequence of claim 3 which encodes a human basic FGF
protein analog with reduced affinity for heparin binding.
5. The DNA sequence of claim 4 encoding a human basic FGF protein
analog, comprising substituting one or more positively charged
amino acid residues located in a heparin binding domain
encompassing residues 128 through 138 with a neutral or negatively
charged amino acid.
6. The DNA sequence of claim 5 wherein the neutral or negatively
charged amino acid is selected from the group consisting of serine,
threonine or glutamic acid.
7. The DNA sequence of claim 5 wherein the location and composition
of the substituted amino acid is selected from the group consisting
of serine.sub.128, glutamic acid.sub.128, threonine.sub.129,
serine.sub.128/threonine.sub.129, and serine.sub.138.
8. The DNA sequence of claim 2 which encodes a human basic FGF
protein analog wherein one or more cysteine residues are replaced
by a neutral amino acid and said protein analog exhibits the
biological activity of native basic FGF.
9. The DNA sequence of claim 8 wherein the neutral amino acid is
serine or alanine.
10. The DNA sequence of claim 9 wherein the substituted cysteine
residue is at position 78, 96 or a combination thereof.
11. The DNA sequence of claim 5 which encodes a human basic FGF
protein analog wherein said analog binds to a receptor for FGF and
has reduced ability to induce a biological response.
12. The DNA sequence of claim 3 which encodes an amino-terminal
deletion analog of FGF having FGF antagonist activity.
13. The DNA sequence of claim 12 wherein said deletion spans
residues 1 through 24 of human basic FGF.
14. The DNA sequence of claim 12 encoding a human basic FGF analog
further comprising one or more positively charged amino acid
residues located in a heparin binding domain encompassing residues
128 through 138 substituted with a neutral or negatively charged
amino acid.
15. The DNA sequence of claim 3 which is operably linked to control
sequences for expression.
16. The DNA sequence of claim 15 wherein the control sequences
include a transcription termination signal.
17. The DNA sequence of claim 3 which is transformed into a
recombinant host cell.
18. A recombinant vector containing the DNA sequence of claim 3 and
effective in expressing FGF or an analog thereof.
19. The vector of claim 18 which is selected from the group
consisting of plasmids pUC9-TSF11 and pUC9delH3-pTSF-3.
20. The vector of claim 18 wherein the DNA sequence encoding an FGF
analog is operably linked to control sequences compatible with
bacteria.
21. The vector of claim 18 wherein the DNA sequence encoding an FGF
analog is operably linked to control sequences compatible with
mammalian hosts.
22. Recombinant host cells transformed with the vector of claim
18.
23. Bacterial cells transformed with the vector of claim 20.
24. Mammalian cells transformed with the vector of claim 21.
25. A method for producing FGF protein analogs which comprises
culturing host cells harboring the DNA of claim 3 and recovering
the FGF protein analog.
26. The method of claim 25 wherein the host cells are
bacterial.
27. The method of claim 25 wherein the host cells are
mammalian.
28. A human basic FGF protein analog having reduced affinity for
heparin binding comprising substituting one or more positively
charged amino acid residues located in a heparin binding domain
encompassing residues 128 through 138 with a neutral or negatively
charged amino acid.
29. A human basic FGF protein analog wherein the cysteine at
positions 78, 96, or a combination thereof, is replaced by a
neutral amino acid and said analog exhibits the biological activity
of native, human basic FGF.
30. The human basic FGF protein analog of claim 29 which is
bFGF-C78/96S.
31. An antagonist of human basic FGF.
32. The FGF antagonist of claim 31 wherein the first 24 amino
terminal residues of basic FGF are deleted.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This is a continuation-in-part of U.S. Ser. No. 070,797,
filed Jul. 7, 1987, which is a continuation-in-part of U.S. Ser.
No. 050,706, filed May 15, 1987, which is a continuation-in-part of
U.S. Ser. No. 869,382 filed May 30, 1986, which is a
continuation-in-part of U.S. Ser. No. 809,163, filed Dec. 16, 1985,
which is a continuation-in-part of U.S. Ser. No. 775,521, filed
Sep. 12, 1985.
TECHNICAL FIELD
[0002] The invention relates to recombinant production of growth
factors important for constructing vascular systems in healing
tissues and inhibiting abnormal persistent angiogenesis. In
particular, analogs of genes encoding human basic and acidic
fibroblast growth factors (FGF) are cloned and expressed.
BACKGROUND ART
[0003] The process of healing when tissue is subjected to trauma,
such as wounding or burns, is an extremely complex one, but it is
known to be mediated by a number of protein factors. These factors
are essential to the growth and differentiation of the cells which
serve to replace the tissue destroyed. A number of candidate
factors have been identified on the basis of the ability of
extracts from various tissues, such as brain, pituitary, and
hypothalamus, to stimulate the mitosis of cultured cells. Numerous
shorthand names have been applied to the active factors in these
extracts, including platelet-derived growth factor (PDGF),
macrophage-derived growth factor (MDGF), epidermal growth factor
(EGF), tumor angiogenesis factor (TAF), endothelial cell growth
factor (ECGF), fibroblast growth factor (FGF), hypothalamus-derived
growth factor (HDGF), retina-derived growth factor (RDGF), and
heparin-binding growth factor (HGF). (See, for example, Hunt, T.
K., J Trauma (1984) 24:S39-S49; Lobb, R. R., et al, Biochemistry
(1984) 23:6295-6299).
[0004] Folkman, J., et al, Science (1983) 221:719-725, reported
that one of the processes involved in wound healing, the formation
of blood vessels, is profoundly affected in tumors by heparin. From
this and other studies, it is clear that heparin specifically binds
to protein(s) associated with a number of these growth factor
activities, and therefore heparin has been used as a purification
tool. It has been shown that the affinity of some growth factors
for heparin is independent of overall ionic charge, since both
positively and negatively charged factors are bound (Maciag, T., et
al, Science (1984) 225:932-935; Shing, Y., et al, Science (1984)
223:1296-1299; Klagsbrun, M., et al, Proc Natl Acad Sci (USA)
(1985) 82:805-809). The capacity to bind or not to bind to heparin
is one measure of differentiation between the activities in the
various extracts. For example, EGF and PDGF do not bind strongly to
heparin; in fact, EGF does not bind to heparin at all. The other
factors above do show strong heparin binding. However, it is
believed that acidic brain FGF, ECGF, RDGF, and HGF-alpha are in
fact the same factor. Similarly, it is also believed that pituitary
FGF, cationic brain FGF, TAF, and HGF- are the same protein. (Lobb,
R. R., et al (supra)). A summary and comparison of thirteen
endothelial growth factors which have been purified using heparin
affinity is found in Lobb, R., et al, J Biol Chem (1986)
261:1924-1928.
[0005] Using heparin affinity chromatography, basic fibroblast
growth factors exhibiting a potent mitogenic activity for capillary
endothelium have been isolated from rat chondrosarcoma (Shing, Y.,
et al, supra) and from bovine cartilage (Sullivan, R., et al, J
Biol Chem (1985) 260:2399-2403). Thomas, K. A, et al, Proc Natl
Acad Sci (USA) (1984) 81:357-361, U.S. Pat. No. 4,444,760, purified
two heterogeneous forms of an acidic bovine brain fibroblast growth
factor having molecular weights of 16,600 and 16,800 daltons.
Gospodarowicz and collaborators have shown the presence in both
bovine brains and pituitaries of basic fibroblast growth factor
activities and purified these proteins using heparin-affinity
chromatography in combination with other purification techniques
(Bohlen, P., et al, Proc Natl Acad Sci (USA) (1984) 81:5364-5368;
Gospodarowicz, D., et al (ibid) 6963-6967). These factors also have
molecular weights of approximately 16 kd, as does a similar factor
isolated from human placenta (Gospodarowicz, D., et al, Biochem
Biophys Res Comm (1985) 128:554-562).
[0006] The complete sequence for basic FGF derived from bovine
pituitary has been determined (Esch, F., et al, Proc Natl Acad Sci
(USA) (1985) 82: 6507-6511). Homogeneous preparations were obtained
and showed potent mitogenic activity in in vitro assays with
endothelial cells (basic FGF has an ED.sub.50 of 60 pg/ml).
[0007] Acidic FGF has an ED.sub.50 of about 6,000 pg/ml. An
N-terminal sequence for acidic FGF derived from bovine brain tissue
was determined by Bohlen, P., et al, EMBO J (1985) 4:1951-1956.
Gimenez-Gallego, G., et al, determined the N-terminal sequences for
both acidic and basic FGF prepared from human brain, and compared
them to the bovine sequences (Biochem Biophys Res Comm (1986)
135:541-548). Their results are consistent with those disclosed
herein. Also, the complete amino acid sequence of bovine
brain-derived acidic FGF was determined from the isolated protein
(Gimenez-Gallego, G., et al, Science (1985) 230:1385-1388; Esch,
F., et al, Biochem Biophys Res Comm (1985) 133:554-562). These two
determinations are in agreement with the exception of a single
amino acid. However, Esch et al later reported that their sequence
is in agreement with that of Gimenez-Gallego et al. The complete
amino acid sequence of human acidic FGF was deduced from the gene
(Jaye, M., et al, Science (1986) 233:541-545 and the complete human
protein sequence was also determined by Gimenez-Gallego, G., et al,
Biochem Biophys Res Comm (1986) 138:611-617 and Harper, J. W., et
al, Biochem (1986) 25:4097-4103).
[0008] The FGF proteins described above and other growth factors
are clearly effective in promoting the healing of tissue subjected
to trauma (see, e.g., Sporn, M. B., et al, Science (1983)
219:1329-1331; Davidson, J. M., et al, J.C.B. (1985) 100:1219-1227;
Thomas, K. A., et al, Proc Natl Acad Sci (USA) (1985)
82:6409-6413). Davidson, et al, (supra) specifically discloses the
efficacy of FGF in wound healing. The basic FGF native proteins
have been alleged to be useful in treatment of myocardial
infarction (Svet-Moldavsky, G. J., et al, Lancet (Apr. 23, 1977)
913; U.S. Pat. Nos. 4,296,100 and 4,378,347). In addition, human
basic FGF has been found to increase neuronal survival and neurite
extension in fetal rat hippocampal neurons (Walicke, P., et al,
Proc Natl Acad Sci (USA) (1986) 83:3012-3016), suggesting that this
factor may also be useful in the treatment of degenerative
neurological disorders, such as Alzheimer's disease and Parkinson's
disease.
[0009] The FGF proteins described above provide an effective means
to promote the repair of traumatized tissue as a result of
wounding, surgery, burns, fractures or neurological degeneration.
However, data is accumulating regarding certain properties of these
growth factors which suggests that agonists of FGF may be more
therapeutically effective than the native FGF proteins for tissue
repair, and in certain circumstances that FGF antagonists may also
be useful therapeutically.
[0010] For example, agonists of FGF which have greater biological
activity as compared to native FGF would be more desirable for use
in the wound healing indications described above. In contrast,
antagonists of FGF would be extremely useful in therapies where
neovascularization is a dominant pathology and it would be
therapeutically useful to inhibit the process of angiogenesis.
Therefore, it would also be desirable to construct FGF analogs
which antagonize the effects of native FGF thereby inhibiting
angiogenesis.
[0011] It is considered desirable to provide modifications to the
native FGF DNA sequences reported for these growth factors in order
to isolate the regions of the protein responsible for the distinct
biological activities or regions important in the interactions of
the factor with the cellular environment. Having determined the
appropriate region or site of the specific interaction, structural
analogs can be created which preserve certain activities, e.g.
wound healing activity, while reducing or eliminating undesirable
functions, such as the sequestration of FGF in the extracellular
matrix.
[0012] It would also be desirable to insure the availability of
these FGF protein analogs in large quantities and in a form free
from any toxic or infectious impurities. The human form of the
protein is preferred, and perhaps required, for therapeutic use.
Since the DNA sequences encoding the proteins for both human acidic
and basic FGF have been cloned and expressed by recombinant DNA
techniques, site-directed mutagenesis may be employed to produce a
variety of acidic and basic FGF analogs. The invention herein
provides the means whereby acidic and basic FGF analogs can be
obtained in practical quantities and in pure, uncontaminated
form.
DISCLOSURE OF THE INVENTION
[0013] The invention provides the tools for synthesis and
manipulation of fibroblast growth factor analogs useful in
effecting accelerated healing of wounds, bone fractures, burn
tissue, damaged myocardial tissue, degenerated neurological tissue,
or other trauma. Concurrently, fibroblast growth factor
antagonists, such as angiogenesis inhibitors, which would be useful
for treatment of diseases common to ophthalmology, dermatology and
rheumatology where neovascularization is a dominant pathology, and
in certain neoplasms that include, but are not limited to, the most
highly angiogenic, such as brain tumors, are also provided. Cloning
and expression of the genes encoding these analogs are provided by
the methods and materials of the invention.
[0014] In one aspect, the invention relates to recombinant DNA
sequences encoding analogs of human acidic and basic FGF (human
aFGF and human bFGF). In other aspects, the invention relates to
recombinant vectors bearing these DNA sequences, to host cells
transformed with such vectors and harboring these DNA sequences,
and to the recombinant proteins produced by these transformed
cells. In yet other aspects, the invention relates to methods of
producing these fibroblast growth factor analogs using recombinant
techniques.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1 and 2 show the native DNA sequences encoding, and
the deduced amino acid sequences of, human basic FGF and acidic
FGF, respectively.
[0016] FIG. 3 shows a comparison of the amino acid sequences for
human acidic and basic FGF and the various regions targeted for
alteration, including potential heparin-binding domains and
receptor-binding regions.
[0017] FIG. 4 shows the construction of a synthetic tryptophan
operon promoter and operator regulatory sequence, and a restriction
site map of plasmid pTRP-233.
[0018] FIG. 5 is a flow chart of the construction of plasmid
pUC9delH3-pTSF-3.
[0019] FIG. 6 is an illustration of the procedure used to insert
any of the FGF analog gene sequences into the expression vector
pUC9delH3- pTSF-3.
[0020] FIG. 7 shows the results of wild type bFGF as compared to
the double cysteine substituted FGF analog, bFGF-C78/96S, using a
high performance liquid chromatography (HPLC) heparin affinity
column. FIG. 7 shows the elution of 10 ug of reduced (FIG. 7a) and
nonreduced (FIG. 7b) bFGF-C78/96S from a heparin HPLC column
developed with a NaCl gradient (0.6 M-3.0 M). A similar experiment
using purified wild type bFGF under reduced (FIG. 7c)
and-nonreduced (FIG. 7d) conditions is provided for comparison.
MODES OF CARRYING OUT THE INVENTION
[0021] A. The Fibroblast Growth Factors
[0022] Two different bovine (and analogous human) fibroblast growth
factors have been purified to homogeneity by others and partially
or completely sequenced. Both factors are capable of mitogenic
activity in in vitro assays using cultured cells, such as bovine
brain and adrenal cortex-derived capillary endothelial cells, human
umbilical vein endothelial cells, bovine adrenal cortex
steroidogenic cells, granulosa cells, and vascular smooth muscle
cells. In vitro assays employing these cell cultures have been
described by Gospodarowicz, D., et al, J Cell Physiol (1985)
122:323-332; and Gospodarowicz, D., et al, J Cell Biol (1983)
97:1677-1685. More recently, alternative in vitro assays have been
described by Esch et al, Proc Natl Acad Sci (USA) (1985)
82:6507-6511; and by Gospodarowicz, D., et al, J Cell Physiol
(1986) 127: 121-136. Purified bovine basic FGF has been shown to be
angiogenic in vivo in a chicken chorioallantoic membrane assay.
(Gospodarowicz, D. in Hormonal Proteins and Peptides XII:205-230
(Academic Press). Purified bovine acidic FGF has been shown to be
angiogenic in vivo in the same assay (Thomas, K. A., et al, Proc
Natl Acad Sci (supra)).
[0023] Bovine pituitary basic FGF has been completely sequenced by
Esch, Proc Natl Acad Sci USA (supra); the human sequence is shown
in FIG. 1. The reported primary sequence contains 146 amino acids,
beginning with the proline residue numbered "10" in FIG. 1; the
N-terminal portion of this sequence is in agreement with the
sequence previously reported for the N-terminus of the native
bovine protein by Bohlen et al, Proc Natl Acad Sci USA (supra). A
higher molecular weight human basic FGF has been reported from the
human hepatoma cell line, SK-HEP-1, by Sullivan, R. J., et al, J
Cell Biol (1985) 101:108a; by Klagsbrun, M., et al, Proc Natl Acad
Sci USA (1986) 83:2448-2452; and by Klagsbrun, M. et al, Proc Natl
Acad Sci USA (1987) 84:1839-1843. Longer forms of FGF have been
reported by Sommer, A., et al, Biochem Biophys Res Comm (1987)
144:543 (human placental tissue) as well as from pituitary and
human prostatic tissue reported by Uneo, et al, Biochem Biophys Res
Comm (1986) 138:580-588 and Story, et al, Biochem Biophys Res Comm
(1987) 142:702-709, respectively. Translation of the upstream
sequences of FIG. 1 back to a potential ATG translation start codon
in human basic FGF DNA shows that it is likely that an additional
form of the protein containing the amino acids upstream of the
proline shown as residue 10 in FIG. 1 is also produced. The ATG
codon lies nine codons upstream from the codon for the proline
residue. It is reasonably certain that if the methionine encoded by
this ATG serves as the initiating methionine, then it will be
processed off when the gene is expressed in eucaryotic systems.
Such processing may or may not occur when the gene is expressed
recombinantly in bacterial systems. Thus, the "long" form of the
protein expressed in bacteria contains an additional 8 or 9 amino
acid sequence at the N-terminus, for a total of 154 or 155 amino
acids. All of the investigative groups have also shown that much of
this extended FGF is blocked at the N-terminus.
[0024] Proteins having FGF activity in the above-mentioned in vitro
assays and sharing a similar putative N-terminal sequence with the
bovine pituitary basic FGF (the 146 amino acid form) have also been
isolated from bovine brain, adrenal gland, retina, and from human
placenta. The native protein obtained from certain of these tissues
is heterogeneous--a second form missing the putative fifteen
N-terminal amino acids retains activity. (Gospodarowicz, D., Meth
Enz (1987) 147A:106-119.) It is considered, therefore, that bovine
and human basic FGFs exist in at least three forms, a mature form
starting at amino acid 10 in FIG. 1 (a proline), longer forms
containing eight additional amino acids at the N-terminus, and
shorter forms lacking fifteen amino acids of the putative mature
sequences shown. Thus, there is believed to be natively produced
"long" basic FGF containing 154 or 155 amino acids (Abraham, J. A.,
et al, EMBO J (1986) 5:2523-2528), "primary" basic FGF containing
146 amino acids, and "short" basic FGF containing 131 amino acids.
It is also possible that forms extending even further upstream
exist. These FGFs are designated "basic" FGF, because they contain
a high number of basic amino acid residues (lysine, arginine,
histidine) and are therefore cations at neutral pH.
[0025] A protein is defined herein as basic FGF (also referred to
as bFGF) if it shows FGF activity in the foregoing assays, binds to
heparin, is a cation at neutral pH, and reacts immunologically with
antibodies prepared using a synthetic analog of the amino terminal
sequence [tyr.sup.10] FGF (1-10) conjugated to bovine serum albumin
(if appropriate) or to other antibodies raised against bovine (or
human) FGF or synthetic or native peptides thereof. See Baird, A.,
et al, Regulatory Peptides (1985) 10:3.sup.09-317.
[0026] Acidic FGF has been isolated from bovine and human brain by
others, and the complete coding sequence for human acidic FGF was
determined and is shown in FIG. 2.
[0027] The acidic protein also has three known active forms, one
having the 140 amino acid sequence beginning at the phenylalanine
residue numbered "16" in the figure, and a second shorter form
corresponding to amino acids 22-155, and an N-terminal extended
form corresponding to 2-155 (blocked by acetylation) Burgess, et
al, Proc Natl Acad Sci USA (1986) 83:7216. These proteins contain a
disproportionate number of acidic amino acid residues, i.e.,
glutamic and aspartic acids and the proteins are therefore anions
at neutral pH.
[0028] A protein is defined herein as acidic FGF (also referred to
herein as aFGF) if it shows FGF activity in in vitro assays, binds
to heparin, is an anion at neutral pH, and is immunologically
reactive with antibodies prepared against human or bovine acidic
FGF or against synthetic or native peptides thereof.
[0029] Acidic FGF and basic FGF are thus used herein to designate
the foregoing proteins or proteins having amino acid sequences
represented by those shown in FIGS. 1 and 2. Of course, these
definitions are not restricted to the specific sequences shown, but
include analog proteins which contain accidentally or deliberately
induced alterations, such as deletions, additions, extensions, or
exchanges of amino acid residues, so long as the biological
activity of the FGF agonists, as measured by the foregoing in vitro
assay and immunological cross-reactivity assay, is retained.
Analogs of FGF with antagonist activity will, of course, have
altered activity and specificity.
[0030] The various FGF analogs described herein contain
deliberately induced alterations formed by directed mutagenesis
techniques. These analogs retain the general secondary structure of
FGF but have been mutated so as to produce various antagonist and
agonist forms of FGF.
[0031] In designing such analogs, Shing et al (Science (1984) 223:
1269-1299) have demonstrated in vitro that basic FGF binds tightly
to heparin and Maciag, T., et al, Science (1984) 225:932 have
reported that acidic FGF also binds heparin. Thus it is likely that
heparin, heparan sulfate, heparin-like glycosaminoglycans, and
heparan-like glycosaminoglycans, which are present in the
extracellular environment, including the extracellular matrix, may
bind FGF in vivo. Since basic FGF binds in the extracellular matrix
produced by vascular and capillary endothelial cells in vitro
(Baird and Ling, Biochem Biophys Res Comm (1987) 142: 428-435), it
follows that analogs of basic FGF with reduced heparin binding
ability will have enhanced potency, as more FGF will reach its
targeted receptor and will not be sequestered by heparin and
heparin-like compounds in the extracellular environment. These
analogs will be more useful therapeutically as lower dosages of the
particular analog will be required per treatment.
[0032] Baird et al (Rec Prog Horm Res (1986) 42:143-205) have
recently speculated on the regions of basic FGF, residues 26-31 and
residues 115-120 illustrated in FIG. 3, which might mediate the
binding to heparin. The ability of clustered basic residues,
possibly in conjunction with aromatic residues, to mediate heparin
binding has been described previously with respect to other
proteins (Schwarzbauer et al, Cell (1983) 35:421-431; Cardin et al,
Biochem Biophys Res Comm (1986) 134:783-789). Mutations created in
bFGF, as described herein, replace positively charged amino acids
within those targeted regions with neutral or negatively charged
residues, with consideration given towards minimizing change in
secondary structure of the molecule (e.g., alpha helix, beta sheet,
turn motifs). In contrast to the putative heparin binding domains
identified above, which do not appear to be the main functional
heparin binding domains in the present studies, a third region of
bFGF including residues 128-138 which contains a clustering of
basic residues, was targeted as a potential heparin binding domain.
Preferred mutations targeting the heparin binding domains include
bFGF-K128S, bFGF-K128E, bFGF-R129T, bFGF-K134S, bFGF-K138S, and
K128S/R129T. Substitutions of a basic or positively charged residue
with a negatively charged residue such as glutamic acid are
preferred.
[0033] Analogs of bFGF are defined as: bFGF-XYZ where X is the
amino acid in the native human bFGF sequence that is being mutated,
Y is the position of amino acid X, and Z is the amino acid residue
that is being substituted for X at position Y.
[0034] Mutations of bFGF which are found to decrease or eliminate
heparin binding can also be combined with other mutations found to
result in the formation of analogs with either agonist or
antagonist activity.
[0035] It is also within the skill of the art to create additional
FGF analogs following the teaching provided herein, wherein those
residues important for heparin binding are changed to other neutral
amino acids (e.g., serine, alanine, glycine, etc.), or negatively
charged amino acids (e.g., glutamic acid, aspartic acid), or
deleted in order to reduce heparin binding activity as tested by
HPLC heparin-affinity analysis as described herein. Analogs of the
acidic form of FGF can be constructed as described above by
deleting positively charged amino acids or by replacing positively
charged amino acids within the corresponding heparin-binding
domains (23-27, 115-120, 127-137) with neutral or negatively
charged amino acids.
[0036] It has been found that bacterially-produced recombinant
proteins can be difficult to recover in an active form. For
example, it is known that cysteine-containing proteins produced in
bacteria often form incorrect intramolecular cysteines which can
inhibit biological function (see human interleukin-2; Wang et al,
Science (1984) 224: 1431-1433 and human fibroblast interferon; Mark
et al, Proc Natl Acad Sci (USA) (1984) 81: 5662-5666).
Modifications of one or more of the cysteine residues present in
the native FGF proteins may minimize incorrect disulfide bridge
formation, eliminate the need for use of reducing agents to
stabilize the FGF protein, and hence reduce multimerization or
incorrect disulfide bonds thereby increasing the recoverable yield
of the recombinantly produced analog, increasing the uniformity of
the FGF preparation by maintaining it over time in a monomeric
form, improving its shelf stability and reducing its half life when
applied to wounds. Unexpectedly, these analogs have been shown to
have augmented biological activity.
[0037] Generally, the above modifications at cysteine residues are
conducted by changing a single nucleotide within the codon
specifying a particular cysteine, corresponding to an amino acid
substitution in the resulting protein. Cysteine residues occur in
the basic form of FGF at positions 34, 78, 96, and 101, and occur
in the acidic form at positions 31, 98, and 132. Since the
disulfide structure of native FGF is not known, both single and
multiple cysteine substitutions of FGF are exemplified herein.
While these modifications produce a change in the primary structure
of the protein analog, preferred analogs will generally retain the
ability to effect cellular responses normally induced by FGF,
unless the cysteine-substituted analogs are combined with other
antagonist changes.
[0038] These same cysteine substitutions can be made in combination
with other analog substitutions, such as the aforementioned
heparin-binding mutants, to produce yet additional illustrative FGF
analogs. Correspondingly, any of the aforementioned FGF analogs can
be modified to contain one or more of the amino acid substitutions
described below to produce a desired analog.
[0039] Antagonists of bFGF activity would have clinical
applications in a variety of pathologies related to abnormal
persistent angiogenesis (Folkman, J. and Klagsbrun, M., Science
(1987) 235:442-447) including diabetic retinopathy, retrolental
fibroplasia, neovascular glaucoma, rheumatoid arthritis,
hemangiomas, angiofibromas, psoriasis, atherosclerosis and as
contraceptives. In addition, it has been shown that certain solid
tumors require neovascularization in order to sustain growth. Given
the important role FGF plays in the process of angiogenesis, it is
clear that analogs of FGF which are capable of inhibiting its
effect would be useful in treating these diseases therapeutically.
Thus, analogs which bind the FGF receptor yet do not elicit a
biological response or that demonstrate a reduced biological
response will exhibit useful antagonist properties.
[0040] The ability to elaborate a specific cell surface receptor
for basic FGF has been described in a variety of cell types
including baby hamster kidney cells (Neufeld and Gospodarowicz, J
Biol Chem (1985) 260:13860-13868), bovine epithelial lens cells
(Moenner, et al. Proc Natl Acad Sci (USA) (1986) 83:5024-5028),
Swiss 3T3 and a murine skeletal muscle cell line (Olwin and
Hauschka, Biochemistry (1986) 25:3487-3492) and Swiss 3T3 and
aortic endothelial cells (Huang et al, J Biol Chem (1986)
261:11600-11607). In addition binding studies have suggested that
both the basic and acidic forms of FGF can bind to the same high
affinity receptor (Olwin and Hauschka, supra, and Neufeld and
Gospodarowicz, J Biol Chem (1986) 261:5631-5637).
[0041] The interaction of a hormone (e.g., bFGF) with its receptor
results in a tight, specific molecular association. This
association may involve any or all of the known intermolecular
attractive forces such as ion pairing or van der Waals forces. The
specificity and the stability of the association are due to what
may be thought of as "exactness of fit" (the precise
three-dimensional molecular conformations of the two proteins,
receptor and hormone) and "tightness of fit" (the fact that these
molecular structures are composed of precise amino acid sequences
which therefore results in specific intermolecular attractions due
to energetically favorable juxtaposition of amino acid side
chains). Thus, amino acid substitutions, deletions and insertions
within receptor binding regions may effect either molecular
conformation of the region or amino acid side chain interactions
(between hormone and receptor) or both. Changes which stabilize
favorable conformation or enhance amino acid side chain
interactions will result in increased receptor affinity while those
which destabilize favorable conformation or decrease amino acid
side chain interactions will result in decreased receptor affinity.
The former changes are useful in themselves as their introduction
into agonists may result in more potent agonists and their
introduction into antagonists may result in more potent
antagonists. The latter changes are useful in terms of defining
amino acid segments crucial to receptor binding.
[0042] Schubert et al (J Cell Biol (1987) 104:635-643) have shown
that synthetic peptides containing fragments of bFGF (residues
33-77 and 112-129 numbered according to FIG. 3) inhibit binding of
bFGF to its receptor. Therefore, these regions appear to contain
FGF receptor binding sequences. We have introduced amino acid
substitutions into human basic FGF within these putative receptor
binding regions and additional regions adjacent to the latter
(e.g., amino acids 99-111 which exhibit strong homology to the
equivalent amino acid sequence region in acidic FGF). Both charged
(positive and negative) and aromatic amino acids were targeted for
replacement with neutral residues. These substitutions were made
with consideration given towards minimizing changes in the
secondary structure of the resultant protein. Accordingly, the
analogs D99A and R116T appear to exhibit increased receptor
affinity and 3T3 mitogenic activity, respectively, whereas analogs
E105S and Y112A exhibit decreased receptor binding (see Table 3
herein).
[0043] For purposes of the present invention the following terms
are defined below.
[0044] "Agonist" refers to an FGF analog capable of combining with
the FGF receptor and producing a typical biological response. For
example, an FGF agonist might be a protein than can bind to the FGF
receptor but has reduced ability to bind heparin, thereby creating
a more potent therapeutic.
[0045] "Antagonist" refers to an FGF analog that opposes the
effects of FGF by a competitive mechanism for the same receptor
sites. The antagonist has reduced ability to induce secondary
biological responses normally associated with FGF.
[0046] "Site-specific mutagenesis" or "directed mutagenesis" refers
to the use of the oligonucleotide-directed mutagenesis procedure,
which entails using a synthetic oligonucleotide primer that is
complementary to the region of the bFGF gene at the specific codon
or codons to be altered, but which contains single or multiple base
changes in that codon. By this technique, a designer gene may be
produced that results in a specific amino acid being replaced with
any other amino acid of choice. When deletion is desired the
oligonucleotide primer lacks the specific codon. Conversion of, for
example, a specific cysteine, to neutral amino acids such as
glycine, valine, alanine, leucine, isoleucine, tyrosine,
phenylalanine, histidine, tryptophan, serine, threonine or
methionine is a preferred approach. Serine and alanine are the most
preferred replacements because of their chemical analogy to
cysteine. When a cysteine is deleted, the mature analog is one
amino acid shorter than the native parent protein or the
microbially produced wild type bFGF.
[0047] "Purified" or "pure" refers to material which is free from
substances which normally accompany it as found in its native
state. Thus "pure" acidic human FGF (hFGF), for example, refers to
acidic hFGF which does not contain materials normally associated
with its in situ environment in human brain or pituitary. Of
course, "pure" acidic hFGF may include materials in covalent
association with it, such as glycoside residues.
[0048] "Operably linked" refers to a juxtaposition wherein the
components are configured so as to perform their usual function.
Thus, control sequences or promoters operably linked to a coding
sequence are capable of effecting the expression of the coding
sequence.
[0049] "Control sequence" refers to a DNA sequence or sequences
which are capable, when properly ligated to a desired coding
sequence, of affecting its expression in hosts compatible with such
sequences. Such control sequences include at least promoters in
both procaryotic and eucaryotic hosts, and optionally,
transcription termination signals. Additional factors necessary or
helpful in effecting expression may also be identified. As used
herein, "control sequences" simply refers to whatever DNA sequence
may be required to effect expression in the particular host
used.
[0050] "Cells" or "cell cultures" or "recombinant host cells" or
"host cells" are often used interchangeably as will be clear from
the context. These terms include the immediate subject cell, and,
of course, the progeny thereof. It is understood that not all
progeny are exactly identical to the parental cell, due to chance
mutations or differences in environment. However, such altered
progeny are included in these terms, so long as the progeny retain
the desired characteristics conferred on the originally transformed
cell. In the present case, for example, such a characteristic might
be the ability to produce recombinant FGF analogs.
[0051] B. General Description
[0052] Utility and Administration
[0053] The invention provides DNAs encoding growth factor protein
analogs which have two diverse applications. The first application
is similar to FGF in that the analogs augment tissue repair by
encouraging vascularization and/or cell growth or cell survival.
These purified growth factors are generally applied topically to
the traumatized or diseased tissue in order to stimulate
vascularization, regeneration, and healing. Appropriate substrates
are burns, wounds, bone fractures, surgical abrasions such as those
of plastic surgery, or others requiring repair. Because application
of these factors accelerates healing, they also reduce the risk of
infection.
[0054] Indications wherein FGF is of value in encouraging
neovascularization include musculo-skeletal conditions such as bone
fractures, ligament and tendon repair, tendonitis, and bursitis;
skin conditions such as burns, cuts, lacerations, bed sores, and
slow-healing ulcers such as those seen in diabetics; and in tissue
repair during ischaemia and myocardial infarction.
[0055] In addition to analogs which augment wound healing, analogs
of FGF can be constructed which inhibit angiogenesis. Analogs of
FGF which can antagonize the FGF angiogenesis activity would be
clinically useful for treating certain diseases where
neovascularization is the dominant pathology, such as retinopathies
of the eye including diabetic retinopathy and neovascular glaucoma;
skin disorders including psoriasis and retrolental fibroplasia;
chronic inflammation; rheumatoid arthritis; atherosclerosis; and
certain neoplasms that are highly angiogenic, such as the growth of
certain benign and malignant tumors such as hemangiomas and
angiofibromas, and solid tumors.
[0056] Formulations of the recombinantly produced growth factors
using available excipients and carriers are prepared according to
standard methods known to those in the art. The proteins can be
formulated as eyedrops, lotions, gels, powder, dressing, as part of
a controlled release system, or ointments with additional active
ingredients, such as antibiotics, if desired.
[0057] For topical administration, which is the most appropriate
with regard to superficial lesions, standard topical formulations
are employed using, for example, 10 ng/ml-100 mg/ml solutions; the
preferred range is 10 ug/ml-10 mg/ml. Such solutions would be
applied up to 6 times a day to the affected area. In certain
applications, such as burns, a single dose would be preferred. In
other applications, such as ulcers, multiple doses may be
preferred. The concentration of the ointment or other formulation
depends, of course, on the severity of the wound or stage of
disease and the nature of the subject. In most protocols, the dose
is lowered with time to lessen likelihood of scarring. For example,
the most severe wounds, such as third degree burns, are typically
treated with a 100 ug/ml composition, but as healing begins, the
dose is progressively dropped to approximately 10 ug/ml or lower,
as the wound heals. A topical formulation for EGF using BSA as
carrier was disclosed by Franklin, J. D., et al, Plastic and
Reconstruc Surg (1979) 64:766-770.
[0058] For treatment of pathologies related to persistent
angiogenesis wherein FGF inhibitors are to be applied, the
concentration of the formulation is generally 10-fold higher,
regardless of the mode of administration. The higher dosage assures
that the FGF inhibitor is able to compete effectively with
endogenously produced FGF. Thus for topical administration of the
FGF inhibitor used to treat psoriasis and retrolental fibroplasia,
the dosage would be increased 10-fold.
[0059] For arthritis and bone and tissue repair, administration is
preferred locally by means of subcutaneous implant, staples or slow
release formulation implanted directly proximal the target. Surgery
may be required for such conditions as bone injuries, thus making
implantation directly practical. Slow-release forms can be
formulated in polymers, such as Hydron (Langer, R., et al, Nature
(1976) 263:797-799) or Elvax 40P (Dupont) (Murray, J. B., et al, In
Vitro (1983) 19:743-747). Other sustained-release systems have been
suggested by Hsieh, D. S. T., et al, J Pharm Sci (1983) 72:17-22),
and a formulation specifically for epidermal growth factor, but not
preferred in the present invention, is suggested by Buckley, A.,
Proc Natl Acad Sci (USA) (1985) 82:7340-7344.
[0060] As with topical administration, for sustained release
delivery, the concentration of FGF in the formulation depends on a
number of factors, including the severity of the condition, the
stability of FGF at 37.degree. C., the rate of FGF release from the
polymer, and the agonist or antagonist nature of the FGF analog. In
general, the formulations are constructed so as to achieve a
constant local concentration of about 100 times the serum level of
factor or 10 times the tissue concentration, as described by
Buckley et al (Proc Natl Acad Sci (USA) (supra)). Based on an FGF
concentration in tissue of 5-50 ng/g wet weight (comparable to EGF
at 60 ng/g wet weight), release of 50-5000 ng FGF per hour is
acceptable. The initial concentration, of course, depends on the
severity of the wound or advancement of pathology.
[0061] For treatment in diseases common to ophthalmology, such as
retinopathies and neovascular glaucoma, eyedrop formulation or
direct injection into the eye would be two preferred routes of
administration. Liquid formulations for these applications are
generally known in the art and include formulation in buffer or
physiological saline, or other appropriate excipient. Dosage levels
may be supplied between 1 ug/ml and 10 mg/ml from two to four times
a day.
[0062] It is expected that FGF may act in concert, and even
synergistically, with other growth factors such as epidermal growth
factor (EGF), the transforming growth factors (TGF-alpha or TGF-),
insulin-like growth factors (IGF-1 and IGF-2), and/or
platelet-derived growth factor (PDGF). In addition, specifically
for bone repair, it may act in synergy with agonists or antagonists
of parathyroid hormone or calcitonin, since these compounds promote
bone growth and resorption. Therefore, also included within the
compositions and administration protocols of the invention are
embodiments wherein the FGF of the invention is administered in the
same composition with, or in the same protocol with, one or more of
the foregoing factors, thus more effectively to achieve the desired
tissue repair.
[0063] Since FGF is effective in promoting neurite outgrowth, nerve
regeneration, and neuronal survival, it may be useful for treatment
of certain neurological disorders such as Alzheimer's and
Parkinson's diseases, amyotrophic lateral sclerosis, stroke,
peripheral neuropathies, and general aging of the nervous system,
as well as traumatic injury to the spinal cord and peripheral
nerves. Administration of the drug for these indications is
preferably by implant in formulations similar to those set forth
above in connection with rheumatoid arthritis and bone healing. The
drug may also be delivered by means of implants of cell cultures by
means of implants of cell cultures which produce FGF. Treatment of
neurological disorders may also involve transplantation of new
cells or tissues to functionally replace damaged neural tissue
(e.g., adrenal and fetal brain tissue transplants in Parkinsonian
patients). In such cases, the degree of success of transplantation
as well as the degree of function of the transplanted tissue are
enhanced by treating the cell cultures or tissue explants with the
FGF or analog preparations of the invention prior to
transplantation and/or by administration of FGF or FGF analogs of
the invention following transplantation.
[0064] FGF and analogs thereof may also be injected directly into
the spinal fluid or into the brain by means of canulation or by
administration using osmotic minipumps or they may be applied
systemically. For atherosclerosis peripheral neuropathies and the
like, and tumor angiogenesis, systemic administration is preferred,
with administration of the drug delivered initially at the time of
surgery, where appropriate.
[0065] Systemic formulations are generally as are known in the art
and include formulation in buffer or physiological saline, or other
appropriate excipient. Dosage levels for FGF agonist administration
are approximately those of wound healing; however, for tissue
culture, explant maintenance, atherosclerosis or tumor
angiogenesis, it may be supplied at 1.0-100 ng/ml of serum or
culture medium.
[0066] In addition, it has been shown that angiogenic stimuli, such
as those provided by the FGF proteins discussed herein, result in
the release of plasminogen activator (PA) and of collagenase in
vitro (Gross, J. L., et al, Proc Natl Acad Sci (USA) (1983)
80:2623-2627). Therefore, the FGF proteins of the invention are
also useful in treatment of conditions which respond to these
enzymes. While it may be necessary in acute situations (such as the
presence of a blood clot associated with stroke or heart attack)
directly to administer large doses of PA to dissolve the clot, for
treatment of chronic propensity to form embolisms, administration
of FGF to maintain a suitable level of PA in the blood stream may
be desirable. Therefore, for this indication, systemic
administration of the drug, especially an analog with reduced
heparin-binding ability, using conventional means such as
intramuscular or intravenous injection, is preferred.
[0067] The invention provides practical quantities of pure FGF
analogs for use in connection with the foregoing indications.
Specific growth factors are exemplified herein, each of which is
apparently active in at least three forms. Both acidic and basic
analogs are considered to occur in long, primary, and short forms,
as described above. It is considered that the N-terminal methionine
of the long forms is processed off when the protein is produced in
eucaryotic systems, and that the subsequent amino acid residue is
derivatized, probably by acetylation, post-translation.
[0068] While FGF in its various forms does not have a recognized
signal sequence, it must somehow be secreted or retrieved from the
cell, since it acts outside the cells producing it at a
membrane-bound receptor. Therefore, while it may not be secreted by
the recognized constitutive secretion pathway, its secretion is
accomplished by some means, for example by cell lysis or by
exocytosis, by association with a glycosaminoglycan, such as
heparan sulfate or heparin. For most tissues from which FGF is
naturally derived, and for many mammalian expression systems, such
release may be achieved by securing exocytosis with a calcium
ionophore, such as the commonly employed A23187 (CalBiochem),
which, in in vitro conditions, is added to the culture medium at
1-10 uM in the presence of 1 mM CaCl.sub.2. For expression systems
derived from macrophages or monocytes, other activation methods
have been shown to be effective, such as the addition of
lipopolysaccharide (LPS) at 10 ug/ml or the addition of E. coli
endotoxin (Difco) (300 ng/ml). These stimulators have been shown to
release the analogous factor interleukin-1 from macrophages by
March, C. J., et al, Nature (1985) 315:641-647. These techniques
can also be employed in releasing recombinantly produced FGF
proteins when produced intracellularly without added signal
sequences, as described below. Additional stimulators for release
of intracellularly produced proteins include the phorbol esters and
the triglycerides.
[0069] Gene Retrieval
[0070] The general strategy whereby the illustrated FGF-encoding
sequences were obtained is as described in co-pending U.S. Ser. No.
869,382, filed May 30, 1986, Abraham, J. A. et al, EMBO J (1986)
supra, and Abraham, J. A. et al, Science (1986) 233:545-548, all of
which are incorporated herein by reference.
[0071] Expression of FGF Genes
[0072] The DNA sequences described herein can be expressed in
appropriate expression systems. Of course, for the DNAs disclosed
herein, the foregoing protocol for retrieving the genomic or cDNA
FGF sequences need not be repeated, but conventional chemical
synthesis methods can suitably be employed. Alternatively, the gene
encoding basic FGF can be retrieved from the deposited
bacteriophage lambdaBB2 and converted to the human form.
Site-directed mutagenesis permits adjustment of the DNA to obtain
any desired form of the protein. DNA sequences can be provided with
appropriate controls suitable for any host, including bacteria,
yeast, or eucaryotic cells. Exemplary control sequence DNAs and
hosts are given in paragraph C.1 below.
[0073] In particular, complete DNA encoding any of the FGF analogs
described herein can be constructed, for example, using a
combination of recombinant and synthetic methods to obtain each of
the DNA analog sequences of FGF. These gene sequences have been
constructed with convenient restriction sites bounding the FGF
coding sequence so that the entire gene may be inserted on an
.sup..about.503 bp NcoI-HindIII restriction fragment for insertion
into an appropriately digested host vector such that the FGF coding
sequence is operably linked to control sequences present on the
vector. Intracellularly produced forms of the FGF protein analogs
can be obtained by cell lysis, or their release from the cells can
be stimulated by using heterologous signal sequences fused to the
gene sequence using the known relationship of the signal sequence
to cleavage site to obtain-the protein in the desired form.
Particularly preferred are bacterial expression systems which
utilize control systems compatible with E. coli cells, such as
plasmids pUC9-TSF11 and pUC9delH3-pTSF-3. These vectors are derived
from pUC9 (Messing and Vieira, Gene (1982) 19:259-268), which
contains parts of pBR322 and M13mp9 and a multiple cloning site
polylinker.
[0074] The recombinant FGF proteins thus produced are then purified
in a manner similar to that utilized for purification of FGF from
natural sources, but purification is considerably simpler, as the
proteins form a much larger proportion of the starting
material.
[0075] B. Standard Methods
[0076] Most of the techniques which are used to transform cells,
construct vectors, construct oligonucleotides, perform
site-specific mutagenesis, and the like are widely practiced in the
art, and most practitioners are familiar with the standard resource
materials which describe specific conditions and procedures.
However, for convenience, the following paragraphs may serve as a
guideline.
[0077] B.1. Hosts and Control Sequences
[0078] Both procaryotic and eucaryotic systems may be used to
express the FGF analog encoding sequences; procaryotic hosts are,
of course, the most convenient for cloning procedures. Procaryotes
most frequently are represented by various strains of E. coli;
however, other microbial strains may also be used. Plasmid vectors
which contain replication sites, selectable markers and control
sequences derived from a species compatible with the host are used;
for example, E. coli is typically transformed using derivatives of
pBR322, a plasmid derived from an E. coli species by Bolivar, et
al, Gene (1977) 2:95. pBR322 contains genes for ampicillin and
tetracycline resistance, and thus provides multiple selectable
markers which can be either retained or destroyed in constructing
the desired vector. Commonly used procaryotic control sequences
which are defined herein to include promoters for transcription
initiation, optionally with an operator, along with ribosome
binding site sequences, include such commonly used promoters as the
-lactamase (penicillinase) and lactose (lac) promoter systems
(Chang, et al, Nature (1977) 198:1056), the tryptophan (trp)
promoter system (Goeddel, et al, Nucleic Acids Res (1980) 8:4057),
the lambda-derived P.sub.L promoter (Shimatake, et al, Nature
(1981) 292:128) and N-gene ribosome binding site, and the trp-lac
(trc) promoter system (Amann and Brosius, Gene (1985) 40:183).
[0079] In addition to bacteria, eucaryotic microbes, such as yeast,
may also be used as hosts. Laboratory strains of Saccharomyces
cerevisiae, Baker's yeast, are most used although a number of other
strains or species are commonly available. Vectors employing, for
example, the 2 u origin of replication of Broach, J. R., Meth Enz
(1983) 101:307, or other yeast compatible origins of replication
(see, for example, Stinchcomb, et al, Nature (1979) 282:39,
Tschumper, G., et al, Gene (1980) 10:157 and Clarke, L, et al, Meth
Enz (1983) 101:300) may be used. Control sequences for yeast
vectors include promoters for the synthesis of glycolytic enzymes
(Hess, et al, J Adv Enzyme Req (1968) 7:149; Holland, et al,
Biochemistry (1978) 17:4900). Additional promoters known in the art
include the promoter for 3-phosphoglycerate kinase (Hitzeman, et
al, J Biol Chem (1980) 255:2073). Other promoters, which have the
additional advantage of transcription controlled by growth
conditions and/or genetic background are the promoter regions for
alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,
degradative enzymes associated with nitrogen metabolism, the alpha
factor system and enzymes responsible for maltose and galactose
utilization. It is also believed terminator sequences are desirable
at the 3' end of the coding sequences. Such terminators are found
in the 3' untranslated region following the coding sequences in
yeast-derived genes.
[0080] It is also, of course, possible to express genes encoding
polypeptides in eucaryotic host cell cultures derived from
multicellular organisms. See, for example, Axel, et al, U.S. Pat.
No. 4,399,216. These systems have the additional advantage of the
ability to splice out introns and thus can be used directly to
express genomic fragments. Useful host cell lines include VERO,
HeLa baby hamster kidney (BHK), CV-1, COS, MDCK, NIH 3T3, L, and
Chinese hamster ovary (CHO) cells. Expression vectors for such
cells ordinarily include promoters and control sequences compatible
with mammalian cells such as, for example, the commonly used early
and late promoters from Simian Virus 40 (SV40) (Fiers, et al,
Nature (1978) 273:113), or other viral promoters such as those
derived from polyoma, Adenovirus 2, bovine papilloma virus, or
avian sarcoma viruses. The controllable promoter, hMTII (Karin, M.,
et al, Nature (1982) 299:797-802) may also be used. General aspects
of mammalian cell host system transformations have been described
by Axel (supra). It now appears, also that "enhancer" regions are
important in optimizing expression; these are, generally, sequences
found upstream or downstream of the promoter region in noncoding
DNA regions. Origins of replication may be obtained, if needed,
from viral sources. However, integration into the chromosome is a
common mechanism for DNA replication in eucaryotes.
[0081] B.2. Transformations
[0082] Depending on the host cell used, transformation is done
using standard techniques appropriate to such cells. The calcium
treatment employing calcium chloride, as described by Cohen, S. N.,
Proc Natl Acad Sci (USA) (1972) 69:2110, or the RbCl.sub.2 method
described in Maniatis, et al, Molecular Cloning: A Laboratory
Manual (1982) Cold Spring Harbor Press, p. 254 and Hanahan, D., J
Mol Biol (1983) 166:557-580 may be used for procaryotes or other
cells which contain substantial cell wall barriers. For mammalian
cells without such cell walls, the calcium phosphate precipitation
method of Graham and van der Eb, Virology (1978) 52:546, optionally
as modified by Wigler, M., et al, Cell (1979) 16:77.sup.7-785 may
be used. Transformations into yeast may be carried out according to
the method of Beggs, J. D., Nature (1978) 275:104-109 or of Hinnen,
A., et al, Proc Natl Acad Sci (USA) (1978) 75:1929.
[0083] B.3. Vector Construction
[0084] Construction of suitable vectors containing the desired
coding and control sequences employs standard ligation and
restriction techniques which are well understood in the art.
Isolated plasmids, DNA sequences, or synthesized oligonucleotides
are cleaved, tailored, and religated in the form desired.
[0085] The DNA sequences which form the vectors are available from
a number of sources. Backbone vectors and control systems are
generally found on available "host" vectors which are used for the
bulk of the sequences in construction. Typical sequences have been
set forth in .paragraph.C.1 above. For the pertinent coding
sequence, initial construction may be, and usually is, a matter of
retrieving the appropriate sequences from cDNA libraries, genomic
DNA libraries, or deposited plasmids. However, once the sequence is
disclosed it is possible to synthesize the entire gene sequence in
vitro starting from the individual nucleoside derivatives. The
entire gene sequence for genes of sizeable length, e.g., 500-1000
bp may be prepared by synthesizing individual overlapping
complementary oligonucleotides and filling in single stranded
nonoverlapping portions using DNA polymerase in the presence of the
deoxyribonucleotide triphosphates. This approach has been used
successfully in the construction of several genes of known
sequence. See, for example, Edge, M. D., Nature (1981) 292:756;
Nambair, K. P., et al, Science (1984) 223:1299; Jay, Ernest, J Biol
Chem (1984) 259:6311.
[0086] Synthetic oligonucleotides are prepared by either the
phosphotriester method as described by Edge, et al, Nature (supra)
and Duckworth, et al, Nucleic Acids Res (1981) 9:1691 or the
phosphoramidite method as described by Beaucage, S. L., and
Caruthers, M. H., Tet Letts (1981) 22:1859 and Matteucci, M. D.,
and Caruthers, M. H., J Am Chem Soc (1981) 103:3185 and can be
prepared using commercially available automated oligonucleotide
synthesizers. Kinasing of single strands prior to annealing or for
labeling is achieved using an excess, e.g., approximately 10 units
of polynucleotide kinase to 1 nmole substrate in the presence of 50
mM Tris, pH 7.6, 10 mM MgCl.sub.2, 5 mM dithiothreitol, 1-2 mM ATP,
1.7 pmoles [lambda-.sup.32P]-ATP (2.9 mCi/mmole), 0.1 mM
spermidine, 0.1 mM EDTA.
[0087] Once the components of the desired vectors are thus
available, they can be excised and ligated using standard
restriction and ligation procedures.
[0088] Site specific DNA cleavage is performed by treating with the
suitable restriction enzyme (or enzymes) under conditions which are
generally understood in the art, and the particulars of which are
specified by the manufacturer of these commercially available
restriction enzymes. See, e.g., New England Biolabs, Product
Catalog. In general, about 1 ug of plasmid or DNA sequence is
cleaved by one unit of enzyme in about 20 ul of buffer solution; in
the examples herein, typically, an excess of restriction enzyme is
used to insure complete digestion of the DNA substrate. Incubation
times of about one hour to two hours at about 37.degree. C. are
workable, although variations can be tolerated. After each
incubation, protein is removed by extraction with
phenol/chloroform, and may be followed by ether extraction, and the
nucleic acid recovered from aqueous fractions by precipitation with
ethanol. If desired, size separation of the cleaved fragments may
be performed by polyacrylamide gel or agarose gel electrophoresis
using standard techniques. A general description of size
separations is found in Methods in Enzymology (1980)
65:499-560.
[0089] Restriction cleaved fragments may be blunt ended by treating
with the large fragment of E. coli DNA polymerase I (Klenow) in the
presence of the four deoxynucleotide triphosphates (dNTPs) using
incubation times of about 15 to 25 min at 20 to 25.degree. C. in 50
mM Tris pH 7.6, 50 mM NaCl, 6 mM MgCl.sub.2, 6 mM DTT and 0.1-1.0
mM dNTPs. The Klenow fragment fills in at 5' single-stranded
overhangs but chews back protruding 3' single strands, even though
the four dNTPs are present. If desired, selective repair can be
performed by supplying only one of the, or selected, dNTPs within
the limitations dictated by the nature of the overhang. After
treatment with Klenow, the mixture is extracted with
phenol/chloroform and ethanol precipitated. Treatment under
appropriate conditions with S1 nuclease or BAL-31 results in
hydrolysis of any single-stranded portion.
[0090] Ligations are performed in 15-50 ul volumes under the
following standard conditions and temperatures: for example, 20 mM
Tris-Cl pH 7.5, 10 mM MgCl.sub.2, 10 mM DTT, 33 ug/ml BSA, 10 mM-50
mM NaCl, and either 40 uM ATP, 0.01-0.02 (Weiss) units T4 DNA
ligase at 0.degree. C. (for "sticky end" ligation) or 1 mM ATP,
0.3-0.6 (Weiss) units T4 DNA ligase at 14.degree. C. (for "blunt
end" ligation). Intermolecular "sticky end" ligations are usually
performed at 33-100 ug/ml total DNA concentrations (5-100 nM total
end concentration). Intermolecular blunt end ligations are
performed at 1 uM total ends concentration.
[0091] In vector construction employing "vector fragments", the
vector fragment is commonly treated with bacterial alkaline
phosphatase (BAP) or calf intestinal alkaline phosphatase (CIP) in
order to remove the 5' phosphate and prevent self-ligation of the
vector. Digestions are conducted at pH 8 in approximately 10 mM
Tris-HCl, 1 mM EDTA using about 1 unit of BAP or CIP per ug of
vector at 60.degree. C. for about one hour. In order to recover the
nucleic acid fragments, the preparation is extracted with
phenol/chloroform and ethanol precipitated. Alternatively,
religation can be prevented in vectors which have been double
digested by additional restriction enzyme digestion and separation
of the unwanted fragments.
[0092] For portions of vectors derived from CDNA or genomic DNA
which require sequence modifications, site specific mutagenesis may
be used (Zoller, M. J., and Smith, M. Nucleic Acids Res (1982)
10:6487-6500 and Adelman, J. P., et al, DNA (1983) 2:183-193). This
is conducted using a primer synthetic oligonucleotide primer
complementary to a single stranded phage DNA to be mutagenized
except for limited mismatching, representing the desired
mutation.
[0093] The size of the oligonucleotide primer is determined by the
requirement for stable hybridization of the primer to the region of
the gene in which the mutation is to be induced, and by the
limitations of the currently available methods for synthesizing
oligonucleotides. The factors to be considered in designing
oligonucleotides for use in oligonucleotide-directed mutagenesis
(e.g., overall size, size of portions flanking the mutation site)
are described by Smith, M. and Gillam, S. in Genetic Engineering:
Principles and Methods, Plenum Press (1981) 3:1-32. In general the
overall length of the oligonucleotide will be such as to optimize
stable, unique hybridization at the mutation site with the 5' and
3' extensions from the mutation site being of sufficient size to
avoid editing of the mutation by the exonuclease activity of the
DNA polymerase. Oligonucleotides used for mutagenesis in accordance
with the present invention usually contain from about 18 to about
45 bases, preferably from about 23 to about 27 bases. They will
usually contain at least about three bases 3' of the altered or
missing codon.
[0094] The method for preparing the modified bFGF genes generally
involves inducing a site-specific mutagenesis in the bFGF gene at a
specific codon using a synthetic nucleotide primer which omits the
codon or alters it so that it codes for another amino acid. It must
be recognized that when deletions are introduced, the proper
reading frame for the DNA sequence must be maintained for
expression of the desired protein.
[0095] The primer is hybridized to single-stranded phage such as
M13, fd, or deltaX174 into which a strand of the bFGF gene has been
cloned. It will be appreciated that the phage may carry either the
sense strand or antisense strand of the gene. When the phage
carries the antisense strand the primer is identical to the region
of the sense strand that contains the codon to be mutated except
for a mismatch with that codon that defines a deletion of the codon
or a triplet that codes for another amino acid. When the phage
carries the sense strand the primer is complementary to the region
of the sense strand that contains the codon to be mutated except
for an appropriate mismatch in the triplet that is paired with the
codon to be deleted. Conditions that may be used in the
hybridization are described by Smith, M. and Gillam, S., supra. The
temperature will usually range between about 0.degree. C. and
70.degree. C., more usually about 10.degree. C. to 50.degree. C.
After the hybridization, the primer is extended on the phage DNA by
reaction with DNA polymerase I, T.sub.4 DNA polymerase, reverse
transcriptase, or other suitable DNA polymerase. The resulting
dsDNA is converted to closed circular dsDNA by treatment with a DNA
ligase such as T.sub.4 DNA ligase. DNA molecules containing
single-stranded regions may be destroyed by S1 endonuclease
treatment.
[0096] The resulting double-stranded DNA is transformed into a
phage-supporting host bacterium. Cultures of the transformed
bacteria are plated in top agar, permitting plaque formation from
single cells which harbor the phage.
[0097] Theoretically, 50% of the new plaques will contain the phage
having, as a single strand, the mutated form; 50% will have the
original sequence. The resulting plaques are washed after
hybridization with kinased synthetic primer at a wash temperature
which permits binding of an exact match, but at which the
mismatches with the original strand are sufficient to prevent
binding. Plaques which hybridize with the probe are then picked,
cultured, and the DNA recovered.
[0098] C.4. Verification of Construction
[0099] In the constructions set forth below, correct ligations for
plasmid construction are confirmed by first transforming E. coli
strain MC1061 obtained from Dr. M. Casadaban (Casadaban, M., et al,
J Mol Biol (1980) 138:179-207) or other suitable host with the
ligation mixture. Successful transformants are selected by
ampicillin, tetracycline or other antibiotic resistance or using
other markers depending on the mode of plasmid construction, as is
understood in the art. Plasmids from the transformants are then
prepared according to the method of Clewell, D. B., et al, Proc
Natl Acad Sci (USA) (1969) 62:1159, optionally following
chloramphenicol amplification (Clewell, D. B., J Bacteriol (1972)
110:667). Several mini DNA preps are commonly used, e.g., Holmes,
D. S., et al, Anal Biochem (1981) 114:193-197 and Birnboim, H. C.,
et al, Nucleic Acids Res (1979) 7:1513-1523. The isolated DNA is
analyzed by dot blot analysis as described by Kafatos, F. C., et
al, Nucl Acid Res (1977) 7:1541-1552, restriction enzyme analysis,
or sequenced by the dideoxy nucleotide method of Sanger, F., et al,
Proc Natl Acad Sci (USA) (1977) 74:5463, as further described by
Messing, et al, Nucleic Acids Res (1981) 9:309, or by the method of
Maxam, et al, Methods in Enzymology (1980) 65:499.
[0100] C.5. Hosts Exemplified
[0101] Host strains used in cloning and procaryotic expression
herein are as follows:
[0102] For cloning and sequencing, and for expression of
construction under control of most bacterial promoters, E. coli
strains such as MC1061, DH1, RR1, B, C600hfl, K803, HB101, JA221,
and JM101 were used.
[0103] D. Illustrative Procedure The following examples are
intended to illustrate but not to limit the invention. The DNA
encoding the FGF starting material was obtained initially by
screening a bovine genomic library and obtaining a pivotal probe,
followed by retrieval of additional DNA. However, it would not be
necessary to repeat this procedure, as the sequence of the pivotal
probe is now known and could thus be constructed chemically in
vitro. In addition, bacteriophage harboring bovine aFGF and bFGF
and human aFGF and bFGF sequences are deposited at the American
Type Culture Collection. Thus, the DNA sequence used as the
starting material for the mutagenesis in the following examples is
available from a variety of sources.
EXAMPLE 1
Construction of pTrp-233 Bacterial Expression Plasmid
[0104] 1. Construction of the Synthetic Tryptophan Operon Promoter
and Operator Regulatory Sequence
[0105] The ten oligodeoxynucleotides shown in FIG. 4 were
synthesized by the phosphotriester method and purified. 500 pmole
of each oligodeoxynucleotide except 1 and 10 were phosphorylated
individually in 20 ul containing 60 mM Tris-HCl, pH 8, 15 mM DTT,
10 mM MgCl.sub.2, 20 uCi of [lambda-.sup.32 P]-ATP and 20 units of
polynucleotide kinase (P/L Biochemicals) for 30 min. at 37.degree.
C. This was followed by the addition of 10 ul containing 60 mM
Tris-HCl, pH 8, 15 mM DTT, 10 mM MgCl.sub.2, 1.5 mM ATP and 20
additional units of polynucleotide kinase followed by another 30
min incubation at 37.degree. C. Following incubation the samples
were incubated at 100.degree. C. for 5 min. 500 pmole of
oligodeoxynucleotides 1 and 10 were diluted to 30 ul in the above
buffer without ATP.
[0106] 16.7 pmole of each oligodeoxynucleotide constituting a
double stranded pair (e.g. oligodeoxynucleotides 1 and 2, 3 and 4
etc. FIG. 4) were mixed and incubated at 90.degree. C. for 2 min
followed by slow cooling to room temperature. Each pair was then
combined with the others in the construction and extracted with
phenol/chloroform followed by ethanol precipitation. The
oligodeoxynucleotide pairs were reconstituted in 30 ul containing 5
mM Tris-HCl, pH 8, 10 mM MgCl.sub.2, 20 mM DTT, heated to
50.degree. C. for 10 min and allowed to cool to room temperature
followed by the addition of ATP to a final concentration of 0.5 mM.
800 units of T4 DNA ligase were then added and the mixture
incubated at 12.5.degree. C. for 12-16 hours.
[0107] The ligation mixture was extracted with phenol/chloroform
and the DNA ethanol precipitated. The dried DNA was reconstituted
in 30 ul and digested with EcoRI and PstI for 1 hour at 37.degree.
C. The mixture was extracted with phenol/chloroform and ethanol
precipitated followed by separation of the various double stranded
DNA segments by electrophoresis on an 8% polyacrylamide gel,
according to the method of Laemmli et al, Nature (1970) 227:680.
The DNA fragments were visualized by wet gel autoradiography and a
band corresponding to approximately 100 bp in length was cut out
and eluted overnight as described. The excised synthetic DNA
fragment was ligated to plasmids M13-mp8 or M13-mp9 (Messing and
Vieira, supra) similarly digested with EcoRI and PstI, and
submitted to dideoxynucleotide sequence analysis (Sanger et al.
supra) to confirm the designed sequence shown in FIG. 4. This
designed sequence contains the promoter (-35 and -10 regions) and
operator regions of the tryptophan operon (trp) as well as the
ribosome binding region of the tryptophan operon leader peptide.
Analogous sequences to that shown in FIG. 4 have been proven to be
useful in the expression of heterologous proteins in E. coli
(Hallewell, R. A., and Emtage, S., Gene (1980) 9:27-47, Ikehara,
M., et al. Proc Natl Acad Sci (USA) (1984) 81:5956-5960).
[0108] 2. Construction of the Synthetic trp Promoter/Operator
Containing Plasmid, pTrp-233
[0109] Plasmid pKK233-2 (Amann, E. and Brosius, J., supra) was
digested to completion with NdeI followed by the filling in of the
termini by the method of Maniatis et al, Molecular Cloning, Cold
Spring Harbor Laboratories, 1982 at p. 394, with 5 units of E. coli
polymerase I, Klenow fragment (Boehringer-Mannheim, Inc.) and the
addition of dATP, dCTP, dGTP and TTP to 50 uM. This was incubated
at 25.degree. C. for 20 min. Following phenol/chloroform extraction
and ethanol precipitation, the NdeI-digested DNA was ligated and
transformed into E. coli (Nakamura, K. et al, J Mol Appl Genet
(1982) 1: 289-299). The resulting plasmid lacking the NdeI site was
designated pKK-233-2-Nde.
[0110] Twenty nanograms of plasmid pKK-233-2-Nde was digested to
completion with EcoRI and PstI followed by calf intestinal
phosphatase treatment (Boehringer Manheim) in accordance with
Maniatis et al., supra at pp. 133-134. Fifty nanograms of the
synthetic trp promoter/operator sequence obtained from M13 RF.,
(described above) by digesting with EcoRI and PstI, were mixed with
ten nanograms of EcoRI-PstI digested pKK-233-2-Nde and ligated with
T4-DNA-ligase as described followed by transformation into E. coli
JA221 1pp-/I'lacI. Transformants were screened for the presence of
plasmid DNA containing the 100 bp EcoRI-PstI synthetic trp
promoter/operator; the correct plasmid was then isolated and
designated pTrp-233.
EXAMPLE 2
Construction of Plasmid pTSF11
[0111] A. Human Basic Fibroblast Growth Factor
[0112] The bovine basic FGF cDNA was used to develop hybridization
probes to isolate basic FGF clones from human cDNA and genomic
libraries as described in U.S. Ser. No. 869, 382, supra, Abraham,
J.A. et al, Science (1986) supra, and Abraham, J. A. et al, The
EMBO Journal (1986) supra, all of which are incorporated herein by
reference.
[0113] There are only two amino acid differences between basic
bovine FGF and human FGF, at position 123, where the bovine protein
has Ser and the human protein has Thr, and at position 137, where
the bovine protein has Pro and the human has Ser. These differences
are the result of a single nucleotide difference in each case;
therefore bovine cDNA may conveniently be modified by site directed
mutagenesis as described below to encode the human protein, and,
indeed, standard site-specific mutagenesis techniques were used to
alter these codons. The lambda BB2 clone (ATCC No. 40196) was
digested with EcoRI and the 1.4 kb region spanning the bFGF
protein-encoding portion was ligated into the EcoRI site of M13mp8
and phage carrying the insert in the correct orientation were
recovered. The in vitro mutagenesis was carried out in the presence
of three oligonucleotides: the "universal" primer, a 17-mer; the
mutagenic 16-mer 5'-GAAATACACCAGTTGG-3'; which alters the coding
sequence at codon 123, and the mutagenic 17-mer
5'-ACTTGGATCCAAAACAG-3', which alters the sequence at codon 137.
The mutagenized phage was also subjected to a second round of in
vitro primer-directed mutagenesis to create a HindIII site 34 bp
downstream from the translation termination codon using the
mutagenic 25-mer, 5'-TTTTACATGAAGCTTTATATTTCAG-3'. The resultant
mutated DNA was sequenced by dideoxynucleotide sequence analysis
(Sanger et al, supra) to confirm that the desired mutagenesis had
occurred, and the approximately 630 bp fragment spanning the
FGF-coding region was excised with HindIII and ligated into HindIII
digested pUC13 to obtain the intermediate plasmid pJJ15-1.
[0114] B. Construction of Gene with Synthetic Coding Region for
N-terminal End of FGF Gene
[0115] In order to lower the G+C content of the 5' end (the first
125 base pairs) of the coding region contained in pJJ15-1, a
synthetic DNA fragment was constructed with the sequence shown
below using the following synthetic oligonucleotides. The
oligonucleotides were annealed in pairs, ligated together
sequentially, and ligated into HindIII cut m13mp9. The sequence of
the synthetic 125 bp insert in mp9 was confirmed by dideoxy
sequencing. The NdeI to HhaI subfragment of the synthetic insert
was isolated, joined to the 377 base pair, HhaI-to-HindIII DNA
fragment from JJ15-1 that spans approximately the carboxy-terminal
three quarters of the basic FGF coding sequence, and then ligated
into the NdeI and HindIII sites of the expression vector pTrp-233
to yield the plasmid pTFS11.
[0116] Oligonucleotides:
1 Number Sequence 1670 5'-pAGCTTCATATGGCTGCTGGTTCTATCACTACC 1623R
5'-pCTGCCAGCTCTGCCAGAAGACGGTGGTT 1624R
5'-pCTGGTGCCTTCCCACCAGGTCACTTCAA 1625R
5'-pAGACCCAAAACGTCTGTACTGCAAAAAC 1680 5'-pGGTGGTTTCTTCCTGCGCA 1679
5'-pTAGAACCAGCAGCCATATGA 1622 5'-pTCTTCTGGCAGAGCTGGCAGGGTAGTGA 1619
5'-pACCTGGTGGGAAGGCACCAGAACCACCG 1626
5'-pAGTACAGACGTTTTGGGTCTTTGAAGTG 1673
5'-pAGCTTGCGCAGGAAGAAACCACCGTTTTTGC
[0117] Construction of Synthetic Gene for the Amino Terminal Region
of bFGF:
[0118] HindIII NdeI
2 10 20 30 40 50 AGCTTCATATG GCTGCTGGTT CTATCACTAC CCTGCCAGCT
CTGCCAGAAG AGTATAC CGACGACCAA GATAGTGATG GGACGGTCGA GACGGTCTTC 60
70 80 90 100 ACGGTGGTTC TGGTGCCTTC CCACCAGGTC ACTTCAAAGA CCCAAAACGT
TGCCACCAAG ACCACGGAAG GGTGGTCCAG TGAAGTTTCT GGGTTTTGCA HhaI 110 120
130 CTGTACTGCA AAAACGGTGG TTTCTTCCTG CGCA GACATGACGT TTTTGCCACC
AAAGAAGGAC GCGTTCGA
[0119] A plasmid map of pTSF11 is given in FIG. 5 of the
accompanying drawings.
EXAMPLE 3
Preparation of Expression Vector for Mutagenized Gene Inserts
[0120] The HindIII site of the polylinker region of plasmid pUC9
was removed so as to facilitate subcloning mutated DNA into the
final expression vector illustrated in FIG. 5. Approximately 5 ug
of pUC9 (New England Biolabs) was digested with HindIII (20 units;
New England Biolabs) according to the manufacturers instructions in
0.05 ml. The reaction was then supplemented with 0.5 mM dNTPs and
the Klenow fragment of DNA Polymerase I (5 units; Boehringer
Manheim) and incubated for 30 minutes at 15.degree. C. The reaction
was then extracted twice with an equal volume of phenol/chloroform
(1/1), twice with chloroform, made 0.2M NaCl, and then precipitated
with two and a half volumes of ethanol. The precipitate was
collected by centrifugation (15,000 g in a Microfuge at 4.degree.
C.), lyophilized, and then incubated in 0.1 ml with 1X kinase
ligase buffer, 1 mM ATP, and T4 DNA ligase (20 units; New England
Biolabs) for 4 hours at 12.degree. C.
[0121] An aliquot of the reaction (0.01 ml) was then used to
transfect competent MC1061 cells. The transfected bacteria were
grown overnight on L agar plates supplemented with 100 ug/ml
ampicillin. DNA was isolated from 6 colonies by the alkaline lysis
procedure and tested for the loss of the HindIII site. A bacteria
containing the plasmid, pUC9delH3-1, was isolated. The plasmid DNA
was prepared and 10 ug was digested in 0.4 ml with PvuI (20 units;
New England Biolabs) and EcoRI (50 units; New England Biolabs) for
2 hours according to the manufacturers instructions. The reaction
was then extracted twice with an equal volume of phenol/chloroform
(1/1) and twice with an equal volume of phenol and then
precipitated with isopropanol. The precipitate was collected by
centrifugation, washed with 70% ethanol, lyophilized, resuspended
in 0.008 ml water and the .about.2.07 kb PvuI-EcoRI fragment of
pUC9delH3-1 (designated fragment A) containing the origin of
replication was isolated by acrylamide gel electrophoresis.
[0122] Concurrently pTSF11 DNA (10 ug) was incubated with PvuI (10
units) and EcoRI (10 units) in 0.15 ml for 1 hour at 37.degree. C.
according to the manufacturers directions and collected as
described above. The .about.1.3 kb PvuI-EcoRI fragment of pTSF11
containing the Trp promoter/operator region, FGF coding region and
the transcription termination sequences, designated fragment B of
pTSF11, was isolated by polyacrylamide gel electrophoresis and
ligated to the .about.2.07 kb PvuI-EcoRI fragment A of pUC9delH3-1,
and used to transfect competent E. coli HB101 cells. The bacteria
were grown overnight on L agar plates supplemented with 100 ug/ml
ampicillin. Plasmid DNA from one recombinant, pUC9delH3-pTSF-3, was
isolated and shown to contain the expected restriction map (HindIII
cuts the plasmid only once; the sizes of HindIII-EcoRI,
HindIII-PvuI and HindIII-PstI fragments are approximately 560 and
2900, 800 and 2700, and 560 and 2900 bp respectively. DNA from the
plasmid pUC9delH3-pTSF-3 was isolated and 200 ug incubated in 1.0
ml with 100 units of HindIII, 100 units of EcoRI, 5 mM spermidine
for 4 hours at 37.degree. C. according to the manufacturers
instructions. The reaction was butanol extracted to reduce the
volume to 0.7 ml and then extracted with phenol/chloroform and
chloroform as described above. The DNA was collected by ethanol
precipitation and the .about.2.9 kb HindIII-EcoRI fragment
containing the ampicillin resistance gene, the origin of
replication and the two transcription stop signals, designated
fragment C of pUC9delH3-pTSF-3, was isolated by two sequential runs
on polyacrylamide gels. This vector fragment serves as the
preferred vector for expressing any of the DNA which has been
altered by in vitro mutagenesis. The construction of this vector is
illustrated in FIG. 5.
[0123] Plasmid pUC9-pTSF11, a vector closely resembling plasmid
pUC9delH3-pTSF-3 but containing an intact HindIII site in the
multiple site polylinker region, can also be used as a preferred
vector for expressing both recombinantly produced FGF (all forms)
and any of the analogs of the present invention. This vector was
constructed by individually digesting plasmids pUC9 and pTSF11 with
PvuI and EcoRI, isolating the .about.2.07 kb PvuI-EcoRI vector
fragment from pUC9 and the .about.1.3 kb PvuI-EcoRI fragment
containing the trp promoter/operator region, FGF coding region, and
the transcription termination sequences from pTSF11, and ligating
the two isolated fragments. This vector can then be used to express
the FGF analog gene sequences as taught with pUC9-pTSF11 by
inserting the HindIII-EcoRI DNA cassettes into the appropriately
digested vector and transforming E. coli bacterium.
EXAMPLE 4
Generalized Procedure for Production of FGF Mutants
[0124] The following protocol can be used to construct all of the
DNA sequences encoding the FGF analogs described herein. Plasmid
FGFt7910 was constructed by ligating the .about.603 bp
EcoRI-HindIII DNA fragment of pTSF11 (comprising the Trp promoter
region and the DNA encoding human bFGF) into the EcoRI-HindIII
sites of an M13mp9 vector. Once the single-stranded DNA of FGFt7910
was isolated, in vitro mutagenesis, as described by Zoller and
Smith, supra, may be performed utilizing one or more of the
synthetic oligonucleotides designated in any of the tables
herein.
[0125] The conditions for site specific mutagenesis can be
generalized as follows. One ug of the single stranded DNA is
hybridized with 5 ng of the phosphorylated mutagenic
oligonucleotide(s) (23 mer to 25 mer encoding the appropriate
mutation) and 1 ng of the M13 universal sequencing primer (17 mer
purchased from P.L. Biochemicals) for 5 to 15 minutes at 55.degree.
C. in 0.01 ml solution of 10 mM Tris-HCl, pH 7.5, and 10 mM
MgCl.sub.2. The reaction is cooled to room temperature and then
added to 0.01 ml of 0.12 mM dXTPs, 5 units Klenow fragment of DNA
polymerase I (Boehringer Mannheim), 20 units of T4 DNA ligase (New
England Biolabs), and incubated for 4-6 hours at 15.degree. C. An
aliquot (0.002 ml) of the reaction is then added to competent JM101
bacteria and plated overnight on L agar plates at 37.degree. C. The
DNA of the resulting M13 clones is transferred to each of two
nitrocellulose filters, baked under vacuum at 80.degree. C. for 2
hours and then incubated for 2 hours at 42.degree. C. in
pre-hybridization solution: 6.times.SSC (1.times.SSC is 150 mM
NaCl, 15 mM sodium citrate, pH 7.0), 0.1% sodium dodecyl sulfate,
2.times.Denhardt's (0.05% ficoll, 0.05% polyvinylpyrrolidone, 0.05%
bovine serum albumin) solution) and 0.4 mg/ml of denatured salmon
sperm DNA. The filters are then incubated for 3 hours at 42.degree.
C. with fresh pre-hybridization solution containing the appropriate
mutagenic oligonucleotide which has been 5'-end labeled with
[lambda-.sup.32P]-ATP and T4 polynucleotide kinase. The filters are
then washed twice with 4.times.SSC at room temperature for 15
minutes, once for 15 minutes at 65.degree. C., once at room
temperature in TMACL solution (3M tetramethylammonium chloride, 50
mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.1% SDS) and once at 65 C. in
TMACL solution and then used to expose X-ray film overnight at room
temperature. Clones corresponding to dark positives on the X-ray
film are then picked from the original plate, the DNA is isolated
and then analyzed for the mutated sequence by dideoxy sequencing.
If two oligonucleotides are being used to produce a double mutant
then one filter is screened with one oligonucleotide and the other
filter is screened with the second oligonucleotide. Double mutants
will have a positive signal with both oligonucleotides.
[0126] The DNA replicative form of the mutated M13 clone is then
prepared, digested with EcoRI and HindIII, and the DNA fragment
encoding the mutated FGF is isolated by agarose gel
electrophoresis. The DNA fragment is then ligated to Fragment C of
pUC9delH3-pTSF-3 (described in Example 3 and illustrated in FIG.
6), transfected into competent HB101 cells, and grown overnight on
L agar plates supplemented with 100 ug/ml ampicillin. Colonies are
selected, grown in L broth supplemented with 100 ug/ml ampicillin
and then the plasmid DNA is isolated from the bacteria. The DNA is
then used to transform competent E. coli B cells (Luria and
Delbrck, Arch Biochem (1942) 1:111).
EXAMPLE 5
Preparation of Basic FGF Analog bFGF-C34/101S
[0127] In this example, cysteine residues at positions 34 and 101
of the human basic FGF protein were changed to serine residues
thereby producing a double mutation. Approximately 2 micrograms
each of the mutagenic 23-mer 5'-ACGTCTGTACTCCAAAAACGGTG-3' (#2222);
which alters the sequence at codon 34, and the mutagenic 23-mer
5'-TACAGACGAGTCTTTCTTTTTTG-3' (#2323); which alters the sequence at
codon 101 were incubated in 50 ul of 1.times.kinase/ligase buffer
(7 mm Tris-HCl pH 7.6, 10 mm MgCl.sub.2, 5 mm dithiothreitol) with
1 mM ATP and 5 units T4 polynucleotide kinase for 30 minutes at
37.degree. C. The phosphorylated oligonucleotides were diluted
two-fold into 1mM Tris-HCl.sub.1, pH 8.0 and 1 mM EDTA.
[0128] One ug of the single stranded M13 template FGFt 7910 was
incubated with 20 ng-each of the phosphorylated oligonucleotides
2222 and 2323 and 1 ng of the universal M13 sequencing primer (New
England Biolabs) in 10 ul of 10 mm Tris-HCl pH 7.5 and 10 mm
MgCl.sub.2 for 20 minutes at 55.degree. C. and then at room
temperature for 10 minutes. The reaction was then supplemented with
0.5 mM dXTPs, 5 units of the Klenow fragment of DNA polymerase I, 1
mM ATP, and 20 units T4 DNA ligase and incubated at 15.degree. C.
for 5 hours. 2 ul of the reaction was then used to transform
competent JM101 bacteria. The transformed cells were plated
overnight at 37.degree. C., and the resulting M13 DNA was
transferred to nitrocellulose filters as described above. One ug of
each of the oligonucleotides (#2222 and #2323) was phosphorylated
as described above except 1 mCi of [lambda-.sup.32P]-ATP (New
England Nuclear #NEGO35C, approximately 5 mCi/nmole) was
substituted for cold ATP. The radioactive probes were then added
separately to the duplicate filters and processed as described
above. M13 clones corresponding to positive signals from the
resulting autoradiographs were isolated and the single stranded M13
DNA prepared by the method of Sanger et al, supra. The resulting
M13 DNA template (#8725) was analyzed by dideoxy sequencing and
shown to contain the expected changes.
[0129] The double stranded replicative form (RF) of the M13
template #8725 was isolated by the method of Birnboim and Doly,
Nucl Acids Res (1979) 7:1513-1519. In this procedure, fifty ul of
M13 phage #8725, isolated from infected JM101, were used to
inoculate 50 ml of a JM101 culture (saturated culture,
20.times.diluted into J broth) which were then grown for 6 hours at
37.degree. C. The bacteria were harvested by centrifugation and the
DNA isolated as described by Birnboim and Doly, supra.
Approximately 5 ug of the RF DNA was cut in 0.4 ml of
1.times.HindIII buffer as described by the manufacturer with 40
units each of HindIII and EcoRI for 2 hours at 37.degree. C. The
reaction was then extracted twice with equal volumes of phenol/
chloroform (1/1) and twice with chloroform and then ethanol
precipitated. The resulting DNA was collected by centrifugation,
washed with 70% ethanol, lyophilized, and resuspended in 20 ul of 1
mM Tris-HCl, pH 8.0, and 1 mM EDTA. The resulting EcoRI-HindIII
fragment was isolated by agarose gel electrophoresis using
GENECLEAN (BI0101 Inc.; La Jolla, Calif.) according to the
manufacturer's instructions. Approximately 50 ng of the
EcoRI-HindIII insert was ligated to the EcoRI-HindIII vector
fragment C of pUC9delH3-pTSF-3 and used to transform competent
MC1061 cells. The bacteria were processed as described in Example 7
in order to purify the analog and then the purified analog was
tested for its ability to stimulate adrenal cortex endothelial
(ACE) cells as described in Example 8.
[0130] Other mutants containing cysteine-to-serine substitutions
have been constructed and expressed in bacteria. These
constructions contain from one to four Cys.fwdarw.Ser
substitutions. All of these substitutions result in the recovery of
an FGF protein with varying levels of activity. The specific
constructions are listed below in Table 1. Each of these FGF analog
proteins has been isolated using a heparin affinity column.
3 TABLE 1 bFGF Analog.sup.+ Oligonucleotide.sup.# Number* 1)
bFGF-C78S 5'-pCAAAGGAGTGTCTGCAAACCGTT 2217 2) bFGF-C96S
5'-pAGCTTCTAAATCTGTTACAGACG 2218 3) bFGF-C78/96S 2218/2217 4)
bFGF-C34/78/ 2217/2218/ 96/101S 2222/2323 25
5'-pACGTCTGTACTCCAAAAACGGTG 2222 5'-pTACAGACGAGTCTTTCTTTTTTG 2323
5) bFGF-C34/78/96S 2222/2218/2217 6) bFGF-C78/96/101S
2217/2218/2323 7) bFGF-C34/78/101S 2222/2217/2323 8) bFGF-C34/78S
2222/2217 9) bFGF-C34/101S 2222/2323 .sup.+Analogs of bFGF are
defined as: bFGF-XYZ where X is the amino acid in the native human
bFGF sequence that is being stated, Y is the position of amino acid
X, and Z is the amino acid residue that is being substituted for X
at position Y. Multiple mutations are indicated. Mutations that
involve the deletion of a region of the native bFGF protein are
indicated with parenthesis #(X-Z) with the deleted region defined
by the amino acids included in residues X to Z.
.sup.#Oligonucleotide used for in vitro mutagenesis. *Number of the
oligonucleotide used for the mutagenesis.
[0131] NOTE: This legend is applicable to all of the following
tables and analog descriptions. The one-letter code depicting
specific amino acids is as follows:
4 Three-Letter One-Letter Amino Acid abbreviation symbol Alanine
Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D
Asparagine or Asx B aspartic acid Cysteine Cys C Glutamine Gln Q
Glutamic acid Glu E Glutamine or Glx Z glutamic acid Glycine Gly G
Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K
Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S
Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V
[0132] The bFGF analogs described below in Table 2 were tested for
their ability to stimulate bovine adrenal cortex endothelial cell
proliferation. As indicated below, the double mutant bFGF-C78/96S
has enhanced activity compared to wild type bFGF. Any alteration of
cysteines at the conserved positions 34 and 101, that is, positions
conserved throughout the family of FGF-related molecules including
human and bovine aFGF, bovine and xenopus bFGF, murine int-2, human
hst and human KS3, significantly decreased the activity of the
resulting analog.
5TABLE 2 Activity of bFGF and Various Cysteine Analogs in the ACE
Cell Proliferation Assay FGF ANALOG % ACTIVITY WILD TYPE bFGF 100
C78S* 53 C96S* 95 C78/96S* 159 C34/101S 2 C34/78/101S.sup.+ 2
C78/96/101S.sup.+ 23 C34/78/96/101S.sup.+ 7 *Average of 2
independent assays. .sup.+Average of 3 independent assays.
EXAMPLE 6
Heparin Binding Assay
[0133] The interaction of bFGF analogs with heparin is
characterized by the ionic strength (NaCl concentration) of a
Tris-HCl buffered solution required to elute the protein from
heparin-Sepharose resin. This analysis determines the NaCl
concentration required to remove the bFGF analogs that were bound
to the heparin-Sepharose resin.
[0134] A heparin-5PW column was prepared by Bio-Rad Laboratories
(Richmond, Calif.) by introducing heparin onto Bio-Gel TSK-50
resin. The column (75.times.7.5 mm I.D.; 4-6 mg/ml of heparin) was
used with two Beckman model 110B Solvent Delivery Modules; a
Beckman model #421 Controller, and a Kratos Spectraflow absorbance
detector model #757. Samples were loaded onto the column in 0.5 M
NaCl, 20 mM Tris HC1, pH 7.5 and then eluted with a gradient of
0.6-3.0 M. Protein was monitored by absorbance at 214 nm. The
conductivity of various samples was tested and compared to buffered
NaCl standards to determine NaCl concentrations along the gradient.
Data from the absorbance detector was collected and analyzed using
Access*Chrom (Nelson Analytical, Inc., Cupertino).
[0135] The cysteine-substituted FGF analogs of Example 5 were
analyzed by heparin-HPLC. The analog bFGF-C78/96S elutes as a
single species with (FIG. 7a) and without (FIG. 7b) dithiothreitol
treatment. This is in contrast to wild type bFGF which elutes as a
single species with dithiothreitol treatment (FIG. 7c) but as a
heterogeneous species in the absence of a reducing agent (FIG. 7d).
In addition, the double mutant does not exhibit any heterogeneity
when analyzed by reverse phase-HPLC or by size exclusion
chromatography as is the case for wild type bFGF.
[0136] The single cysteine-substituted mutants, C78S and C96S, when
analyzed as above, will also reduce the heterogeneity of the
resulting product as compared to wild type bFGF.
[0137] The same supernatant fraction is tested for mitogenic
activity using the endothelial cell proliferation assay or the
Balb/c 3T3 thymidine uptake assay (Hauschka et al J Biol Chem
(1986) 261:12665-12674) described below.
EXAMPLE 7
Isolation of Recombinant Human bFGF and Analogs of bFGF, and
Characterization of the In Vitro Activity
[0138] This will describe the procedure for isolating approximately
100 ug of recombinant bFGF from bacteria. The method can be scaled
up to obtain larger quantities. Bacteria containing the appropriate
plasmid are grown overnight in L broth supplemented with 100 ug/ml
ampicillin. 0.2 ml of the culture are inoculated into 100 ml of
1.times.M9 salts (Maniatis et al, supra) 0.4% glucose, 2 ug/ml
thiamine, 200 ug/ml MgSO.sub.4.7 H.sub.2O, 0.5% casamino acids, 100
uM CaCl.sub.2 and 100 ug/ml ampicillin and grown on a shaker at
37.degree. C. The culture is supplemented with 20 ug/ ml indole
acrylic acid upon reaching an optical density of 0.1 at a
wavelength of 550 nm. The bacteria are harvested upon reaching an
optical density of 1.0 by centrifugation (5000 rpm, 4.degree. C.,
15 minutes), quick frozen in a dry ice/ ethanol bath and then
stored at -80.degree. C. The bacterial pellet is resuspended in 10
ml of 0.02 M Tris-HCl, pH 7.5, 0.6M NaCl, 1 mM PMSF, 80 ng/ml
aprotinin, and 10 ug lysozyme and incubated at 4.degree. C. for 15
minutes. The mixture is then sonicated 5 times at a setting of 3
using a Sonicator Cell Disruptor (Heat Systems). The reaction is
then incubated with DNAseI (100 units) and RNAseA (100 units) for
15 minutes at 4.degree. C. and then centrifuged for 15 minutes at
4.degree. C. at 10,000 rpm. The supernatant is then loaded onto an
8.0 ml heparin-Sepharose column (Pharmacia) which has been
prewashed with 3.0 M NaCl, 10 mM Tris-HCl, pH 7.5 and equilibrated
with 0.6 M NaCl, and 0.02 M Tris-HCl, pH 7.5. The column is washed
with 0.6 M NaCl until no detectable protein, as judged by
absorbance at 280 nm, is eluting off the column. The column is then
washed with 1.0 M NaCl and the bound material eluted off with 2.0 M
NaCl, 20 mM Tris-HCl, pH 7.5. This purification scheme can be
performed in buffers in the presence or absence of 5 mM
dithiothreitol.
EXAMPLE 8
Mitogenic Assays
[0139] FGF analogs are tested for either agonist or antagonist
activity with respect to wild type FGF in an adrenal cortex
capillary endothelial (ACE) cell proliferation assay as described
by Gospodarowicz et al (J Cell Physiol (1985) 122:323-332).
Individual analogs were tested as follows. Approximately
1.times.10.sup.4 cells were plated in 2 ml of DME 16 supplemented
with 10% calf serum, 50 units/well of penicillin and 50 units/well
of streptomycin in a Falcon 6-well plate. Appropriate dilutions (1
pg/ml to 1 ug/ml final concentration) of each sample, as well as
wild type (bovine pituitary basic) FGF were added in 10 ul volumes
to the cells. As a negative control, 6 wells without added FGF
samples were run simultaneously. The plates were incubated at
37.degree. C. for 48 hours and cell samples were re-added to the
appropriate well and incubated for an additional 48 hours at
37.degree. C. Cells were then trypsinized, collected and counted in
a Coulter counter.
[0140] Balb/c 3T3 cells, obtained from ATCC, were used to test for
the ability of bFGF preparations to stimulate DNA synthesis
essentially by the method described by Hauschka et al (1986) supra.
Cells were seeded onto 96 well plates at a density of about
20,000/well in 0.2 ml Dulbecco's modified Eagle's medium (DME;
GIBCO) containing 4.5 g/liter glucose, 2.2 g/liter NaHCO.sub.3, 50
units/ml penicillin, 50 ug/ml streptomycin, and 10% calf serum
(HYCLONE) and allowed to grow to confluency (2-3 days) in a 5%
CO.sub.2, 95% Air incubator at 37.degree. C. Cultures were switched
to serum free medium containing 0.01% bovine serum albumin, after
which 0.01 ml of appropriate dilutions of test substance were
added. Cultures were incubated at 37.degree. C. for an additional
16 hr, after which the medium was changed to serum free medium
containing 0.01% bovine serum albumin plus 50 uCi/ml of [.sup.3H]
thymidine. Plates were then incubated for 2 hours after which TCA
precipitable counts were determined as follows. The 96 well plate
was placed on ice and the medium carefully removed, washed twice
with cold PBS, followed by incubation with 10% trichloroacetic acid
for 20 min at 4.degree. C. Remaining radioactivity was solubilized
in 0.1 N NaOH, and counted.
[0141] These assays were used to test the FGF analogs for their
respective agonist or antagonist activity toward wild type basic
FGF. The ability of FGF analogs to serve as antagonists to basic
FGF is characterized by mixing appropriate quantities, such as
1-1000 ng, of the particular analog with 1 ng of basic FGF and
testing the mixture in the above-described assays.
EXAMPLE 9
Construction of Receptor Binding FGF Analogs
[0142] A number of oligonucleotides were constructed and tested in
an FGF receptor competitive binding assay. The specific mutants are
provided below and include single amino acid substitutions, double
amino acid substitutions and deletion mutations.
6 Analog Oligonucleotide Number bFGF-K35S
5'-pGTCTGTACTGCTCAAACGGTGGTT 2553 bFGF-R42L
5'-pTTTCTTCCTGCTCATCCACCCCG 2327 bFGF-D46A 5'-pCATCCACCCCGCCGGCCGA-
GTGG 2221 bFGF-R48L 5'-pCCCCGACGGCCTAGTGGACGGGG 2454 bFGF-R48A
5'-pACCCCGACGGCGCAGTGGACGGGG 2555 bFGF-D50A
5'-pCGGCCGGAGTGGCCGGGGTCCGCG 2224 bFGF-V52K
5'-pGAGTGGACGGGAAACGCGAGAAGAG 2491 bFGF-R53L
5'-pGGACGGGGTCCTCGAGAAGAGCG 2220 bFGF-K55M 5'-pGGTCCGCGAGATGAGCGAC-
CCAC 2223 bFGF-K55I 5'-pGGTCCGCGAGATAAGCGACCCACA 2567 bFGF-D57A
5'-pCGAGAAGAGCGCCCCACACATCA 2225 bFGF-H59N
5'-pGAGCGACCCAAACATCAAACTAC 2383 bFGF-R90T 5'-pAGAAGATGGAACTTTACTA-
GCTTC 3088 bFGF-D99A 5'-pATGTGTTACAGCAGAGTGTTTCT 2381 bFGF-E100A
5'-pGTTACAGACGCCTGTTTCTTTTTTG 2549 bFGF-E100S
5'-pGTGTTACAGACAGTTGTTTCTTTTT 2380 bFGF-E105S
5'-pGTTTCTTTTTTTCACGATTGGAGT 2556 bFGF-R106L
5'-pCTTTTTTGAACTATTGGAGTCTA 2494 bFGF-E108A
5'-pTGAACGATTGGCATCTAATAACTA 2554 bFGF-Y112A
5'-pAGTCTAATAACGCAAATACTTACCG 2450 bFGF-N113S
5'-pCTAATAACTACAGTACTTACCGG 2452 bFGF-R116T
5'-pCAATACTTACACTTCAAGGAAATA 3091 bFGF-R118L
5'-pCAATACTTACCTGTCAAGGAAAT 2483 bFGF-K119S
5'-pACCGGTCAAGGTCTTACACCAGTTG 2548 bFGF-(41-43)
5'-pGGTGGTTTCTTCCACCCCGACGGC 2336 bFGF-(49-51)
5'-pCCCGACGGCCGAGTCCGCGAGAAG 2335 bFGF-(62-64)
5'-pCCACACATCAAACAAGCAGAAGAG 2334 bFGF-(83-85)
5'-pGCAAACCGTTACAAAGAAGATGGA 2333 bFGF-(105-107)
5'-pTGTTTCTTTTTTGAGTCTAATAAC 2332 bFGF-(112-114)
5'-pGAGTCTAATAACTACCGGTCAAGG 2337
[0143] The FGF analogs were produced as described in Example 7
using an appropriate expression vector, such as plasmid
pUC9delH3-pTSF11-3 or pUC9-pTSF11, and isolated by
heparin-Sepharose chromatography.
[0144] A competitive binding assay was established to determine the
relative affinity of FGF analogs compared to that of recombinant
basic FGF for the FGF receptor. Analogs having high affinity for
the FGF receptor and reduced mitogenic activity are designated
potential FGF antagonists.
[0145] The assay involved binding saturating concentrations of
[.sup.125I]-basic recombinant FGF (10 ng/ml) to Balb/c 3T3 cells in
the presence of various concentrations of unlabeled FGF or analogs.
The binding was conducted at 4.degree. C. for 3-4 hours to
establish equilibrium. The cells were then washed 12.times.with a
0.1% gelatin, 2 N NaCl balanced salt solution containing 50 mM
Hepes to maintain the pH at 7.5. Cells were solubilized in 1 N NaOH
and cell-associated radioactivity measured. Following this
procedure, the non-specific binding was kept at or below 5%. The
affinity of an analog for the FGF receptor was determined relative
to that of bovine pituitary FGF by taking the ratio of the
concentration of analog that inhibits specific-binding by 50% over
the concentration of FGF that inhibits specific binding by 50%. A
ratio of less than 1 indicated that the analog has a higher
affinity for the FGF receptor than FGF and a ratio of greater than
1 indicated that the analog has a lower affinity for the FGF
receptor than FGF.
[0146] A number of analogs that had reduced mitogenic activity in
the ACE assay had equal or higher affinity for the FGF receptor
compared to bovine pituitary FGF. Those mutants that had less than
5% of wild type activity in the ACE assay but had equal or higher
affinity for the FGF receptor include: R31S, K35S, D46A, R48L,
D50A, V52K, R53L, R90T, E100S, E100A, R106L, R116T, R118L, K119S.
These compounds may be useful as antagonists.
[0147] These analogs were also tested for mitogenic ability using
either one of the previously described assays. The results are
provided below in Table 3.
7TABLE 3 Activities of FGF Analogs FGF-Rc 3T3 Mito. ACE Mito. Comp.
Heparin Analog Heparin -/+ Heparin -/+ Heparin -/+ Elution hFGF(b)
100%/100% 100%/100% 1.0/1.0 1.58 M (EC.sub.50) (630 pg/ml) (160
pg/ml) (10 ng/ml) hFGF(b) 50%/100% 1.62 M 25-155 hFGF(a) 5%/25%
0.01%/10% 0.5/ 1.23 M bba* <0.2%/100% 0.3/ 1.43 M K27M 0.3/0.2
D28K 8.8%/ 1.0/1.3 KKR# 8.5%/8.5% 0.11%/4.2% .about.1.5 M R31S
100%/100% 0.45%/17.8% 1.0/0.8 K35S 2.6%/55.5% 0.1/0.05 1.23 M D46A
2.8%/ 0.5/ R48L 1.7%/ 0.16/2.0 R48A 8.9%/84.2% 0.32/0.3 D50A 0.37%/
2.5/2.5 V52K 0.72%/>20% 0.5/0.4 R53L 0.5%/ 0.22/0.25 1.56 M K55I
1%/8% <0.1%/4.2% 2.0/1.6 K55M 90%/ 0.4/0.4 D57A 0.27% H59N 315%/
42%/120% 0.4/0.4 0.13/0.08 R90T 77%/216% 2.68%/95.4% 0.6/0.5 1.58 M
hFGF(b) 100%/100% 100%/100% 1.0/1.0 1.58 M (EC.sub.50) (630 pg/ml)
(160 pg/ml) (10 ng/ml) K95T 100%/170% 44.9%/ 1.58 M D99A 150%/280%
19.2%/ 0.04/0.03 1.53 M E100S 0.03%/ 1.1/0.5 E100A 0.75%/ 0.8/1.0
E105S 12.6%/100% 0.3 50/40 R106L 20%/200% 0.27%/ 0.3/0.13 1.55 M
R106T 210%/250% 9.1%/62.5% 1.58 M E108A 180%/ 30%/ 2.5/2.5 Y112A
0.2%/0.2% 0.01%/4.3% 100/100 1.49 M N113S 160%/ 7.7%/ 1.0/1.0 R116T
50%/300% 0.64%/ 0.2/ 1.53 M R118L 1.8%/ 0.5/0.4 K119S 48%/250%
0.6%/41% 0.35/0.25 1.58 M K128S 210%/ 68%/ 0.3/0.25 1.38 M K128E
200%/140% 13%/ 1.04 M R129T 87%/190% 11.9%/ 1.3/0.5 1.45 M R129L
1.3%/ KR128, 129ST 185%/ 8.4%/ 2.0/0.8 1.14 M K134S 0.6 M K138S 1.0
M C78S 53%/123% 0.3/0.25 1.57 M C96S 95%/96% 0.3/0.8 1.58 M C78,
96S 118%/160% 159%/151% 0.2/0.1 1.58 M C34, 101S 2%/ C34, 78, 101S
65%/144% 2%/72% 0.45/0.5 1.50 M C78, 96, 101S 23%/125% C34, 78, 96,
101S 7%/119% 1.0/0.5 1.51 M *bba is hFGF(b) with hFGF(a)
substitutions for aa 95-155. #KKR has neutral substitutions (M, S
or T) for K27, K30 & R31. @C-4-S has 4 S substitutions for each
of the 4 Cs.
[0148] Legend:
[0149] The data indicate the activities of the various FGF analogs
relative to the activity of wild type FGF. The first row indicates
the actual ED.sub.50 value for wild type FGF in each assay (in
parentheses). The ED.sub.50 is the concentration at which the
analog elicits a half-maximal response in the assay and is
therefore a measure of potency. For analogs exhibiting activity
lower than wild type activity, higher concentrations are required
to elicit activity equivalent to wild type FGF. Therefore, the
ED.sub.50 values of such analogs are higher than wild type (more
analog required to elicit half-maximal stimulation).
[0150] For analogs exhibiting activity higher than wild type
activity, less analog is required to elicit activity equivalent to
wild type activity. Therefore, the ED.sub.50 values of such analogs
are lower than wild type (less analog required to elicit
half-maximal stimulation). The ED.sub.50 values for the various
analogs in each assay are indicated as a percentage of wild type
activity (the ED.sub.50 of wild type FGF divided by the ED.sub.50
of the analog times 100%). Therefore, analogs exhibiting values
greater than 100% appear to have activity greater than wild type
FGF in the assay, while analogs exhibiting values less than 100%
appear to have activity less than wild type FGF in the assay.
[0151] "3T3 Mito./Heparin -/+":
[0152] The data reflect the activity observed in the 3T3/Balb/c
cell mitogenic assay (thymidine uptake) relative to the activity
observed for wild type FGF in this assay. Activity values obtained
in the absence or presence of 1 ug/ml heparin are indicated to the
left and right of the slash respectively.
[0153] "ACE Mito./Heparin -/+":
[0154] The data reflect the activity observed in the Adrenal
Cortical Endothelial cell proliferation assay relative to the
activity observed for wild type FGF in this assay. Activity values
obtained in the absence or presence of 1 ug/ml heparin are
indicated to the left and right of the slash respectively.
[0155] "FGF-Rc Comp./Heparin -/+":
[0156] The data reflect the relative ability of the analog to bind
the FGF receptor as measured in the competitive binding assay. The
values are the ratio of the ED.sub.50 of the analog to the
ED.sub.50 of wild type FGF in this assay. Therefore, analogs which
exhibit values less than 1.0 appear to have an affinity for the
receptor which is greater than that of wild type FGF. Analogs which
exhibit values greater than 1.0 appear to have an affinity for the
receptor which is less than that of wild type FGF. Activity values
obtained in the absence or presence of 1 ug/ml heparin are
indicated to the left and right of the slash respectively.
[0157] Analogs which exhibit near wild type receptor activity and
exhibit low relative activity in the mitogenic assays are potential
antagonists.
[0158] "Heparin/Elution":
[0159] The data indicate the approximate salt (NaCl) concentration
at which the analog elutes from an heparin-TSK column during high
performance liquid chromatography. Analogs exhibiting values less
than 1.58 M appear to have reduced heparin binding as judged by
this procedure. Analogs exhibiting values app. equal to 1.58 (1.53
to 1.63) appear to have affinity for heparin which is
insignificantly changed from that of wild type bFGF as judged by
this procedure.
EXAMPLE 10
Reduced Heparin Binding FGF Analogs
[0160] FGF analogs were constructed wherein mutagenesis was
targeted for the region of the basic FGF molecule which may be
involved in binding heparin and heparin-like compounds. The
analogs, with the specific oligonucleotide sequences which
correspond to the amino acid to be changed, are listed below.
8 Analog Oligonucleotide Number bFGF-K27M
5'-pAGGTCACTTCATGGACCCAAAACG 2487 bFGF-K30A
5'-pTCAAAGACCCAGCACGTCTGTACT 2566 bFGF-R31S
5'-pAAGACCCAAAAATCTCTGTACTGCA 2568 bFGF-D28K
5'-pGTCACTTCAAAAAGCCAAAACGTCT 2480 bFGF-R118L
5'-pCAATACTTACCTGTCAAGGAAAT 2483 bFGF-K35S 5'-pGTCTGTACTGCTCAAACGG-
TGGTT 2553 bFGF-K128S 5'-pATGTGGCACTGTCTCGAACTGGGCA 2545 bFGF-K128E
5'-pATGTGGCACTGGAGCGAACTGGGCA 3332 bFGF-R129T
5'-pGGCACTGAAAACTACTGGGCAGT 3087 bFGF-K128E/ R129T bFGF-K134S
5'-pCTGGGCAGTATTCTCTTGGATCCAA 3212 bFGF-K138S
5'-pAACTTGGATCCTCTACAGGACCTGG 3215
[0161] These gene sequences were inserted into an appropriate
expression vector as taught previously and the resulting protein
tested for reduced heparin binding activity. The results provided
in Table 3 indicate that the region of bFGF encompassing residues
128-138 is a targeted heparin binding region as amino acid
substitutions in this region has led to a decrease in heparin
binding as measured by elution from the heparin-SPW resin.
EXAMPLE 11
Preparation of an FGF Antagonist by N-terminal Deletion
[0162] The blunted NdeI-HindIII FGF fragment from pUC9delH3-PTSF-3
was subcloned into the SmaI-HindIII site of M13mp18. An oligo was
used to introduce a new NdeI site in the FGF molecule at amino acid
25 using in vitro mutagenesis. The new NdeI site serves as both a
new restriction site for subcloning the FGF fragment and also as a
new translational start site for the shortened form of FGF. The
mutagenic oligo used has the sequence:
[0163] 5'-TTG GGT CTT TGA AGT GCA TAT GTG GGA AGG CAC CAG
[0164] The shortened FGF was subcloned into PTSF-delbeta-gal for
expression as an NdeI-HindIII fragment and the resulting plasmid
designated bFGF(25-155). Protein sequence confirmed that the
N-terminus of the protein is histidine. pTSF-delbeta-gal was
constructed by digesting pTSF11 with PvuII and EcoRI, thereby
deleting approximately one-half of the beta-gal promoter
operator.
[0165] The N-terminal deletion analog, bFGF(25-155) was purified by
heparin-Sepharose chromatography as above. This analog exhibits
agonist activity in the 3T3 mitogenic assay with an.sub.50ED
similar to that of bFGF. Although stimulation in the 3T3 assay
peaks at approximately 1 ng/ml for both wild type and bFGF(25-155)
the level of stimulation for the analog (determined in the absence
of heparin) is not as great as observed for wild type bFGF. Thus,
bFGF(25-155) displays characteristics of a partial agonist. In
addition, concentrations of the bFGF(25-155) analog greater than 1
ng/ml result in apparent inhibition of activity; whereas for wild
type bFGF, activity in the 3T3 assay peaks at approximately 1 ng/ml
and is not significantly reduced even at 1 ug/ml, the activity of
bFGF(25-155) at 10 ng/ml is approximately 15% that for wild type
bFGF and at 100 ng/ml, bFGF(25-155) essentially lacks activity.
[0166] The FGF analog bFGF(25-155) is an FGF antagonist as
determined by its ability, at concentrations of 1 ng/ml or greater,
to inhibit the activity of wild type FGF in the absence of heparin.
The activity elicited by 1 ng/ml wild type bFGF is reduced
approximately 50% in the presence of 1 ng/ml bFGF(25-155), reduced
approximately 75% in the presence of 10 ng/ml bFGF(25-155), and
reduced by more than 95% in the presence of 100 ng/ml bFGF(25-155).
That this inhibition is competitive is demonstrated by the shift in
the ED.sub.50 for wild type bFGF observed in the presence of
bFGF(25-155). The ED.sub.50 for wild type bFGF is less than 1 ng/ml
in the absence of the analog bFGF(25-155), approximately 10 ng/ml
in the presence of 10 ng/ml bFGF(25-155), and approximately 100
ng/ml in the presence of 100 ng/ml bFGF(25-155). These data suggest
that bFGF(25-155) binds the FGF receptor, probably with an affinity
similar to that of wild type FGF, but bFGF(25-155) exhibits altered
(reduced) activity. Thus, in the absence of heparin, the FGF analog
bFGF(25-155) is a competitive inhibitor of wild type FGF and is
therefore an antagonist.
[0167] While the FGF analog bFGF(25-155) has demonstrated FGF
antagonist activity, it retains partial agonist activity. In
addition, the agonist activity of this analog is enhanced, and the
antagonist activity inhibited, by the presence of heparin.
Therefore it is desirable to make additional alterations in the
sequence to further reduce the activity of the analog without
significantly reducing its affinity for the FGF receptor. The
reason for the reduced activity of bFGF(25-155) is not known,
however, it is presumed that the integrity of the N-terminal
segment of wild type FGF is necessary for full activity. Therefore
it is possible that further deletions and/or amino acid
substitutions in the N-terminal region exclusve of the receptor
binding domain will further diminish activity without reducing
receptor binding. For example, deletions in the region of amino
acids 25 to 33 may accomplish this end. Another approach would be
to delete or alter amino acids in other, non-receptor binding
domains such as amino acids 78-98 or 130-155. Finally, since the
antagonist activity of this analog is inhibited by heparin,
introducing substitutions which reduce heparin binding may reduce
agonist activity and increase relative antagonist activity. These
approaches may be used in combination and are considered within the
scope of the present invention.
EXAMPLE 12
Construction of Expression Vectors and Stable Expression of FGF
Analogs in Mammalian Cells
[0168] The DNA sequences encoding FGF are most conveniently used to
produce the recombinant proteins in a variety of hosts, as set
forth in .ang.C.1 above. However, expression in mammalian systems
is an alternative to bacterial expression as the mammalian host is
capable of post translational processing analogous to that
experienced by the natively produced protein.
[0169] To construct the vectors, the cloned FGF-encoding analog is
excised with EcoRI and HindIII, provided with EcoRI or other
appropriate linkers if necessary, and then inserted into an
appropriate host vector such as pHS1 or its derivatives as
described below.
[0170] Construction of Host Vectors
[0171] pHS1
[0172] The plasmid pHS1 is suitable for expression of inserted DNA
in mammalian hosts. It contains 840 bp of the hMT-II sequence from
p84H (Karin, M., et al, Nature (1982) 299: 297-802) which spans
from the HindIII site at position -765 of the hMT-II gene to the
BamHI cleavage site at base +70. To construct pHS1, plasmid p84H
was digested to completion with BamHI, treated with exonuclease
BAL-31 to remove terminal nucleotides, and then digested with
HindIII. The desired 840 bp fragment was ligated into pUC8 (Vieira,
J., et al, Gene (1982) 19: 259-268) which had been opened with
HindIII and HincII digestion. The ligation mixture was used to
transform E. coli HB101 to Amp.sup.R, and one candidate plasmid,
designated pHS1, was isolated and sequenced by dideoxy sequencing.
pHS1 contains the hMT-II control sequences upstream of a polylinker
containing convenient restriction sites.
[0173] The workable host plasmid pHS1 can be further modified to
contain additional control elements besides the metallothionein
promoter. In particular, the enhancer elements of viral systems,
such as SV40, can be included, as well as termination signals
associated with the 3' untranslated regions of other proteins such
as hGH.
[0174] Viral Enhancer
[0175] A pair of host expression vectors containing the SV40
enhancer in operable linkage to the MT-II promoter was constructed
by inserting an 1120 bp SV40 DNA fragment into the HindIII site
preceding the MT-II promoter sequences in pHS1. The SV40 DNA
fragment spans the SV40 origin of replication and includes
nucleotide 5171 through nucleotide 5243 (at the origin), the
duplicated 72 bp repeat from nucleotide 107-250, and continues
through nucleotide 1046 on the side of the origin containing the 5'
end of late viral mRNAs. This HindIII 1120 bp fragment is obtained
from a HindIII digest of SV40 DNA (Buchman, A. R., et al, DNA Tumor
Viruses, 2d ed (J. Tooze, ed.), Cold Spring Harbor Laboratory, New
York (1981), pp. 799-841), and cloned into pBR322 for
amplification. The cloning vector was cut with HindIII, and the
1120 bp SV40 DNA fragment isolated by gel electrophoresis and
ligated into HindIII-digested, CIP-treated, pHS1. The resulting
vectors, designated pHS1-SV(9) and pHS1-SV(10), contain the
fragment in opposite orientations preceding the MT-II promoter. In
pHS1-SV(9), the enhancer is about 1600 bp from the 5' mRNA start
site; in the opposite orientation it is approximately 980 bp from
the 5' mRNA start site. Both orientations are operable, but the
orientation wherein the enhancer sequences are proximal to the
start site provides higher levels of expression. It is believed
that deletions which place the enhancer 250-400 bp upstream of the
transcription start are optimal.
[0176] Additional vectors were constructed which place the SV40
enhancer 3' terminus 190 bp, 250 bp, and 360 bp respectively
upstream from the 5' end of the MT promoter TATA box. The
constructions were based on the mapping of the upstream regulatory
regions of the human MT promoter described by Karin, M., et al,
Nature (1984) 308:513-519. All constructions retain the sequences
containing the duplicated sites for regulation by heavy metals, but
the constructions with the 190 bp and 250 bp separations do not
retain the sequence for glucocorticoid regulation which is further
upstream from these sites.
[0177] These vectors, designated pHS'-SVl90, pHS'-SV250, and
pHS'-SV360 are prepared as follows; all constructions are identical
except for the length of sequence containing the metallothionein
promoter and upstream region which is supplied as a fragment
excised from pHS1.
[0178] For pHS'-SV190, pHS1 is digested with SacII, blunted, and
ligated to KpnI linkers. The DNA is then digested with EcoRI and
KpnI to liberate the appropriate portion of the MT-II control
sequences. Similarly, for pHS'-SV250, pHS1 is digested with HgaI,
blunted, ligated to KpnI linkers and digested with EcoRI and KpnI;
for pHS'-SV360, DdeI is used in the initial digestion.
[0179] An intermediate vector containing the SV40 enhancer is
prepared by inserting the HindIII/KpnI fragment of SV40 (which
extends from position 5171 to position 294 and which contains the
enhancer element 50 bp from the KpnI site) into KpnI/HindIII
digested pUC19 to obtain pUC-SV. (pUC19 contains three convenient
restriction sites in the polylinker region, in order, HindIII,
KpnI, and EcoRI.) The finished vectors are obtained by inserting
the KpnI/EcoRI fragments prepared as described above into
KpnI/EcoRI digested pUC-SV.
[0180] All of the foregoing modified vectors, thus, take advantage
of the SV40 enhancer element. Other viral enhancers could, of
course, be used in an analogous manner.
[0181] Transcription Termination Sequences
[0182] To provide transcription termination control sequences, DNA
representing the coding sequence and 3' untranslated sequence of
human growth hormone was ligated into pHS1. The intermediate vector
can provide the hGH 3' untranslated sequence to coding sequences
subsequently ligated into the vector in place of the hGH coding
sequence.
[0183] The genomic sequences encoding hGH were isolated from p2.6-3
(DeNoto, et al, Nucleic Acids Res (1981) 19:3719) by digestion with
BamHI, which cuts at the 5' end of the first exon, and EcoRI, which
cuts 3' of the functional gene, followed by polyacrylamide gel
purification. The isolated fragment was ligated into BamHI/EcoRI
digested pHS1 and the ligation mixture transformed into E. coli
MC1061 to Amp.sup.R. Successful transformants were screened by
restriction analysis, and a strain containing the desired plasmid,
pMT-hGHg, was further propagated to prepare quantities of plasmid
DNA.
[0184] In a manner similar to that described above for constructing
pHS1-SV(9) or pHS1-SV(10), but substituting for pHS1, pMT-hGHg, a
pair of vectors containing the hGH gene under the control of the MT
promoter, and operably linked to SV40 enhancer, and designated,
respectively, phGHg-SV(9) and phGHg-SV(10), were obtained. The
ligation mixtures were used to transform E. coli 1061 to Amp.sup.R,
and the correct constructions verified.
[0185] Construction of Expression Vectors
[0186] phGHg-SV(10) is then used as a host vector to accommodate
the DNA sequences encoding any of the FGF analogs. phGHg-SV(10) is
digested with BamHI and SmaI, blunted with Klenow, and treated with
CIP to excise the hGH coding sequence. This opened vector is
ligated to an NdeI(blunt)/HindIII(blunt) FGF analog fragment to
obtain the desired expression vector pFGF-SV(10).
[0187] In addition, other host vectors may be used to obtain
expression of these sequences, including pHS1 and pHS1 modified to
contain the various configurations of SV enhancer as above
described. Insertion is by analogous means, using BamHI/EcoRI
digestion of the host vector. Also, DNA modified to encode any of
the "long", "primary" or "short" forms of the acidic or basic FGF
analogs may be employed.
[0188] These vectors are generically designated PMT-FGF for the
purposes of the discussion below.
[0189] Production of FGF by Mammalian Recombinants
[0190] Chinese hamster ovary (CHO)-K1 cells are grown on medium
composed of a 1:1 mixture of F12 medium and DME medium with 12%
fetal calf serum. The competent cells are co-transformed with
pMT-FGF and pSV2:NEO (Southern, P., et al, J Mol Appl Genet (1982)
1:327-341). pSV2:NEO contains a functional gene conferring
resistance to the neomycin analog G418. In the transformation, 500
ng of pSV2-NEO and 5 ug of pMT-FGF are applied to a 16 mm dish of
cells in a calcium phosphate-DNA co-precipitate according to the
protocol of Wigler, M., et al, Cell (1979) 16:
[0191] 777-785, with the inclusion of a two minute "shock" with 15%
glycerol after four hours of exposure to the DNA. A day later, the
cells are subjected to 1 mg/ml G418 to provide a pool of
G418-resistant colonies, which are assayed for FGF production and
then can be cloned out.
[0192] Successful transformants, also having a stable inheritance
of pMT-FGF, are plated at low density for purification of clonal
isolates. Small amounts of these isolates are grown in multi-well
plates after exposure to 10.sup.-4 M zinc chloride for convenient
assay of FGF production. FGF determinations are made by standard
ELISA or radio-immunoassays against the antisera prepared against
the appropriate FGF protein analog using standard methods. Clonal
isolates which produce large amounts of the desired FGF analogs are
selected.
[0193] The cells, which have been shown to produce FGF analogs
under suitable conditions, are seeded at {fraction (1/10)}
confluency in basal medium supplemented with 10% fetal calf serum,
incubated overnight, and then induced for FGF production by
addition of zinc chloride in the concentration range of
1.times.10.sup.-4 M to 3.times.10.sup.-4 M. FGF levels rise for
7-10 days, under optimal inducing conditions, 2.times.10.sup.-4 M
ZnCl.sub.2.
[0194] If desired, the FGF analog can be obtained from the lysed
cells and purified according to the procedures set forth above for
the native protein, or by other standard methods known in the
art.
[0195] In addition, as discussed above, secretion of the FGF
protein analogs produced by the foregoing constructs can be
achieved by exocytosis initiated by a calcium ionophore or other
suitable stimulant. While it is not expected that proteins produced
by CHO cells, specifically, would be released by LPS or phorbol
ester stimulation, for example, by substituting for CHO cells, cell
lines derived from macrophage as recombinant hosts, such secretion
can be effected. Also, by altering the construction so as to
provide a signal sequence secretion using the normal constitutive
pathways could also be effected using CHO or other mammalian cell
hosts. Effecting secretion has some advantages, of course, since
the protein purification task becomes much simpler.
[0196] On or before 9 September 1985, Applicants deposited with the
American Type Culture Collection (ATCC), Rockville, Md., USA, the
lambda phage lamdaBB2 which was assigned ATCC accession number
40196. On or before Sep. 12, 1986, conditions of deposit for
lambdaBB2 (ATCC 40196) was converted to conform to those specified
under the Budapest Treaty on the International Recognition of the
Deposit of Microorganisms (Budapest Treaty). Availability of the
deposited strain is not to be construed as a license to practice
the invention in contravention of the rights granted under the
authority of any government in accordance with its patent laws.
Sequence CWU 1
1
* * * * *