Note: Descriptions are shown in the official language in which they were submitted.
~34063~
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TITLE OF THE INVENTION
MUTANT ACIDIC FIBROBLAST GROWTH FACTOR
20 BRIEF DESCRIPTION OF THE DRAWINGS
Figure I is a diagram of the pKK223-3
plasmid containing a gene for mutant r-aFGF.
Figure 2 compares the relative stabilities
of recombinant wild-type haFGF and the various
2s mutants, with heparin (A) and without heparin (B).
- 13~0~3~
- 2 -
BACKGROUND OF THE INVENTION
The discovery of substances that control the
growth of animal cells, especially human cells, and
the mechanism by which they work is currently one of
the major focuses of biomedical research concerned
with tissue repair and wound healing. Fibroblast
growth factors (FGFs), mitogens for various cell
types including many cells of mesodermal origin, have
been identified and it has been suggested that they
may induce mitosis which will result in tissue
repair. Fibroblast mitogenic activity was first
observed with extracts of tissue from the central
nevous system. Brain-derived fibroblast mitogens
were first described by Trowell et al., J. Exp. Biol.
16: 60-70 (1939) and Hoffman, Growth 4: 361-376
(1940). It was subsequently shown that pituitary
extracts also had potent mitogenic activity for
fibroblastoid cells, Amelin, Proc. Natl. Acad. Sci.
USA 70: 2702-2706 (1973). Partial purification of
both brain and pituitary fibroblast growth factor
revealed mitogenic activity for a variety of cell
types of differentiated cells including vascular
endothelial cells, Gospodarowicz _et al., Natl. Cancer
Inst. Monogr. 48: 109-130 (1978) Fibroblast growth
factor was originally thought to be a single peptide
derived from the limited proteolysis of myelin basic
protein. It has recently been shown that FGF exists
in two forms, acidic FGF (aFGF) and basic FGF (bFGF),
and both forms can be isolated and purified from
mammalian brain, Thomas and Gimenez-Gallego, TIBS
il: 81-84 (195). Numerous cell types respond to
stimulation with either purified aFGF or bFGF to
synthesize DNA and divide, including primary
- 3 -
fibroblasts, vascular and corneal endothelial cells,
chondrocytes, osteoblasts, myoblasts, smooth muscle,
glial cells and neuroblasts, Esch et al., Proc. Natl.
Acad. Sci. USA 82: 6507-6511 (1985); Kuo et al., Fed.
Proc. 44: 695 (1985); Gensburger et al., C.R. Acad.
Sc. Faris 303: 465-468 (1986). Pure bovine brain-
derived aFGF not only acts as a potent mitogen for
vascular endothelial cells in culture but also induces
blood vessel growth in vivo, Thomas, et al. Proc.
Natl. Acad. Sci. USA 82: 6409-6413 (1985). The
mitogenic activity of purified aFGF can also be used
to promote wound healing, Thomas, U.S. Patent
4,444,760.
Acidic fibroblast growth factor was origin-
ally purified to homogeneity from bovine brain based
on its mitogenic activity for BALB/c 3T3 fibroblasts,
Thomas et al., Proc. Natl. Acad. Sci. USA 81: 357-361
(1984). This brain-derived growth factor has been
repurified and renamed in multiple laboratories based
both on its: mitogenic activity for vascular
endothelial and astroglial cells (endothelial cell
growth factor and astroglial growth factor 1), source
(retinal-derived growth factor, eye-derived growth
factor II, and perhaps brain-derived growth factor),
and binding to heparin-Sepharose (class 1
heparin-binding growth factor or heparin-binding
growth factor alpha), Thomas and Gimenez-Gallego TIBS
_11: 81-84 (1986). The amino acid sequence of bovine
aFGF has been determined, recognized to be highly
homologous to basic FGF and related to the fibroblast
mitogens interleukin 1-alpha and 1-beta,
Gimenez-Gallego et al., Science 230: 1385-1388
(1985). The complete amino acid sequence of
- 4 -
human aFGF has been determined from the purified
protein, Gimenez-Gallego et al., Biochem. Biophy.
Res. Comm. 138: 611-617 (1986), and from the gene,
Jaye _et al., Science 233: 541-545 (1986).
Native aFGF purified from brain or
recbmbinant-derived aFGF (r-aFGF) requires the
co-administration of heparin to optimally stimulate
Balb/c 3T3 fibroblasts and vascular endothelial cells
in culture. Human brain-derived and recombinant aFGF
are only about 1% to 5~ as active on these cells in
culture in the absence of heparin compared to optimal
activity in the presence of heparin. While the doses
required for maximal aFGF activity are relatively
low, it might be desirable to administer aFGF with no
heparin since heparin could conceivably elicit
detrimental side effects. Pure human aFGF, in
addition to the standard conditions that destroy the
activity of most proteins, extremes of heat, pH and
the presence of proteases, is also labile to
lyophilization and oxidation. The pure aFGF becomes
cross-linked through intrachain or interchain
disulfide bonds by oxidation and can be recovered in
active form by disulfide reduction with 20 mM
dithiothreitol. Heparin can inhibit intermolecular
disulfide bond mediated aggregation of aFGF. This
heterogeneous glycosaminoglycan has also been noted
to stabilize aFGF from heat denaturation and
proteolytic degradation by trypsin. Consequently,
either exogenous or endogenous heparin is required
for the _in vivo activity associated with tissue
repair. The pt~esent invention provides unique
~3~~ri3~
-5-
mutated forms of recombinant-derived aFGF which have
an increased biological activity in the absence of
heparin compared to native aFGF.
OBJECT OF THE INVENTION
It is, accordingly, an object of the present
invention to convert by mutation recombinant bovine
and human aFGF genes to genes capable of encoding
proteins which are more active in the absence of
heparin than the native or recombinant protein.
Another object is to incorporatethe specific genes
into appropriate cloning vectors. A further object
is to transform an appropriate host with each of the
recombinant vectors and to induce expression of the
specific mutated aFGF genes. Another object is to
isolate and purify biologically active bovine and
human mutated aFGF. These and other objects of the
present invention will be apparent from the following
description.
SUMMARY OF THE I NVETTT I ON
Novel genes coding for mutated bovine and
human aFGF are constructed. The unique genes are
derived from genes encoding recombinant native bovine
and human aFGF by specific point mutation. Each gene
construct is inserted into an expression vector which
is used to transform an appropriate host. The trans-
formed host cells produce unique mutated recombinant
aFGF, human or bovine, which is purified and has
enhanced or improved biological activity in the
absence of heparin compared to the unmutated forms.
~~~0~3~
- 6 -
DETAILED DESCRIPTION
Acidic fibroblast growth factor exists in
various microheterogeneous forms which are isolated
from the various tissue sources and cell types known
to contain aFGF. Microheterogeneous forms as used
herein refer to a single gene product, that is a
protein produced from a single gene unit of DNA,
which is structurally modified following translation.
These structural modifications, however, do not result
in any significant alterations of biological activity
of the peptide. Biological activity and biologically
active are used interchangably and are herein defined
as the ability of native, recombinant or mutant
recombinant aFGF to stimulate DNA synthesis in
quiescent Balb/c 3T3 fibroblasts as described in
Example 7, to stimulate any of the cell types
described above or to carry out any of the functions
described in the art. The modifications may take
place either in vivo or during the isolation and
purification process. In vivo modification results
in, but is not limited to, acetylation at the
N-terminus, proteolysis, glycosylation or
phosphorylation. Proteolysis may include
exoproteolysis wherein one or more terminal amino
acids are sequentially, enzymatically cleaved to
produce microheterogeneous forms which have fewer
amino acids than the original gene product.
Proteolysis may also include endoproteolytic
modification that results from the action of
endoproteases which cleave the peptide at specific
locations within the amino acid sequence. Similar
modifications can occur during the purification
_,_
process which also results in the production of
microheterogeneous forms. The most common
modification occuring during purification is
proteolysis which is generally held to a minimum by
the use of protease inhibitors. Under most conditions
a mixture of microheterogeneous forms are present
following purification of native aFGF. Native aFGF
refers to aFGF isolated and purified from tissues or
cells that contain aFGF.
The invention is contemplated to include alI
animal. microheterogeneous forms of acidic fibroblast
growth factor. The preferred embodiments include
bovine and human microheterogeneous forms of aFGF.
The most preferred microheterogeneous forms of bovine
aFGF include a 154 amino acid form, a 140 amino acid
form and a 134 amino acid form. The 140 amino acid
form is shown in Table I, Gimenez-Gallego et al.,
Science 230: 1385-1388 (1985), and is the most
preferred of the bovine species.
30
I~~~~3
TABLE I
Amino Acid Sec;'uence of Bovine aFGF
1 to zo
PheAsnLeuProLeuGlyAsnTyrLysLysProLysLeuLeuTyrCysSerAsnGlyGlyTyrPheLeuArgIleCeu
30 40 50
ProAspGlyThrValAspGlyThrlysAspArgSerAspGlnHisIleGlnLeuGlnLeuCysAlaGluSerIleGlyG
lu
60 70 80
1 5
ValTyrIleLysSerThrGluThrGlyGlnPheLeuAlaMetAspThrAspGlyLeuLeuTyrGlySerGlnThrProA
sn
90 100
GluGluCysLeuPheLeuGluArgLeuGluGluAsnHisTyrAsnThrTyrIleSerLysLysHisAlaGluLysHisT
rp
, to lzo 130
PheValGlyLeuLysLysAsnGlyArgSerLysLeuGlyProArgThrHisPheGlyGlnLysAlaIleLeuPheLeuP
ro
140
LeuProValSerSerAsp
The nucleotide sequence of the 140 amino acid form,
recombinant, of bovine aFGF is shown in Table II.
- 9 -
TABLE II
Nucleotide Seguence of Bovine aFGF
1 20 40 60 80
AATTCATGTTCAATCTGCCACTGGGTAATTACAAAAAGCCAAAGCTTCTTTACTGCTCTAACGGTGGTTACTTTCTCCG
C
GTACAAGTTAGACGGTGACCCATTAATGTTTTTCGGTTTCGAAGAAATGACGAGATTGCCACCAATGAAAGAGGCG
100 120 140 160
ATCCTGCCAGATGGTACCGTGGACGGCACCAAAGATCGTTCTGATCAACATATTCAACTGCAGCTGTGCGCCGAATCTA
T
1 O
TAGGACGGTCTACCATGGCACCTGCCGTGGTTTCTAGCAAGACTAGTTGTATAAGTTGACGTCGACACGCGGCTTAGAT
A
180 200 220 240
CGGTGAAGTTTACATCAAATCTACCGAAACTGGTCAATTCCTTGCCATGGACACTGATGGCCTGCTGTACGGATCCCAG
A
GCCACTTCAAATGTAGTTTAGATGGCTTTGACCAGTTAAGGAACGGTACCTGTGACTACCGGACGACATGCCTAGGGTC
T
260 280 300 320
CCCCAAACGAGGAGTGCCTTTTCCTGGAGCGCCTGGAGGAAAACCATTACAACACCTACATCTCTAAAAAGCATGCTGA
G
GGGGTTTGCTCCTCACGGAAAAGGACCTCGCGGACCTCCTTTTGGTAATGTTGTGGATGTAGAGATTTTTCGTACGACT
L
340 360 380 400
AAACATTGGTTCGTAGGCCTTAAGAAAAATGGCCGCTCTAAACTGGGCCCTCGTACTCACTTTGGTCAAAAAGCTATCC
T
TTTGTAACCAAGCATCCGGAATTCTTTTTACCGGCGAGATTTGACCCGGGAGCATGAGTGAAACCAGTTTTTCGATAGG
A
420 440
GTTCCTGCCACTGCCAGTGAGCTCTGACTAATAGATATCG
CAAGGACGGTGACGGTCACTCGAGACTGATTATCTATAGCAGCT
The 154 amino acid form includes the following
additional amino acids; Ala-Glu-Gly-Glu-Thr-Thr-Thr-
phe-Thr-Ala-Leu-Thr-Glu-Lys, with the carboxyl
terminus Lys attached to the amino terminus Phe at the
first position of the 140 amino acid form. The amino
terminal alanine residue of the 154 amino acid form
~.~~~~3
-lo-
of the bovine aFGF may be acetylated. The 134 amino
acid form is identical to the 140 amino acid form
except that the first 6 amino acids of the amino
terminus have been removed. When native aFGF is
isolated the relative amounts of these microhetero-
geneous forms vary depending on the process used but
generally contain at least two of these forms.
Human aFGF exhibits a similar microhetero-
geneity to that of bovine aFGF. The most preferred
microheterogeneous forms of human aFGF include a 154
amino acid form, a 140 amino acid form and a 139 amino.
acid form. The human 140 amino acid form differs from
the bovine form by eleven amino acids, as shown in
TABLE VIII. The 154 amino acid form contains the
exact sequence of the human 140 amino acid form plus
the 14 additional amino acids associated with the
bovine 154 amino acid form, with one exception. The
amino acid at the fifth position of the N-terminus or
at the -10 position as determined from the 140 amino
acid Phe N-terminus in the human form is isoleucine
and is substituted for the threonine in the bovine
form. The additional 14 amino acid human N-terminal
sequence is; Ala-Glu-Gly-Glu-Ile-Thr-Thr-Phe-Thr-
Ala-Leu-Thr-Glu-Lys. The additional amino acids of
the 154 amino acid form are numbered from the
N-terminal Ala, -14, to the carboxyl terminal Lys,-1.
The amino terminal alanine resiude at the -14
position may be acetylated. A third form of human
aFGF contains 139 amino acids and is equivalent to
the human 140 amino acid form with the amino terminal
phenylalanine residue removed. The amino terminal
asparagine residue may be deamidated to aspartic acid
~.~3~~~~~~
- 11 -
in the 139 amino acid form of human aFGF. The 140
and 139 amino acid forms are the most preferred forms
of the human microheterogeneous forms. The 140 amino
acid form is shown in Table III, Gimenez-Gallego et
al., Biochem. Biophys. Res. Comm. 138: 611-617
(1986).
m71 TT T T T T
Amino Acid Sequence of Human aFGF
1 lp 20
PheAsnLeuProProGlyAsnTyrLysLysProLysLeuLeuTyrCysSerAsnGlyGlyHisPheLeuArgIleLeu
30 40 50
ProAspGlyThrValAspGlyThrArgAspArgSerAspGlnHisIleGlnLeuGlnLeuSerAlaGluSerValGlyG
lu
60 70 80
ValTyrIleLysSerThrGluThrG1y61nTyrLeuAlaMetAspThrAspGlyLeuleuTyrGlySerGlnThrProA
sn
90 100
GluGluCysLeuPheLeuGluArgLeuGluGluAsnHisTyrAsnThrTyr~IleSerLysLysHisAlaGlulysAsn
Trp
110 120 130
2 S
PheValGlyLeuLysLysAsnGlySerCysLysArgGlyProArgThrHisTyrGlyGlnlysAlaIleLeuPheleuP
rn
140
LeuProValSerSerAsp
The nucleotide sequence of the 140 amino acid form,
recombinant, of human aFGF is shown in Table IV.
~~~~~3
- 12 -
TABLE IV
Nucleotide Secruence of Human aFGF
1 20 40 60 80
AATTCATGTTCAATCTGCCACCGGGTAATTACAAAAAGCCAAAGCTTCTTTACTGCTCTAACGGTGGTCACTTTCTCCG
C
GTACAAGTTAGACGGTGGCCCATTAATGTTTTTCGGTTTCGAAGAAATGACGAGATTGCCACCAGTGAAAGAGGCG
100 120 140 160
I O
ATCCTGCCAGATGGTACCGTGGACGGCACCAGAGATCGTTCTGATCAACATATTCAACTGCAGCTGTCCGCCGAATCTG
T
TAGGACGGTCTACCATGGCACCTGCCGTGGTCTCTAGCAAGACTAGTTGTATAAGTTGACGTCGACAGGCGGCTTAGAC
A
180 200 220 240
CGGTGAAGTTTACATCAAATCTACCGAAACTGGTCAATACCTTGCCATGGACACTGATGGCCTGCTGTACGGATCCCAG
A
Z S
GCCACTTCAAATGTAGTTTAGATGGCTTTGACCAGTTATGGAACGGTACCTGTGACTACCGGACGACATGCCTAGGGTC
T
260 280 300 320
CCCCAAACGAGGAGTGCCTTTTCCTGGAGCGCCTGGAGGAAAACCATTACAACACCTACATCTCTAAAAAGCATGCTGA
G
GGGGTTTGCTCCTCACGGAAAAGGACCTCGCGGACCTCCTTTTGGTAATGTTGTGGATGTAGAGATTTTTCGTACGACT
C
340 360 380 400
AAAAATTGGTTCGTAGGCCTTAAGAAAAATGGCAGCTGTAAACGCGGCCCTCGTACTCACTATGGCCAAAAAGCTATCC
T
TTTTTAACCAAGCATCCGGAATTCTTTTTACCGTCGACATTTGCGCCGGGAGCATGAGTGATACCGGTTTTTCGATAGG
A
2 5 420 440
GTTCCTGCCACTGCCAGTGAGCTCTGACTAATAGATATCG
CAAGGACGGTGACGGTCACTCGAGACTGATTATCTATAGCAGCT
The preferred procedure for obtaining a gene
for mammalian aFGF is to synthesize the gene because
this allows optimization of translated protein and
ease of mutagenesis. The gene may be synthesized
based on the amino acid sequence of a
microheterogeneous form of aFGF obtained
~~ ~~ ~E3r~
- 13 -
from any animal including man. The preferred method
is to use the bovine amino acid sequence for aFGF and
chemically point mutate the base sequence, to produce
the genes for other species, Linemeyer et al.
Biotechnol. 5:960-965 (1987).
_ The synthetic genes are based on the
determined bovine amino acid sequence described by
Gimenez-Gallego et al., Science 230: 1385-1388 (1985)
and the human amino acid sequence as described by
Gimenez-Gallego et al. Biochem. Biophys. Res. Comm.,
138: 611-617 (1986) and shown in Tables I and III.
The unique nucleotide sequence of the 140 amino acid
form of bovine aFGF is derived from reverse
translation of the amino acid sequence by a technique
similar to that of Itakura et al., Science 198:
1056-1063 (1977). The various novel nucleotide
sequences corresponding to the native amino acid
sequence of bovine aFGF are shown in the following
table:
30
13~~~b~r~
- 14 -
TABLE V
5 10 15 20
1 Phe AsnLeuProLeuGlyAsnTyrLysLysProLysLeuLeuTyrCysSerAsnGlyGly
~
TTQ AAQCTNCCNCTNGGNAAQTAQAAPAAPCCNAAPCTNCTNTAQTGQTCNAAQGGNGGN
TTP TTP TTPTTP AGQ
25 30 35 40
1 5 Tyr Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly Thr Lys Asp Arg Ser
Asp Gln
TAQ TTQ CTN CGN ATQ CTN CCN GAQ GGN ACN GTN GAQ GGN ACN AAP GAQ CGN TCN GAQ
CAP
TTP AGP ATA TTP AGP AGQ
45 50 55 60
His Ile Gln Leu Gln Leu Cys Ala Glu Ser Ile Gly Glu Val Tyr Ile Lys Ser Thr
Glu
CAQ ATQ CAP CTN CAP CTN TGQ GCN GAP TCN ATQ GGN GAP GTN TAQ ATQ AAP TCN ACN
GAP
ATA TTP TTP AGQ ATA ATA AGQ
65 70 75 80
2 5 Thr Gly Gln Phe Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr
Pro Asn
ACN GGN CAP TTQ CTN GCN ATG GAQ ACN GAQ GGN CTN CTN TAQ GGN TCN CAP ACN CCN
AAQ
TTP TTP TTP AGQ
~.~~0~~~~
- is -
10
85 90 95 100
Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser
Lys _
GAP GAP TGQ CTN TTQ CTN GAP CGN C1N GAP GAP AAQ CAQ TAQ AAQ ACN TAQ ATQ TCN
AAP
TTP TTP AGP TTP ATA AGQ
1 s 105 110 115 120
Lys His Ala Glu Lys His Trp Phe Val Gly Leu Lys Lys Asn Gly Arg Ser Lys Leu
Gly
AAP CAQ GCN GAP AAP CAQ TGG TTQ GTN GGN CTN AAP AAP AAQ GGN CGN TCN AAP CTN
GGN
TTP AGP AGQ TTP
2 0 125 130 135 140
Pro Arg Thr His Phe Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser
Asp
CCN CGN ACN CAQ TTQ GGN CAP AAP GCN ATQ CTN TTQ CTN CCN CTN CCN GTN TCN TCN
GAQ
AGP ATA TTP TTP TTP AGQ AGQ
2 S where Q = C or T,
P = A or G, and
N = A, T, C, or G
r
- 16 -
The bovine gene is constructed with a leader
portion containing a single restriction enzyme
cleavage site and an N-terminal methionine codon for
a translational start site. The gene also contains a
tail containing tandem translational stop codons and
two restriction enzyme cleavage sites. The redundancy
of the genetic code allows a choice of base sequences
which in turn allows for the incorporation of unique
restriction enzyme cleavage sites throughout the
gene. The preferred bovine gene base sequence with
the location of the restriction enzyme cleavage sites
is shown in the following table:
20
30
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v ~O m
~ Q
Q f-
J N = M
U a
c~ c~
N U L
~
T U' L V
V
U 1- H- Q
Q 1-
n
7 O~ 01 F-
V' Q
U
a
~
U' V Q V
U'
7 U' O F-
U L
h-
~ d M
U U
O C U O T V ~p
U' U'
~ a
Q ~ ~j U' Q
U
O F- 7 U'
V
L
a VU' J U
.~3~~~~j
- 19 -
The gene sequence for each strand of the
double-stranded molecule is randomly divided into 8
nucleotide sequences. The oligonucleotides are
constructed with overlapping ends to allow the
formation of the double-stranded DNA. The following
table contains one of a multitude of oligonucleotide
arrangements that is used to produce the bovine aFGF
gene.
20
30
~..~~Of39
- 20 -
TT T1T T1 !1T T
OLIGO-1 10 20 30 40 50 58
5' AATTCATGTT CAATCTGCCA CTGGGTAATT ACAAAAAGCC AAAGCTTCTT TACTGCTC 3'
OLIGO-2 10 20 30 40 45
I O 5' AGAAGCTTTG GCTTTTTGTA ATTACCCAGT GGCAGATTGA ACATG 3'
OLIGO-3 10 20 30 40 5D 60
5' TAACGGTGGT TACTTTCTCC GCATCCTGCC AGATGGTACC GTGGACGGCA CCAAAGATCG 3'
1 S OLIGO-4 10 20 30 40 50 59
5' TGCCGTCCAC GGTACCATCT GGCAGGATGC GGAGAAAGTA ACCACCGTTA GAGCAGTAA 3'
OLIGO-5 10 20 30 40 46
5' TTCTGATCAA CATATTCAAC TGCAGCTGTG CGCCGAATCT ATCGGT 3'
OLIGO-6 10 20 30 40 50 60 65
5' GTAAACTTCA CCGATAGATT CGGCGCACAG CTGCAGTTGA ATATGTTGAT CAGAACGATC TTTGG 3'
OLIGO-7 10 20 30 40 50 60 67
2 S 5' GAAGTTTACA TCAAATCTAC CGAAACTGGT CAATTCCTTG CCATGGACAC TGATGGCCTG
CTGTACG 3'
- 21 -
OLIGO-8 10 20 30 40 50 60 62
5' GATCCGTACA GCAGGCCATC AGTGTCCATG GCAAGGAATT GACCAGTTTC GGTAGATTTG AT 3'
OLIGO-9 10 20 30 40 50 52
5' GATCCCAGAC CCCAAACGAG GAGTGCCTTT TCCTGGAGCG CCTGGAGGAA AA 3'
1 ~ OLIGO-10 10 20 30 40 50 58
5' GTTGTAATGG TTTTCCTCCA GGCGCTCCAG GAAAAGGCAC TCCTCGTTTG GGGTCTGG 3'
OLIGO-11 10 20 30 40 48
5' CCATTACAAC ACCTACATCT CTAAAAAGCA TGCTGAGAAA CATTGGTT 3'
1 5 OLIGO-12 10 20 30 40 46
5' GGCCTACGAA CCAATGTTTC TCAGCATGCT TTTTAGAGAT GTAGGT 3'
OLIGO-13 10 20 30 40 50 53
5' CGTAGGCCTT AAGAAAAATG GCCGCTCTAA ACTGGGCCCT CGTACTCACT TTG 3'
OLIGO-14 10 20 39 40 50 55
5' GCTTTTTGAC CAAAGTGAGT ACGAGGGCCC AGTTTAGAGC GGCCATTTTT CTTAA 3'
OLIGO-15 10 20 30 40 50 56
2 S 5' GTCAAAAAGC TATCCTGTTC CTGCCACTGC CAGTGAGCTC TGACTAATAG ATATCG 3'
OLIGO-16 10 20 30 40 50
5' TCGACGATAT CTATTAGTCA GAGCTCACTG GCAGTGGCAG GAACAGGATA 3'
~.,3~~~3~
- 22 -
The oligonucleotides illustrated in Table VII are
presented merely as an example of oligonucleotide
subunits and should not be construed as limiting
thereto. The composite base sequence showing the
overlap and arrangement of the oligonucleotides is
illustrated in Table II.
The bovine gene is assembled in 2 steps:
first, the half corresponding to the N-terminal
portion of the protein; and second, the C-terminal
half. Generally, the oligonucleotides are kinased
with T4 polynucleotide kinase in the presence of .
either ATP or 32P-labelled ATP. In the first
reaction of each step the oligonucleotides which make
up one strand of the gene are kinased with the
exception of the most 5' oligonucleotide. In the
second reaction the oligonucleotides which make up
the second strand are kinased, with the exception of
the most 5' oligonucleotide. When kinased oligo-
nucleotides are used, about 1~ of the added oligo-
nucleotide is 32P-labelled for later identification
of the products. Annealing is carried out in an
appropriate buffer, such as one containing but not
limited to about 60 mM TRIS, about pH 7.6, about 5 mM
dithiothreitol (DTT), about 10 mM MgCl2, and about
uM ATP at about 90°C for about 4 minutes followed
by a rapid transfer to about 60°C and a slow cooling
to about 30°C. Ligation is carried out in an
appropriate buffer, such as one containing, but not
30 limited to, about 60 mM TRIS, about pH 7.6, about 10
mM DTT, about 10 mM MgCl2, about 1 mM ATP, and about
0.03 units T4 DNA ligase at about 20°C for about 1 and
1/2 hour.
1~~~~3
- 23 -
The ligated oligonucleotides are purified by
polyacrylamide gel electrophoresis following ethanol
precipitation. The oligonucleotides are redissolved
in a buffer containing about 20 ul of about 80%
formamide, about 50 mM TRIS-borate) about pH 8.3,
about 1 mM ethylenediaminetetraacetic acid (EDTA),
about 0.1% (w/v) xylene cyanol, and about 0.1% (w/v)
bromophenol blue. Each sample is heated at about
90°C for about 3 minutes and electrophoresed in about
a 10% urea-polyacrylamide gel at about 75 watts for
about 5 hours. The 231 base N-terminal bands are
removed, combined and eluted at about 4°C in about
0.5 M ammonium acetate containing about 1mM EDTA at
about pH 8. The 209 base C-terminal bands are treated
in the same manner.
The synthetic gene sequences coding for
either the N-terminal or the C-terminal portions of
the aFGF are incorporated into the pBR322 plasmid.
It is especially desired and intended that there be
included within the scope of this invention, the use
of other plasmids into which the aFGF gene can be
incorporated and which will allow the expression of
the aFGF gene. Reannealed oligonucleotides, about
300 fmole and about 100 fmole of the recovered 231
base pair N-terminus are each ligated to about 100
fmole of agarose gel-purified about 3.9 kilo base
(kb) EcoRI-BamHI pBR322 for the N-terminus. The 209
by C-terminus is constructed in the same manner using
B~I-SalI pBR322. Ligation is carried out in a
buffer containing about 25 mM TRIS, about pH 7.8,
about 1 mM DTT, about 10 mM MgCl2, about 0.4 mM
ATP, with about 1 unit of T4 DNA ligase for about 1
hour at about 20°C. Each half-gene ligated vector ~s
A3~O~a~l
- 24 -
used to transform competent bacterial cells, such as
E. coli RR1 (Bethesda Research Laboratories, BRL)
following suppliers procedures. The transformed cells
are selected for growth in ampicillin and screened for
the presence of either the 231 base pair (bp) EcoRI-
BamHI insert or the 209 by BamHI-SalI insert by
restriction analysis of mini-lysate plasmid
preparations.
The DNA sequence of clones containing the
appropriate sized inserts is determined using Maxam
and Gilbert, Proc. Natl. Acad. Sci. USA 74: 560-564 .
(1977) chemical DNA sequence techniques.
The final full-length aFGF synthetic gene was
cloned by cleaving the N-terminal half clone with
restriction enzymes BamHI and SalI, treating with
alkaline phosphatase and ligating this to the gel
purified 209 by BamHI-SalI insert of the C-terminal
half clone. This ligated material was used to
transform competent RR1 cells as before.
Expression of the synthetic aFGF gene is
accomplished by a number of different promoter-
expression systems. It is desired and intended that
there be included within the scope of this invention,
the use of other promoter-expression systems for the
expression of the intact aFGF gene. The preferred
construct uses the E. coli tac promoter, a hybrid
between regions of the trp promoter and the lac
promoter as described by deBoer _et al., Proc. Nat.
Acad. Sci. USA 80: 21-25 (1983). Plasmid pKK223-3
(Pharmacia) which contains the tac promoter and rrnB
rRNA transcription terminator was modified to remove
the pBR322-derived SalI restriction enzyme site. The
rrnB rRNA terminator has been shown to allow
.~~~~~Je3~
- 25 -
expression by strong promoters, Gentz et al., Proc.
Natl. Acad. Sci. USA 78: 4936-4940 (1981); Brosius,
Gene 27: 161-172 (1984).
The pKK223-3 plasmid DNA is cleaved with
restriction enzymes to produce a 2.7 kb DNA fragment
to generate clone pKK2.7. The synthetic aFGF gene is
cleaved from its pBR322 vector and transferred to the
pKK2.7 plasmid after restricting pKK2.7 with EcoRI
and SalI. The resulting recombinant, shown in figure
1, is transformed into E. coli JM105 (Pharmacia) or
DH5 (BRL) cells and expressed.
Site specific mutagenesis is an efficient
way to convert the amino acid sequence of one
mammalian species of aFGF to the aFGF amino acid
sequence of another species. The following
description relates to the site specific mutagenic
conversion of bovine aFGF, 140 amino acid form
(numbered in accordance with the native form), to
h~an aFGF, it is to be understood, however, that the
process can be used to convert any mammalian species
aFGF to that of any other species. The only
limitation on the conversion is that the amino acid
sequences of both aFGFs must be known. The following
table lists the amino acids which must be substituted
and the location on the bovine aFGF amino acid map,
Table VI, at which the substitutions are made:
~.f3~ ~~3a
- 26 -
TABLE VIII
Amino Acid Substituted Amino Acids
Location Human aFGF for Bovine aFGF
5 Pro Leu
21 His Tyr
35 Arg Lys
4~ Ser Cys
51 Val Ile
64 Tyr Phe
106 Asn His
116 Ser Arg
11~ Cys Ser
119 Arg Leu
125 Tyr Phe
As with the bovine gene sequence eight
oligonucleotides representing the human gene sequence
are constructed by the same procedure as that used
for the bovine oligonucleotides. The following table
contains one of a multitude of oligonucleotide
arrangements that is used to produce the human aFGF
gene.
1~~~~~~
- 27 -
T T L1 T L~ T V
OLIGO-1
5' CTGCCACCGGGTAATTAC 3'
OLIGO-2
5' CGGTGGTCACTTTCTCCG 3'
OLIGO-3
5' CGGCACCAGAGATCGTTC 3'
OLIGO-4
5' GCAGCTGTCCGCCGAATCTGTCGGTGAAG 3'
OLIGO-5
5' CTGGTCAATACCTTGCCATGG 3'
OLIGO-6
5' GCTGAGAAAAATTGGTTCG 3'
OLIGO-7
5' GGCCGCGTTTACAGCTGCCATTTTTCTTAAGG 3'
OLIGO-8
5' CGTACTCACTATGGCCAAAAAGCTATCC 3'
~.~~~~3:~
_ 2g ._
The cloned synthetic bovine gene for aFGF is
converted to a human synthetic gene for aFGF by a
series of directed point mutations. Oligonucleotide-
directed mutagenesis of the cloned gene allows the
alteration of the base sequence of bovine aFGF so
that-the resulting amino acid sequence contains the
substituted amino acids shown in Table VIII and is
human aFGF. A deletion is made in the bovine gene to
remove the amino terminal phenylalanine for the
production of the human 139 amino acid microhetero-
geneous form of aFGF. A point mutation is carried .
out to replace the second position asparagine with
aspartic acid. Alternatively, the asparagine is
deamidated to aspartic acid. The methods for carrying
out these procedures are described below or are known
in the art. The oligonucleotide-directed mutagenesis
is carried out using standard procedures known to the
art, Zoller and Smith, Methods in Enzymology, 100:
468-500 (1983); Norris et al.) Nucleic Acids Research,
11: 5103-5112 (1983); and Zoller and Smith, DNA, 3:
479-488 (1984). The point mutations of the bovine to
human conversion are carried out by the standardized
oligonucleotide-directed mutagenesis and are shown in
the following Table. The location of the base
mutagenesis can be seen in Table X.
:~3~0~3
- 29 -
TABLE X
Base Substituted Base Corresponding
Location Human aFGF for Bovine aFGF Human Amino Acid
22 C T Pro
6g C T His
112 G A Arg
148 C G Ser
159 G A Val
199 A T Tyr
324 A C Asn
354 A C Ser
358 G C Cys
364 G T Arg
365 C G Arg
382 A T Tyr
To expedite the mutagenesis of the bovine
aFGF gene it is transfered to a standard vector,
M13mp19, a single-stranded DNA bacteriophage vector.
The bovine pKK-aFGF plasmid is cleaved with EcoRI and
SalI and the resulting 440 by fragment is gel
purified. Vector M13mp19 RF DNA is cleaved with the
same two endonucleases and the ends are subsequently
dephosphorylated with bacterial alkaline phosphatase.
The vector DNA and the aFGF gene fragment DNA are
ligated and the mixture is used to transform E. coli
DH5 cells. A phage clone containing the bovine aFGF
gene is selected, M13mp19-baFGF.
The human oligomers shown in Table IX are
phosphorylated and annealed individually to M13mp19-
baFGF single-stranded phage DNA. Closed-circular
double-stranded molecules are prepared with T4 DNA
13~~~~~
- 30 -
ligase and DNA polymerase I klenow fragment. The
preparations were each used to transform competent
JM105 cells and the resulting transformant plaques
are selected by hybridization with the appropriate
oligomer which is labeled using polynucleotide
kinase. Single-stranded DNA is isolated from the
phage clone containing the human oligmer 4 mutations
and the above procedure is repeated using the human
oligomer 5 to generate a clone containing both the
oligomer 4 and 5 mutations.
In the following procedures the bovine-to- .
human sequence mutations in these M13-based clones
were combined into one pBR322-based clone. RF DNAs
were prepared from clones containing the base changes
specified by human oligomers 1, 2, 6, and 8. The DNA
of the human 1 mutant clone was cleaved with EcoRI,
the ends were dephosphorylated with bacterial alkaline
phosphatase, and the DNA was cleaved with HindIII.
The human 2 mutant DNA was cleaved with HindIII,
treated with phosphatase, and then cleaved with BamHI.
The human 6 mutant DNA was cleaved with BamHI,
phosphatase treated, and subsequently cleaved with
ApaI. Likewise, the human 8 mutant DNA was cleaved
with ApaI, the ends were dephosphorylated, and the
DNA was cleaved with SalI. These four DNA
preparations were electrophoresed through 2% agarose
and the fragments of 45 bp, 190 bp, 135 bp, and 70 by
from the mutant DNAs containing human 1, 2, 6, and 8
mutations, res ectivel
P y, were eluted from the gel.
Volumes of each fragment are collectively ligated to
a gel-purified 3.7 ~b EcoRI-SalI fragment from pBR322
with T4 DNA ligase and used to transform competent _E.
coli DH5 cells (BRL) as described by the supplier. A
13~~J~3~3
- 31 -
clone containing the mutations specified by all four
mutant oligomers is selected by hybridiz-
ation with radiolabeled probes prepared from each of
the oligomers. The 140 by KpnI-BamHI DNA fragment
isolated from cleaved RF DNA of the human 3 mutant
M13 clone is ligated to endonuclease cleavage products
of this human 1-2-6-8 mutant DNA and transformed into
DH5 competent cells to generate a clone with the human
1-2-3-6-8 mutations. BamHI-PstI digestion fragments
of this latter clone are ligated to the BamHI-PstI
digestion fragments of RF DNA from the human 4-5 _
M13-based clone and the ligation mixture is used to
transform DH5 competent cells. A clone containing
the human 1-2-3-4-5-6-8 mutations is selected by
oligomer hybridization and the aFGF gene EcoRI-SalI
DNA fragment of this recombinant plasmid is ligated
to phosphatase-treated EcoRI-SalI-cleaved RF DNA of
M13mp18 (BRL). Competent DH5 cells are transformed
with this ligated DNA and the transformed cells are
plated on JM105 host cells to generate an M13 clone.
The single-stranded phage DNA of this clone was
annealed with the human 7 oligomer and an M13 clone
containing all the desired mutations was obtained
following the procedure described above. The human
aFGF clone is designated M13mp18-haFGF.
Pure aFGF in the absence of heparin becomes
less active presumably due to the generation of
incorrectly stabilized intramolecular disulfide bonds
and aggregates formed by intermolecular disulfide
bonds. The covalent disulfide bonds are formed
between two cysteine residues either in two separate
polypeptide chains, interchain disulfide bond, or in
different positions within a single chain, intrachaia
disulfide bond. In the case of enzymatic
~~4~~~~
- 32 -
oxidative iodination, the active molecules can be
recovered by reduction with 20 mM dithiothreitol in
the presence of 3 M guanidinium chloride at a pH of
about 9.1. The present invention utilizes
site-directed mutagenesis for the specific
substitution or deletion of amino acids capable of
forming extraneous intramolecular or intermolecular
covalent bonds and oxidation susceptable amino
acids. Substitution as used herein refers to a
deliberate change in the DNA base sequence of aFGF
such that a desired amino acid is substituted for an .
undesired amino acid. The undesired amino acid may
be one which forms unwanted covalent bonds,
especially disulfide bonds, or one which is
air-oxidizable either of which may decrease the
biological activity of the molecule. A deletion as
used herein refers to a deliberate change in the DNA
base sequence of aFGF resulting in the elimination of
the unwanted amino acid. The primary amino acid
associated with intramolecular and intermolecular
covalent bond formation is cysteine while the amino
acids which are oxidization prone include cysteine,
methionine and tryptophan. The cysteine residue or
residues may be replaced with any amino acid which
will not form disulfide bonds. The prefered amino
acid for the substitution of cysteine is serine. The
oxidation prone amino acids are replaced with any
amino acid which is oxidation resistant, this
includes, but is not restricted to, alanine, valine,
leucine and isoleucine.
The invention is contemplated to include
site-specific mutations of one or more of the cysteine
residues and any non-terminal methionine residue which
1~~J~3a~
- 33 -
could render native or recombinant aFGF less active or
inactive due to the formation of incorrect intra-
molecular or intermolecular bonds or oxidative
changes. The recombinant and native human and bovine
protein contains two cysteine residues in common
located at positions 16 and 83 and a methionine
residue in common located at position 67 as defined
by the native 140 amino acid form of both bovine and
h~an aFGF. Bovine and human aFGFs each contain a
third cysteine residue at positions 47 and 117,
respectively. The common cysteine residues are the
most likely to form a disulfide bond since the
location of cysteine residues in disulfide bonds is
highly conserved in homologous proteins. Thus the
third cysteine residues that are in different
locations in bovine and human aFGFs are very likely
not found in disulfide linkages in the fully active
proteins. It will be understood that the novel
mutant aFGFs of the present invention will not only
include the forms substituted at the non-common
cysteine residues but also those that have all
cysteines substituted or deleted, those in which any
one or two of the cysteines have been substituted or
deleted and those in which methionine has been
substituted or deleted. The substitution or deletion
of any one, especially the unique cysteine, all
cysteines, two of the three cysteines or methionine
in the human or bovine aFGF by site-directed
mutagenesis may after the formation of unwanted
intramolecular and intermolecular disulfide bonds and
oxidized forms.
~3 ~0~~~~
- 34 -
Site-specific mutagenesis is carried out on
preferably bovine or human r-aFGF produced from
genomic DNA, cDNA or by construction of genes for one
or more of the microheterogeneous forms of the
protein based on the microheterogeneous forms of aFGF
from mammalian species including man. Genomic DNA is
extracted from mammalian brain or pituitary cells and
prepared for cloning by either random fragmentation
of high molecular weight DNA following the technique
of Maniatis et al., Cell _15: 687-701 (1978) or by
cleavage with a restriction enzyme by the method of
Smithies et al., Science 202: 1284-1289 (1978). The
genomic DNA is then incorporated into an appropriate
cloning vector, generally E. coli lambda phage, see
Maniatis et al., Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York (1982).
To obtain cDNA for aFGF, poly (A)-containing
~A is extracted from cells that express aFGF by the
method of Aviv and Leder, Proc. Natl. Acad. Sci. _69:
1408-1412 (1972). The cDNA is prepared using reverse
transcriptase and DNA polymerase using standard
techniques, as described in Maniatis _et _al., Molecular
Cloning, a Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, New York (1982). The
cDNA is tailed and cloned into an appropriate vector,
usually pHR322, by a technique similar to that of
Wensink, et al., Cell 3: 315-325 (1974).
The clonal genomic DNA or cDNA libraries are
screened to identify the clones containing aFGF
sequences by l.fbridization with an oligonucleotide
probe. The sequence of the oligonucleotide
hybridization probe is based on the determined amino
1~4~~3~
- 35 -
acid sequence of aFGF. Maniatis et _al., supra,
Anderson and Kingston, Proc. Natl. Acad. Sci. USA
80: 6838-6842 (1983) and Suggs _et _al., Proc. Natl.
Acad. Sci. USA 78: 6613-6617 (1981) describe various
procedures for screening genomic and cDNA clones.
The. preferred procedure is to specifically point
mutate the synthesized bovine and human genes as
described above.
Site-specific mutagenesis is carried out on
a human or bovine aFGF single-stranded bacteriophage
recombinant clone, such as M13mp18-haFGF or .
M13mp19-baFGF following the procedures of Zoller, and
Smith, Methods in Enzym. 100: 468-500 (1983), Norris
et al., Nucleic Acids Res. 11: 5103-5112 (1983), and
Zoller and Smith, DNA 3: 479-488 (1984). Three
oligonucleotides for each species are designed to
specify serine codons in place of each of the cysteine
codons of the human aFGF gene at positions 16, 83 and
117 and at positions 16, 47 and 83 for the bovine
gene. An oligonucleotide is designed to specify a
leucine codon in place of the methionine codon of the
human or bovine aFGF at position 67. The human
oligomers synthesized are shown in the following
table with the mutated bases underlined.
mnnr ~ vT
Cysteine 1 (16) 5'CCGTTAGAGGAGTAAAGAAGC 3'
Cysteine 2 (83) 5'GGAAAAGGGACTCCTCG 3'
Cysteine 3 (117) 5'CCGCGTTTAGAGCTGCC 3'
Methionine (67) 5' CCATCAGTGTCCAGGGCAAGG 3'
13~~~3~~
- 36 -
Similar oligomers are identified for the appropriate
regions of the bovine aFGF gene and the specific
mutations carried out as described below.
The human oligomers are phosphorylated and
annealed individually to M13mp18-haFGF or
M13mp19-baFGF single-stranded DNA. A second strand
of DNA is synthesized using the annealed oligomer as
primer. Each cysteine mutated gene is used to
transform an appropriate host such as competent E.
coli DH5 cells. The transformed cells are plated on
a lawn of an acceptable host for the M13 virus such _
as E. coli JM105 cells. The transformed plaques are
selected by hybridization with the appropriately
labeled oligomer. Conditions of hybridization are
optimized for each probe to prevent retention of
hybrids containing single base changes.
Single-stranded DNA is isolated from phage clones
containing each of the cysteine-to-serine mutations
for DNA sequence analysis using the method of Sanger
et al., Proc. Natl. Acad. Sci. USA 74: 5463-5467
(1977). RF DNAs are prepared for each clone, cleaved
with EcoRI and SalI and purified by agarose gel
electrophoresis. The purified 440 by inserts are
individually ligated to the 2.7 kb EcoRI-SalI DNA
fragment of the pKK2.7 tac promoter expression
vector. The ligated DNAs are used to transform
competent DH5 cells and clones containing DNA with
the mutated cysteine codons are selected by hybridiza-
Lion to the appropriate oligomer. Each aFGF gene
insert is sequences by the method of Maxam and
Gilbert, Methods in Enzymololgy 65: 499-560 (1980).
The clones containing the single base change from the
original human DNA are designated: pKK-haFGF (Ser
~.34~~~:~
- 37 -
16), pKK-haFGF (Ser 83) and pKK-haFGF (Ser 117);
while the bovine DNA; is designated pKK-baFGF (Ser
16), pKK-baFGF (Ser 47) and pKK-baFGF (Ser 83), for
the location of the substitution in the protein.
Substitution of any two or all three of the
cysteine residues is accomplished by multiple point
mutations or by combining restriction fragments of
either human or bovine recombinant wild-type and the
(Ser 16), (Ser 47), (Ser 83) and (Ser 117) mutant
synthetic genes, cloned in M13mp19 for bovine and
M13mp18 for human, and subcloned in pKK2.7 as
described above. It is to be understood that the
multiple mutations can be carried out with either the
bovine or human single mutation aFGF constructs as
described above, however, the following illustration
will include only human aFGF. The pKK-haFGF (Ser
16,32) and pKK-haFGF (Ser 16,32) recombinants are
constructed by introducing the 0.23 Kb EcoRl-BamHl
fragment of M13mp18 (Ser 16) into pKK2.7 followed by
insertion of the 0.2 Kb BamHl-Sall fragments either
from M13mp18 (Ser 83) or from M13mp18 (Ser 117). The
pKK2.7 vector is modified to remove the BamHl site
upstream of the tac promoter while leaving the BamHl
site in the multicloning sequence. Following
digestion with the corresponding restriction enzymes,
subsequent ligation and transformation of an
appropriate host, clones are selected and screened
for those containing plasmids with the expected
molecular weight for the recombinants, about 3.1 Kb.
An appropriate bacterial host may include, but is not
limited to, E. coli DHS, JM105 or AB1899.
~~40~3g
- 38 -
The mutant haFGF (Ser 16,83,117) is
constructed by replacing the 0.13 Kb Sphl-Sall
fragment of pKK-haFGF (Ser 16,83), by the
corresponding fragment of pKK-haFGF (Ser 117) that
encodes for Ser instead of Cys in the 117 position.
The 3 Kb Sphl-Sall fragment of pKK-haFGF (Ser 16,83)
is purified by preparative agarose gel electro-
phoresis, electroelution and ligated to the 0.13 Kb
Sphl-Sall fragment of pKK-haFGF (Ser 117) purified
from a 5$ polyacrylamide gel in the same way. The
purified fragments are ligated and recombinants
selected as described above.
The pKK-haFGF (Ser 83,117) mutant is
constructed by replacing the 0.3 Kb Pstl fragment of
pKK-haFGF, the non-mutated form, with the fragment
pKK-haFGF (Ser 16,83,117) that includes the codons
for Ser instead of Cys at positions 83 and 117 using
the above techniques. Transformants are analyzed by
pstl-Sall digestion to determine the orientation of
the ligated fragments. All genes are sequenced by
the dideoxy method of Sanger et al., Proc. ~latl.
Acad. Sci. USA 74:5463-5467 (1977).
Expression of a mutated aFGF gene is
accomplished by a number of different promoter-
expression systems in a number of different host
cells. It is desired and intended that there be
included within the scope of this invention, the use
of other host cells and promoter-expression systems
for the expression of the intact mutated aFGF gene.
The host cells include bacteria, yeast, insect, and
mammalian cells. The antigens may also be expressed
in viruses. Although the genes can be expressed in
numerous procaryotic cells and various eucaryotic
~~4'~~3~~
_ 39 _
cells the preferred host cell is Escherichia coli.
The expression vectors which can be used for the
expression of the mutated aFGF include, but are not
limited to, pBR322, pPLa2311, pKC30, ptacl2, 7~gt11,
CheY, pASl, pLC24, pSB226, SV40 and pKK223-3 with
pKK223-3 being preferred. Escherichia coli
expression vectors generally allow the translation of
a methionine residue attached to the first amino acid
of the desired protein. It will be understood that
the present invention includes not only mutant r-aFGF
with a terminal methionine but also mutant r-aFGF
which has had the terminal methionine removed
following translation in such cell types as yeast
cells, mammalian cells or bacterial cells. The
expression vector may have included in the DNA
sequence one or more additional cistrons which will
enhance the expression of the aFGF gene, Schoner _et
_al., Proc. Natl. Acad. Sci USA _83: 8506-8510 (1986).
The preferred construct uses the E. coli tac
promoter, a hybrid between regions of the trp
promoter and the lac promoter as described by deBoer
et al., Proc. Nat. Acad. Sci. USA _80: 21-25
(1983). Plasmid pKK 223-3 (Pharmacia) which contains
the tac promoter and rrnB rRNA transcription
terminator was modified to remove the pBR322-derived
SalI restriction enzyme site. The rrnB rRNA
terminator has been shown to allow expression by
strong promoters, Gentz _et _al., Proc. Natl. Acad.
Sci. USA 78: 4936-4940 (1981); Brosius, Gene 27:
161-172 (1984).
The pKK223-3 plasmid DNA is cleaved with
restriction enzymes to produce a 2.7 kb DNA fragment
to generate clone pKK2.7. The synthetic aFGF gene is
L~~~~~
- ~o -
cleaved from its pBR322 vector and transferred to the
pKK2.7 plasmid after restricting pKK2.7 with EcoRI
and SalI. The resulting recombinant, shown in Figure
1, is transformed into E. coli JM105 (Pharmacia) or
DH5 (BRL) cells and expressed.
The preferred enhancing expression vector
will contain a nucleotide sequence, the first
cistron, upstream of the gene encoding the desired
protein, the second cistron. The mutated aFGF will
be the second cistron. The first cistron will
generally contain a Shine-Dalgarno sequence upstream
of the stop codon. An enhancing expression vector
may contain, but is not limited to, the following
nucleotide sequence:
AATTATGTATCGATTAAATAAGGAGGAAT
TACATAGCTAATTTATTCCTCCTTATTAA
(pKK2.7) (cistron 1,2 oligomers) (aFGF)
which is an effective first cistron for enhancing the
expression of wild-type or mutant aFGF. The first
cistron is inserted into the appropriate pKK-haFGF
construct at the EcoRl site. The insertion results
in the loss of the EcoRl cloning site. The
recombinant is transformed into an appropriate host
cell such as those described above and expressed.
This construct results in about a 10-fold increase in
wild-type or mutant aFGF expression. The plasmids
containing the enhancing expression vector are
designated pKK2c-haFGF. The present invention is
contemplated to include clones containing the
enhancing expression vector such as;
pKK2c-haFGF (Ser 16), pKK2c-haFGF (Ser 83),
... ~.~4~0~3~1
- 41 -
pKK2c-haFGF (Ser 117), pKK2c-haFGF (Ser 16,83),
pKK2c-haFGF (Ser 16,117), pKK2c-haFGF (Ser 83,117),
pKK2c-haFGF (Ser 16,83,117).
The mutated expression clones are grown at
about 37°C in an appropriate growth medium, which
consists of about 1% tryptone, about 0.5% yeast
extract, about 0.5% NaCl, about 0.4% glucose and
about 50 ~.g/ml ampicillin. When the optical
density at 550 nm reaches about 0.5, isopropyl-f3-D-
thiogalactopyranoside (IPTG) may be added to give a
final concentration of about 1 mM and growth is -
continued at about 37°C for up to about 24 hours.
The cells from 1 liter of culture medium are
harvested by centrifugation and resuspended in a
washing buffer containing about 100 mM phosphate and
about 5 mg/ml EDTA. After the final resuspension
about 0.1 mg/ml of lysozyme is added and the
suspension is incubated with gentle shaking at about
30°C for about 15 minutes. The cells are collected
by centrifugation and resuspended in a disruption
buffer containing about 100 mM sodium phosphate at
about pH 6.0, about 3 mM EDTA, about 0.03 mM
N-p-toluenesulfonyl-L-phenyl-alanine chloromethyl
ketone (TPCK), about 0.05 mM pepstatin A, about 0.05
mM phenylmethylsulfonyl fluoride (PMSF), about o.05 mM
leupeptin and about 15 ~rg/ml bovine pancreatic
trypsin inhibitor (BPTI). The cells are either
immediately disrupted or frozen and stored at -70°C
and disrupted immediately after thawing by about two
passages through a French pressure cell at about
20,000 psi at about 4°C. The supernatant fluid is
collected following centrifugation and lyophilyzed.
- 42 -
The mutated aFGFs are purified to homogeneity
by a three step chromatography process employing a
cation exchanger matrix followed by a Heparin-
Sepharose affinity matrix followed by reverse phase
high performance liquid chromatography (HPLC). The
lyophilyzed supernatant fluids are resuspended in
phosphate buffer, about 100 mM, about pH 6.0 and
added to a cation exchanger, preferably CM-Sephadex
which has been equilibrated with the same buffer.
The CM-Sephadex is added at a ratio of about 6.5 ml
of settled resin per gram of protein. The resin is .
collected in a scintered glass funnel and washed three
times with phosphate buffered saline, about 100 mM
phosphate and about 150 mM NaCl at a pH of about 6.
The resin is resuspended in the same buffer, packed
in a column, washed and eluted with about 600 mM NaCl
buffer. Heparin-Sepharose is equilibrated with about
10 mM phosphate buffer, pH about 7.2 added to the
eluate at a ratio of about 1 ml of settled resin per
1 mg of protein, gently shaken for about 1 hour at
about 4° and the resin-protein complex collected in a
funnel. The resin is resuspended in the same buffer
and packed in a column at 1-2 column volume per
hour. The column was washed with a buffer containing
about 10 mM phosphate, pH about 7.2 and about 0.8 M
NaCl and eluted with 1.5 M NaCl in the same buffer.
Each protein was collected and further purified by
reverse phase HPLC. Fractions are loaded on an HPLC
reversed phase column, about C3, equilibrated with
about 10 mM trifluoroacetic acid (TFA) and eluted
with a gradier.~- of from about 0 to about 100% 4 mM
TFA, about 0-67% CH3CN in about 30 minutes.
- 43 -
Mitogenic activity of the purified mutated
recombinant aFGFs is determined by incorporation of
3H-thymidine into DNA by cell line fibroblasts,
preferably BALB/c 3T3 A31 (American Type Culture
Collection). Mutant proteins from plasmids pKK-haFGF
(Ser_16) and pKK-haFGF (Ser 83) stimulated
fibroblasts at a level equal to or lower than the
non-mutated human aFGF. Mutant protein pKK-haFGF
(Ser 117) showed a stimulatory activity that is
higher than the non-mutated forms in the absence of
heparin.
A well controlled and very reproducible
mitogenic assay is required to compare the relative
specific mitogenic activities of wild-type haFGF and
the Cys to Ser mutants. Confluent cultures of Balb/c
3T3 cells in serum free culture fluid were stimulated
with consecutive two-fold dilutions over at least 3
log orders of aFGF concentration spanning the
complete rise of the response from background through
peak DNA synthesis. One stimulatory unit is
calculated as the amount of aFGF per ml that
generated a half maximal response. The specific
mitogenic activity is the number of stimulatory units
per mg of pure aFGF. The assay is further
standardized by diluting stock solutions to about 50
~g aFGF/ml of TFA/CH3CN, or less. The dilution
eliminates any concentration effect so that different
samples can be compared.
Conversion of the Cys 117, any two Cys or
all three Cys residues to Ser results in a 7 to 20
fold increase of the specific activity of the protein
in the absence of heparin. Even in the presence of
heparin, all 4 multiple mutants are
- 44 -
are more active than wild-type human r-aFGF with
haFGF (Ser 83,117) being about 2.7-fold more active.
Although heparin stimulates the activity of wild-type
aFGF 20-fold, it potentiates the activity of the
mutants by only about 3- to about 5-fold.
Conversion of either all, or of any two, of
the three Cys residues of human aFGF to Ser results
in a 7 to 20 fold increase of the specific activity
of the protein in the absence of heparin. Even in
the presence of heparin, all four multiple mutants
are more active than non-mutated haFGF, with haFGF
Ser (83,117) being nearly 3-fold more active.
Mutated recombinant aFGF is useful in
promoting the repair or healing of, but not limited
to, soft tissue wounds resulting from burns, cuts or
lacerations, and cutaneous ulcerations along with
musculo-skeletal wounds such as bone fractures,
ligament and tendon tears, and inflammation of bursas
and tendons. Tissue repair as used herein is defined
as the regeneration of tissue following the
stimulation of mesodermal, ectodermal or
neuroectodermal derived cells by aFGF. Mutated
r-aFGF is also useful in promoting the healing and
regeneration of cartilage and cartilageneous tissue.
Administration of mutated aFGF for soft tissue
repair, including corneal tissue, will generally be
by topical, subcutaneous, intravenous or intraocular
application. Soft tissue includes all tissue except
that associated with the musculo-skeletal system as
described above. The novel peptides may be
administered with ~r without hepar~_n, preferably
without heparin, about 0.1 to about 100
ug/cm2/day of this invention, protein, to the
1~~~~3
- 45 -
wound area either topically or subcutaneously about 1
to about 100 ug/cm3/day. The most preferred
application range for topical administration is about
1 to about 10 ug/cm2/day.
Heparin is a sulfated glycosaminoglycan
consisting of equal parts of the sugars D-glucosamine
and D-glucuronic acid which are sulfated to varying
degrees. It is commercially available in an
u~odified form as well as in a solution form for
direct therapeutic utilization. When heparin is
administered with aFGF in topical or subcutaneous
applications the preferred concentration is from
about 3 times to about 30 times the amount (mass) of
aFGF administered per day.
For musculo-skeletal and cartilage repair or
healing, the mutated r-aFGF is preferably administered
at the site of the injury either during surgery or by
injection. Surgical implantation of slow-release
forms of the mutated aFGF will allow for a continued
release of the growth factor for a prolonged period
of time. Methods of formulation o~f mutated aFGF for
slow release are known in the art. Dosage levels for
musculo-skeletal healing will be about 10 to about
100 ug/cm3/day.
Mutant r-aFGF is furthermore useful in
promoting the facilitation of in vivo vascular tissue
repair, such as blood vessel growth (angiogenesis),
and vessel repair (such as the replacement of damaged
endothelial cells) and in stimulating endothelial
cell growth on appropriate substrates for the
production of blood vessels for implantation. In
vivo angiogenesis activity of the novel mutant r-aFGF
peptides is accomplished by the internal
administation,such as
- 46 -
subcutaneously, of about 1 to 1000 ug/cm3/day
with the more preferred amount of about 10 to about
100 ~g/cm2/day. The preferred application range
for surface repair is about 100 ng to about 100
ug/cm2/day with the most preferred application
range being about 1 to about 10 Ng/cm2/day.
Large vessel repair is accomplished by a single dose
of about 0.1 to about 100 ng/cm3 or by continuous
infusion of about 1 to about 1000 pg/cm3/day. In
vitro growth of Endothelial cells on appropriate
substrates for the production of blood vessels is
accomplished by the administration of about 1 to
about 10 ng/ml/day.
Mutant r-aFGF is also useful in the in vivo
induction of plasminogen activator by vascular
endothelial cells for the treatment of thrombotic
attacks. Thrombotic attacks result form the
formation of thrombi within blood vessels which may
result in thrombotic strokes, deep vein thrombosis,
myocardial infarction and other medical conditions
which give rise to necrosis of tissues and often
times death of the patient. Digestion of preformed
clots and the prevention of further clot formation
can be mediated by mutant r-aFGF thereby enhancing
the treatment of thrombotic attacks. Pretreatment
with mutant r-aFGF may also be used to prevent the
formation of clots in animals, including man, which
are at high risk for clot formation. The desirable
dosage range of mutant r-aFGF for the treatment of
thrombotic attack is about long-lOmg/kg/day.
Mutated and wild-type r-aFGF is also useful
in promoting central and peripheral nerve tissue
repair including the maintenance and stimulation of
hippocampal neurons and neurons that are damaged or
destroyed in Alzheimer's disease and motor and
sensory neurons whose destruction causes paralysis.
.. ~~~~~3~
- 47 -
Damaged nervous tissue may be stimulated by mutated
or wild-type aFGF to produce additional neurons by
mitosis of neuroblasts to re-populate the damaged
nerves in the area and to promote neurite outgrowth
from neurons. The peptides may be administered as
described for wound healing of either soft tissue or
musculo-skeletal tissue.
For topical application, various
pharmaceutical formulations are useful for the
administration of the active compound of this
invention. Such formulations include, but are not .
limited to the following: ointments such as
hydrophilic petrolatum or polyethylene glycol
ointment; pastes which may contain gums such as
xanthan gum; solutions such as alcoholic or aqueous
solutions; gels such as aluminum hydroxide or sodium
alginate gels; albumins such as human or animal
albumins; collagens such as human or animal
collagens; celluloses such as alkyl celluloses,
hydroxyalkyl celluloses and alkylhydroxyalkyl
celluloses, for example methylcellulose, hydroxyethyl
cellulose , carboxymethyl cellulose, hydroxypropyl
methylcellulose, and hydroxypropyl cellulose;
poloxamers such as Pluronic o Polyols exemplified
by Pluronic F-127; tetronics such as tetronic 1508;
and alginates such as sodium aliginate. The
pharmaceutical formulations will include one or more
of the mutated aFGF compounds in amounts of about 0.1
to about 100 ug/ml.
For non-topical application the mutant
r-aFGF is administered in combination with
pharmaceutically acceptable carriers or diluents such
..
- 48 -
as, phosphate buffer, saline, phosphate buffered
saline, Ringer's solution, and the like, in a
pharmaceutical composition, according to standard
pharmaceutical practice.
The ability of mutated aFGF to stimulate
division in various cell types including fibroblasts,
vascular and corneal endothelial cells and the like
makes these peptides useful as pharmaceutical agents.
These compounds can be used to treat wounds of mammals
including humans by the administration of the novel
mutated r-aFGF to patients in need of such treatment..
The following examples illustrate the present
invention without, however, limiting the same thereto.
wTUnT ~ ~
Oliqonucleotide Synthesis
Oligonucleotides were synthesized according
to the technique described by Matteucci and
Caruthers, J. Am. Chem. Soc. 103: 3185-3191 (1981);
Beaucage and Caruthers, Tetrahedron Letters 22:
1859-1862 (1981). The base sequences of the
synthesized oligonucleotides are shown in Tables VII,
IX and XI.
L~1TTM~T L' ~
Assembly of the aFGF Gene
The bovine oligonucleotides from Example 1
were assembled as two separate units, the N-terminal
half (231 bp) and the C-terminal half (209 bp). The
two halves were then combined for the intact synthetic
gene, see Table VI. Initially the oligonucleotides
were kinased in the following reaction mixture: 70
mM Tris pH 7.6, 5 mM DTT, 10 mM MgCl2, 33 uM ATP,
- 49 -
0.3 units T4 polynucleotide kinase per ul, and 2.5
pmole oligonucleotide per ~,1. The mixture was
incubated 1.5 hours at 37°C and then an additional
hour after supplementing the mixture with 0.2
units/ul kinase and ATP to give a concentration of
100-mM. For radioactive labelling, the initial
mixture contained 37 nCi/ul of [Y_32P)_ATP.
The annealing and legations were done in two
separate reactions. In each reaction, 100 pmole of
each of the eight oligonucleotides were added. In
one reaction the oligonucleotides which make up one
strand of the C-terminal or N-terminal half gene were
kinased with the exception of the most 5' oligo-
nucleotide. In the second reaction the oligo-
nucleotides which make up the opposite strand were
kinased, again with the exception of the most 5'
oligonucleotide. Thus, in each reaction 3 oligo-
nucleotides were kinased and 5 were not. When
kinased oligonucleotides were used, 1 pmole of the
32P_labelled oligonucleotide was also added for
later identification of the products. Each reaction
contained 200 ul with 70 mM Tris pH 7.6, 5 mM DTT,
10 mM MgCl2, and 30 ~M ATP. The oligonucleotides
were annealed by heating to 90°C for 4 minutes, then
immediately transferring the reaction to 60°C and
allowing it to cool slowly to 30°C. Legation was
done in 400 ul containing 60 mM Tris pH 7.6, 10 mM
DTT, 10 mM MgCl2, 1 mM ATP, and 0.03 units T4 DNA
ligase per ul by incubating at 20°C for 1.5 hours.
Polyacrylamide gel electrophoresis was used
to purify the legated oligonucleotides. The legated
oligonucleotides were precipitated with ethanol,
redissolved in 20 ul of 80% formamide, 50 mM
~.34~~:3°J
- 50 -
TRIS-borate pH 8.3, 1 mM EDTA, 0.1$ (w/v) xylene
cyanol, and 0.1$ (w/v) bromophenol blue. Each sample
was heated at 90°C for 3 minutes and electrophoresed
in a 10$ urea-polyacrylamide gel at 75 watts for 5
hours. The oligonucleotide bands were visualized by
exposing the gel to X-ray film.
The 231 base bands of each reaction for the
N-terminus were cut out of the gel, combined, and
eluted at 4°C in 1 ml of 0.5 M ammonium acetate, 1 mM
EDTA pH 8. The eluted DNA was precipitated with
ethanol and redissolved in 30 ul of 70 mM Tris pH
7.6, 5 mM DTT, and 10 mM MgCl2. The 209 base bands
of the C-terminus were eluted in the same manner.
The gel purified oligonucleotides were
annealed prior to transformation by heating to 90°C
for 4 minutes and slow cooling to 20°C. Assuming a
5$ recovery from the initial starting oligonucleo-
tides, 300 fmole and 100 fmole of recovered annealed
231 by oligonucleotides were each ligated to 100
fmole of agarose gel purified 3.9 kb EcoRI-BamHI
pBR322 fragment DNA in 20 ~1 of 25 mM Tris pH 7.8,
1 mM DTT, 10 mM MgCl2, 0.4 mM ATP, with 1 unit T4
DNA ligase for 1 hour at 20°C. The annealed 209 by
oligonucleotides were ligated to agarose purified 3.9
kb BamHI-SalI pBR322 fragment DNA under the same
conditions as the 231 base pair fragments. The
ligation reactions were diluted 1:5 in H20 and
1 ul of dilution was used to transform 20 ul of
competent E. coli RR1 cells (BRL) as described by the
supplier. The transformants were selected for growth
in ampicillin and screened for the presence of the
231 by EcoRI-BamHI or the 209 by BamHI-SalI insert bZs
restriction analysis of mini-lysate plasmid
preparations.
~.~3~~ ~3
- 51 -
The DNA sequence of clones containing the
appropriate sized inserts was determined using the
chemical DNA sequence techniques of Maxam and
-5
Gilbert, Proc. Natl. Acad. Sci. USA 74: 560-564
(1977). Since none of the 231 by clones had the
correct sequence, a clone containing the correct
sequence was prepared as follows. One clone with the
correct sequence between the KpnI and BamHI sites was
cleaved with KpnI and with SalI, which cleaves in the
pBR322 vector. The 400 by band was gel purified and
ligated to the 3.8 kb KpnI-SalI band of a second
clone containing the correct sequence from the EcoRI
site to the KpnI site of the aFGF gene insert. After
transformation, a resulting clone was sequenced to
ensure the desired sequence had been obtained.
Since a clone containing the correct 209 by
sequence was obtained, no further manipulation of
these clones was required. The final full-length
aFGF synthetic gene was cloned by cleaving the
N-terminal half clone with BamHI and SalI, treating
with alkaline phosphatase, and ligating this to the
gel purified 209 by BamHI-SalI insert of the
~-terminal half clone. This ligated material was
used to transform competent RR1 cells as before.
~Y~1HDT L' '~
Mutagenesis of the Bovine aFGF Gene
to the Human aFGF Gene
To facilitate the mutagenesis of the bovine
aFGF gene, the synthetic gene from Example 2 was
transferred to M13mp19, a single-stranded DNA
bacteriophage vector. Standard mutagenesis procedures
were used as reported by Zoller and Smith, Methods in
~.3~~~ar~
- 52 -
Enzymology, 100: 468-500 (1983); Norris et al.,
Nucleic Acids Research, 11: 5103-5112 (1983); and
Zoller and Smith, DNA, 3: 479-488 (1984). The bovine
pKK-aFGF plasmid was cleaved with EcoRI and SalI and
the resulting 44o by fragment was agarose gel
purified as in Example 2. Vector M13mp19 RF DNA
(BRL) was cleaved with the same two endonucleases and
the ends were subsequently dephosphorylated in 100
ul of 10 mM Tris pH 8.0 buffer with 100 units of
bacterial alkaline phosphatase. A ligation was
performed using 50 ng of the treated vector DNA and -
12 ng of the aFGF gene fragment DNA in 10 ul of 25
~ Tris pH 7.8, 10 mM MgCl2, 1 mM DTT, 0.4 mM ATP,
with 2 units of T4 DNA ligase for 16 hours at 4°C.
The reaction mixture was diluted 1:5 in H20 and 1
ul of dilution was used to transform 20 ul of
competent E. coli DH5 cells (BRL) as described by the
supplier. The cells were plated with E. coli JM105
(Pharmacia) host cells in 0.03% X-gal and 0.3 mM
IPTG; after incubation at 37°C colorless plaques were
isolated. One phage clone containing the bovine aFGF
gene was selected, M13mp19-baFGF.
Eight oligonucleotides were designed to
specify the human sequence and synthesized, see Table
IX. Oligomer 8 contains an additional mutation in
which thymine at site 386 in the bovine gene is
replaced by cytosine in the human gene. This
mutation allows the incorporation of a restriction
site without altering the human aFGF amino acid
sequence.
- 53 -
The human oligomers 1, 2, 3, 4, 6, and 8
were phosphorylated and 15 pmoles of each were
annealed individually to 0.5 pmole of M13mp19-baFGF
single-stranded phage DNA in 10 ul of 20 mM Tris pH
7.5, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT for 10
minutes at 65°C followed by 10 minutes at 23°C.
Closed-circular double-stranded molecules were then
prepared in 20 ul of 20 mM Tris pH 7.5, 10 mM
MgCl2, 25 mM NaCl, 5.5 mM DTT, 0.5 mM ATP, 0.25 mM
dATP, 0.25 mMd CTP, 0.25 mM dGTP, 0.25 mM dTTP, using
1 unit of T4 DNA ligase and 2 units of DNA polymerase
I klenow fragment by incubation at 15°C for 17
hours. The preparations were each used to transform
competent JM105 cells and the resulting transformant
plaques were selected by hybridization with the
appropriate oligomer which had been radio-labeled
using 32P-ATP and polynucleotide kinase. The
conditions of hybridization were optimized for each
probe to prevent formation of hybrids containing
single base changes. Single-stranded DNA was isolated
from the phage clone containing the human oligomer 4
mutations and the above procedure was repeated using
the human oligomer 5 to generate a clone containing
both the oligomer 4 and 5 mutations.
In the following procedures the bovine-to-
human sequence mutations in these M13-based clones
were combined into one pBR322-based clone. RF DNAs
'"'ere prepared from clones containing the base changes
specified by human oligomers 1, 2, 6, and 8. The DNA
of the human 1 mutant clone was cleaved with EcoRI,
the ends were dephosphorylated with bacterial alkaline
phosphatase, and the DNA was cleaved with HindIII.
1.~4Q63~'
- 54 -
The human 2 mutant DNA was cleaved with HindIII,
treated with phosphatase, and then cleaved with BamHI.
The human 6 mutant DNA was cleaved with BamHI,
phosphatase treated, and subsequently cleaved with
ApaI. Likewise, the human 8 mutant DNA was cleaved
with-ApaI, the ends were dephosphorylated, and the
DNA was cleaved with SalI. These four DNA
preparations were electrophoresed through 2% agarose
and the fragments of 45 bp, 190 bp, 135 bp, and 70 by
from the mutant DNAs containing human 1, 2, 6, and 8
mutations, respectively, were eluted from the gel.
Approximately 60 fmoles of each fragment were
collectively ligated to about 60 fmoles of a
gel-purified 3.7 kb EcoRI-SalI fragment from pBR322
in 5 ul of 25 mM Tris pH 7.8, 10 mM MgCl2, 1 mM
DTT, 0.4 mM ATP, with 1.5 units of T4 DNA ligase for
16 hours at 12°C. The reaction mixture was diluted
1~5 in H20 and 1 ~1 of dilution was used to
transform 20 ul of competent E. coli DH5 cells
(BRL) as described by the supplier. A clone
containing the mutations specified by all four mutant
oligomers was selected by hybridization with
radiolabeled probes prepared from each of the
oligomers. The 140 by KpnI-BamHI DNA fragment
isolated from cleaved RF DNA of the human 3 mutant
M13 clone was ligated to endonuclease cleavage
products of this human 1-2-6-8 mutant DNA and
transformed into DH5 competent cells to generate a
clone with the human 1-2-3-6-8 mutations. BamHI-PstI
digestion fragments of this latter clone were ligated
to the BamHI-PstI digestion fragments of RF DNA from
the human 4-5 M13-based clone and the ligation mixture
.~. ~! ~ ~ ;"~ ry
- 55 -
was used to transform DH5 competent cells. A clone
containing the human 1-2-3-4-5-6-8 mutations was
selected by oligomer hybridization and the aFGF gene
EcoRI-SalI DNA fragment of this recombinant plasmid
was_ligated to phosphatase-treated EcoRI-SalI-cleaved
RF DNA of M13mp18 (BRL). Competent DH5 cells were
transformed with this ligated DNA and the transformed
cells were plated on JM105 host cells to generate an
M13 clone. The single-stranded phage DNA of this
clone was annealed with the human 7 oligomer and an
M13 clone containing all the desired mutations was
obtained following the procedure described above. RF
DNA was prepared from this clone and cleaved with
EcoRI and SalI. The resulting 440 by band was gel
purified and ligated to the 2.7 kb EcoRI-SalI DNA
fragment of the pKK2.7 tac promoter expression vector.
This DNA was used to transform competent DH5 cells
thus generating the human pKK-aFGF expression clone
used for production of the human form of aFGF.
EXAMPLE 4
Mutagenesis of the Cysteine Codons of the aFGF Gene
A human aFGF single-stranded bacteriophage
recombinant clone, M13mp18-haFGF, from Example 3 was
mutagenized using procedures reported by Zoller and
Smith, Methods in Enzymoloqy, 100: 468-500 (1983);
Norris et al., Nucleic Acids Research, _11: 5103-5112
(1983)% and Zoller and Smith, DNA, _3: 479-488 (1984).
Three oligonucleotides were designed to specify serine
codons in place of each of the cysteine codons of the
human aFGF gene at positions 16, 83, and 117. The
oligomers synthesized are shown in Table XI with the
mutated bases underlined.
13~~~3
- 56 -
The oligomers were phosphorylated and 15
pmoles of each were annealed individually to 330 ng
of M13mp18-haFGF single-stranded DNA in 10 ul of 20mM
Tris pH 7.5, 10 mM MgCl2, 50 mM NaCl, and 1 mM DTT
for 10 minutes at 65°C followed by 10 minutes at
23°C. A second strand of DNA was synthesized using
the annealed oligomer as primer in 20 ul of 20 mM
Tris pH 7.5, 10 mM MgCl2, 25 mM NaCl, 5.5 mM DTT,
0.5 mM ATP, 0.25 mM dATP, 0.25 dCTP, 0.25 mM dGTP,
0.25 mM dTTP, using 3 units of T4 DNA ligase and 0.4
units of DNA polymerase I klenow fragment by
incubation at 12°C for 17 hours. The three
Preparations were each diluted 1:5 in H20 and 1 ul
of dilution was used to transform 20 ul aliquots of
competent E. coli DH5 cells (Bethesda Research Labs)
as described by the supplier. The transformed cells
where plated with a lawn of E. coli JM105 cells which
act as host cells for the M13 virus. The resulting
transformant plaques were selected by hybridization
with the appropriate oligomer which had been radio-
labeled using 32P-ATP and polynucleotide kinase.
The conditions of hybridization were optimized for
each probe to prevent retention of hybrids containing
single base changes.
Single-stranded DNA was isolated from phage
clones containing each of the cysteine-to-serine
mutations for DNA sequence analysis using the
dideoxynucleotide chain termination method of Sanger
et al., Proc. Natl. Acad. Sci. USA 74:5463-5467
(1977). RF DNAs were then prepared from three clones,
each containing one of the specified mutations, and
after cleavage with EcoRI and SalI the released FGF
i~~U~3~
- s7 -
gene inserts were isolated by agarose gel electro-
phoresis. The purified 440bp inserts were each
ligated to the 2.7kb EcoRI-SalI DNA fragment of the
pKK2.7 tac promoter expression vector in 10 ul of
25mM Tris pH 7.8, 10 mM MgCl2, 1 mM DTT 0.4 mM ATP,
with.3 units of T4 DNA ligase for 2 hours at 14°C.
The ligated DNAs were used to transform competent DH5
cells and clones containing DNA with the mutated Cys
codons were selected by hybridization to the
appropriate oligomer. The FGF gene insert in the
plasmid DNA of these clones was sequenced completely
by the chemical method of Maxam and Gilbert, Methods
in Enzymology 65:499-560 (1980). One clone contained
-
only the single base change from the original human
aFGF expression clone generating a serine codon in
place of a cysteine codon at position 83 and is
designated as pKK-haFGF(Ser 83).
The clones containing each of the other two
cysteine-to-serine mutations also contained additional
non-specified changes. In order to generate the
desired single base mutants the following ligations
and transformations were performed. The 410bp
HindIII-derived DNA fragment of the clone with the
serine codon at position 16 was isolated and ligated
to the 2.7kb HindIII-derived fragment of the original
pKK-haFGF expression clone. The 230bp NcoI-SalI-
derived DNA fragment of the clone containing the
serine codon at position 117 was isolated and ligated
to the 2.9kb NcoI-SalI-derived fragment of pKK-haFGF.
Each of these ligated samples was used to transform
competent DH5 cells; hybridization and sequencing
techniques were used to identify the other two
- 58 -
desired single base mutants designated
pKK-haFGF(Ser 16) and pKK-haFGF(Ser 117). These three
clones were used for production of the Ser 16, Ser
83, and Ser 117 forms of the human aFGF.
Site-directed mutants of human aFGF with two
or three cysteine (Cys) residues converted to serine
(Ser) residues were constructed by combining
restriction fragments of the non-mutated wild-type
and the Ser (16), Ser (83) and Ser (117) mutant
synthetic genes, have been cloned in pKK2.7 and
subcloned in M13mp18, as described above. The
pKK-haFGF (Ser 16,83) and pKK-haFGF (Ser 16,117)
recombinants were constructed first by introducing
the 0.23 Kb EcoRl-BamHl fragment of M13mp18 (Ser 16),
that includes the codon for Ser 16, into pKK2.7
followed by insertion of the 0.2 Kb BamHl-Sall
fragments either from M13mp18 (Ser 83) or from
M13mp18 (Ser 117). Since the pKK2.7 vector contains
two BamHl sites, one in the multicloning sequence and
the second one upstream of the tac promoter, a
modified pKK2.7 vector, in which the second upstream
BamHl site was eliminated, was used in these
constructions. After digestion with the
corresponding restriction enzymes, subsequent
ligation and transformation of AB1899 competent cells
(E. coli Genetic Stock Center), ampicillin resistant
clones were selected and screened for those
containing plasmids with the expected molecular
weight for the recombinants (3.1 Kb).
The mutant haFGF (Ser 16,83,117) was
constructed by replacing the 0.13 Kb Sphl-Sall
fragment of pKK-haFGF (Ser 16,83), by the
- 59 -
corresponding fragment of pKK (Ser 117) that encodes
for Ser instead of Cys in position 117. The 3 Kb
Sphl-Sall fragment of pKK (Ser 16,18) was purified by
preparative agarose gel electrophoresis,
electroelution and ligated to the 0.13 Kb Sphl-Sall
fragment of pKK (Ser 117) purified from a 5%
polyacrylamide gel in the same way. The purified
fragments were ligated and recombinats were selected
for ampicillin resistance after transformation of
AB1899 cells.
For construction of pKK-haFGF (Ser 83,117),
the 0.3 Kb Pstl fragment of pKK haFGF was replaced
with the same fragment of pKK-haFGF (Ser 16,83,117)
that includes the codons for Ser instead of Cys at
positions 83 and 117 using basically the same
strategy. AB1899 transformants selected for
ampicillin resistance were analyzed by Pstl-Sall
digestion to determine the orientation of the ligated
fragments. All mutant genes were sequenced by the
dideoxy method using the Sequence kit of USB Corp.
L'Y'nMD7 L' G
Expression of the Synthetic Bovine aFGF Gene
The intact aFGF genes from Example 4 were
incorporated into a modified pKK223-3 plasmid. The
pKK223-3 plasmid (Pharmacia) contains the tac promoter
which is a hybrid between regions of the trp promoter
and the lac promoter, deBoer et al., Proc. Natl Acad.
Sci. USA 80: 21-25 (1983). This plasmid also contains
the rrnB rRNA transcription terminator, a strong
terminator sequence found to allow expression from
strong promoters, Gentz et al., Proc. Natl. Acad. Sci.
1~~~~3~
- 60 -
USA 78: 4936-4940 (1981); Hrosius, Gene _27: 161-172
(1984). The pKK223-3 plasmid was modified to remove
the pBR322-derived SalI restriction enzyme site. This
was accomplished by cleaving the pKK223-3 plasmid DNA
with NdeI and NarI, blunt-ending the DNA fragment with
Klenow DNA polymerise, and recircularizing the 2.7 kb
DNA fragment to generate clone pKK2.7. The synthetic
aFGF gene was then cleaved from its pBR322 vector and
transferred to pKK2.7 after restricting this
expression vector with EcoRI and SalI. This
construction positions the initiating methionine of
the synthetic gene il bases downstream of the
Shine-Dalgarno ribosome binding site. The resulting
recombinant vectors, as exemplified by Figure 1, were
transformed into E. coli JM105 cells and also into
E. coli DH5 cells.
The expression clones were grown at 37°C in
LB broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl)
containing 0.4% glucose and 50 ug/ml ampicillin.
When the optical density at 550 nm reached 0.5, IPTG
was added to give 1 mM and growth was continued at
37°C for 3 hours. The cells were harvested by
centrifugation at 10,000 x g for 20 minutes and the
cells from 1 liter of culture were resuspended in
glycerol/phosphate buffered saline 1:1 and quickly
frozen in a dry ice/ethanol bath and stored overnight
at -70°C.
EXAMPLE 6
Enhanced Expression Vector
Enhanced )Avels of expression for the
mutated forms of aFGF of Example 4 were provided by
~.~~0~3~
- 61 -
modification of the expression vector of Example 3 to
introduce an additional cistron upstream of the aFGF
encoding sequence. Two oligonucleotides were
synthesized with the sequences as shown at page 40.
When annealed these oligomers supply 5' extensions of
4 bases which are complementary to the extensions
provided by EcoRl cleavage, a 7 codon open reading
frame following the ATG translation initiation codon
and preceding a TAA stop codon, and an additional
Shine-Dalgarno ribosome binding site located within
the open reading frame upstream of the stop codon.
Using 1 pmole of each oligomer, the oligomers were
a~ealed together in 20 ul of DNA ligase buffer by
heating to 70°C for 10 minutes and slow cooling. The
annealed mixture, 0.3 pmole, was ligated to 0.1 pmole
of EcoRl-cleaved pKK-haFGF plasmid DNA in a final
volume of 25 ul containing 3 units of T4 DNA ligase
(Pharmacia) for 2.5 hours at 14°C. The ligated DNA,
5 ng, was used to transform competent E. coli JM105
cells. the transformants were screened by
restriction analysis, as the EcoRl site is lost by
this insertion, and by immunoblot analysis. The
expression vector of one clone, which demonstrated
higher levels of FGF production, was sequenced by the
chemical technique of Maxam and Gilbert, supra, to
verify the correct insertion of the new cistron
sequences. Subsequently, this high expression
p~2c-haFGF vector was transfered to E. coli DH5 by
transformation procedures.
In order to express the haFGF (Ser 117)
mutant gene, for example, in this high expression
vector, the 0.23 kb Ncol-Sall fragment of pKK-haFGF
1J~~,~~j~:~
- b2 -
(Ser 117) was ligated to the 2.5 kb Ncol-Sall
fragment of pKK2c-haFGF and transformed into
competent cells. The other mutated haFGFs were
transferred to the two cistron high expression vector
in a similar manner, replacing appropriate
restriction fragments containing the wild-type
sequences of pKK2c-haFGF with the analogous
restriction fragments of the mutated haFGF.
EXAMPLE 7
Extraction and Purification of Mutated aFGF
The frozen cells from Example 5 were thawed
and resuspended in a quantity sufficient to make 50 ml
with 100 mM phosphate buffer, pH 7.2, 5 mg/ml EDTA
and the cells were collected by centrifugation at
28,000 x g for 5 minutes. The cells were washed a
second time, collected by centrifugation and
resuspended in 50 ml of the same buffer. The
extinctions of the three mutant strain suspensions at
660 nm were strain pKK-haFGF(Ser 117), 103; strain
pKK-haFGF(Ser 16), 108; strain pKK-haFGF(Ser 83) 59.
Each sample received 0.1 mg/ml of lysozyme and was
incubated for 15 minutes with gentle shaking at 30°C.
The cells were collected by centrifugation and
resuspended in 50 ml of breaking buffer consisting of
100 mM phosphate; pH 6.0; 3 mM, EDTA; 0.05 mM, TPCK,
0.05 mM, Pepstatin A, 0.05 mM, Leupeptin and 15
~'g/ml BPTI. Each cell suspension was kept at 4°C
and broken by two passages through a previously
cooled French pressure cell at 20,000 psi at 4°C.
The disrupted cell s~_.spensions were centrifuged for
15 minutes at 15,000 rpm in a SS-34 Sorvall rotor and
1~~~~3
- 63 -
for 60 minutes at 45,000 rpm in a 70 Ti rotor in a
Beckman ultracentrifuge at 4°C. The supernatant
fluid was collected, the extinctions at 280 nm for a
55 ml volume were determined: pKK-haFGF(Ser 117),
44; pIZK-haFGF(Ser 16), 40 and pKK-haFGF(Ser 83), 23
and the samples were frozen at -70°C.
-- The supernatant fluids were thawed by the
addition of 200 ml of 100 mM phosphate buffer, pH 6.0,
containing CM-Sephade~ at a ratio of 6.5 ml of settled
resin per gram of protein (assuming absorbance through
a 1 cm path of a 1 mg/ml protein solution is 1.0).
The sample was collected in a scintered glass funnel
and washed three times with 200 ml of 100 mM phosphate
buffer containing 150 mM NaCl at a pH of 6Ø The
resin cake was resuspended in 200 m1 of the same
buffer, packed in a column at 12 ml x hr 1 per
cm2 crossectional ArGA and washed at the same flow
rate with 150 mM phosphate buffer containing 600 mM
NaCl. The fractions containing the protein eluted
with the 600 mM NaCl buffer were pooled, the pH
adjusted to 7.2 and the conductivity adjusted with
deionized water to 10 uS x cm 1. Heparin-
Sepharose (freshly prepared) equilibrated with 10 mM
phosphate pH 7.2 (conductivity 1.3 uS x cm 1) was
then added at a ratio of 1 ml of settled resin per mg
of protein (using the same assumed extinction
coefficient as above), the suspension gently shaken
for one hour at 4°C, and the resin collected in a
funnel, resuspended in the same buffer and packed in
a column at 1-2 column volume per hour.
The packed column was washed with 10 mM phosphate,
0.8 M NaCl pH 7.2 at the same flow rate until the
extinction of the eluate at 280 nm decreased to a
~.3~~~3
- 64 -
steady value, to within 0.01 optical absorbance units
above the elution buffer and then the buffer changed
to i0 mM phosphate, 1.5 M NaCl pH 7.2. The fractions
containing the protein eluted with the 1.5 M buffer
(monitored by the extinction at 280 nm) were pooled
together and loaded in a C3 reversed phase HPLC
column equilibrated with 10 mM TFA and eluted with a
gradient from 0-67% CH3CN in 30 minutes.
The purification data of the mutant strains
is shown below:
pKK-haFGF(Ser 16)
Fractions 25-31 eluted from the CM-Sephadex
column with the 0.6 M NaCl buffer in a total volume
of 24 ml and a protein content of 3.5 mg were made
125 ml with deionized water (final conductivity: 7
mS/cm) and 4 ml of heparin-Sepharose~added. The
column was run at 6 ml/h. Fractions 55-57 eluted
with 1.5 M NaCl, were injected on the C3 column.
From this column a major peak was collected with a
protein content of 80 ug.
pKK-haFGF(Ser 83)
Fractions 19-33 eluted from the CM-Sephadex
column with the 0.6 M NaCl buffer in a total volume
of 40 ml and a protein content of 4.0 mg were made
150 ml with deionized water (final conductivity:
10 mS/cm) and 4 ml of heparin-Sepharose added. The
column was run at 6 ml/h. Fractions 40-44, eluted
with 1.5 M NaCl, were injected in the C3 column.
- 65 -
From this column a major peak was collected with a
protein content of 80 ug.
pKK-haFGF(Ser 117)
Fractions 19-33 eluted from the CM-Sephadex
column with the .6 M NaCl buffer in a total volume of
57 ml and a protein content of 11.4 mg were made 250
ml with deionized water (final conductivity: 12
mS/cm) and 10 ml of heparin-Sepharose added. The
column was run at 11 ml/h. Fractions 59-62, eluted
with 1.5 M NaCl, were injected in the C3 column.
From this column a major peak was collected with a
protein content of 614 ug.
The protein products of the multiple mutants
were purified by the same procedures. All forms of
aFGF, recombinant wild-type and the mutants were
highly purified since only single 16 kDa bands were
seen following reduction and electrophoresis in SDS
15% polyacrylamide gels at loads 100-fold above the
threshold of detection.
EXAMPLE 7
Biological Activity of Mutated aFGF
Biological activity of the purified r-aFGF
from Example 6 was evaluated using a fibroblast
mitogenic assay modified from Thomas et al., J. Biol.
Chem. 225: 5517-5520 (1980). BALB/c 3T3 A31
f ibroblasts (American Type Culture Collection) were
plated at 3 x 104 cells per 100 ~1 per well in
96-well culture dishes in culture media containing
1Q% heat-inactivated calf serum and incubated in 7%
.~.~~0~1~~
- 66 -
C02 (pH 7.35 + 0.05). The cells became fully
quiescent by replacing the media with 1.0%
heat-inactivated calf serum 6 and again 24 hours
later. At 55 hours after plating, l0 ul of test
sample with or without 5 ug of heparin and 0.11
ug of dexamethasone were added, at 70 hours each
well was supplemented with 0.2 uCi of [methyl-3H)-
thymidine (20 Ci/mmole, New England Nuclear) and 0.3
ug of unlabeled thymidine (Sigma), and at 95 hours
the cells were processed for determination of
radiolabel incorporated into DNA. Each dose-response
point was the average of four determinations. The
results of Ser-117 Mutant, the only mutant form
showing activity equal to or greater than wild type,
are shown in the following table:
25
~.~~0~3
- 67 -
TABLE XII
Mitogenic Responses of BALB/c 3T3 Fibroblasts to
Mutated aFGF
Dose Wild type Ser-117 Mutant
(amt/ml) -heparin +heparin -heparin +heparin
3.16 pg 1449 724 2055 883
10.0 pg 1917 914 2662 1255
31.6 pg 1547 1007 3076 2748
100 pg 2263 2498 4833 8067
316 pg 2647 14945 11505 44193
1.00 ng 3975 54516 22869 66778
3.16 ng 6400 68447 40487 60306
10.D ng 12665 61294 54163 56326
31.6 ng 21843 56552 70670 59854
100 ng 44744 66816 66802 63856
The 4 titration curves are compared at their
half-maximal rise. The WT in the absence of heparin
does not reach a peak so the same peak magnitude is
assumed as seen for the other 3 peaks and the
half-maximal value extrapolated.
~3~~1n3~
_ 6g _.
TABLE XIII
Comparison of Concentrations Necessary for Half
Maximal Stimulation
Sample Heparin Conc. of 1/2 maximal stimulation
~ - 66 ng/ml
+ 0.56 ng/ml
Ser-117 - 2.3 ng/ml
Mutant + 0.20 ng/ml
All dilutions were prepared from a stock
solution containing 1.51 mg/ml of purified
reactants. The Ser-117 mutant is at least as active
as the wild type in the presence of heparin.
The activity of the wild type is about 10-fold more
dependent on heparin than the mutant consequently 90%
of the heparin dependence of WT aFGF is eliminated in
the Ser-117 mutant.
The mitogenic assay used to evaluate
biological activity was modified so that mutated and
wild-type aFGF could be compared. Heat-inactivated
calf serum was replaced with 1% insulin-selenium-
transferin (ITS), 0.4 gm L-histidine, 50 ul of 1M
ethanolamine, 1.25 gm bovine serum albumin with 5.35
mg of linoleic acid per liter of 75% DMED, 25% Ham's
F12 containing both penicillin-streptomycin and
L-glutamine as described above. Full dose-response
assays were dons as described above at consecutive
two-fold dilutions over at least 3 log orders of aFGF
concentration spanning the complete rise of the
response from background through peak DNA synthesis
:I3~~~3
- 69 - __
levels. All concentration points were done in
quadruplicate on confluent Balb/c 3T3 cells in 96
well dishes. One stimulatory unit was calculated as
the amount of aFGF per ml that generated a
half-maximal response. The specific mitogenic
activity is the number of such stimulatory antis per
mg of pure aFGF. All samples of aFGF were prediluted
to 50 ug/ml in the same TFA/CH3CN solvent. The
activities of wild-type and mutated aFGF are compared
in the following table.
20
30
- 70 -
TABLE XIV
With Without Fold
Sample Heparin Heparin Increase
WT 5.37 ng/ml 269 pg/ml 20.0
(0.186 X 106) (3.72 X 106)
Ser 16 33.9 ng/ml 400 pg/ml 84.8
(0.030 x 106) (2.50 x 106)
Ser 83 4.36 ng/ml 251 pg/ml 17.4
(0.229 x 106) (3.98 x 106)
Ser 117 182 ng/ml 240 pg/ml 7.58
(0.549 x 106) (4.17 x 106)
Ser 16,83 800 pg/ml 195 pg/ml 4.10
(1.25 x 106) (5.13 x 106)
Ser 16,117 741 pg/ml 148 pg/ml 5.01
(1.35 x 106) (6.76 x 106)
Ser 83,117 295 pg/ml 100 pg/ml 2.95
(3.39 x 106) (10.0 x 106)
Ser 16,83,117 427 pg/ml 107 pg/ml 3.99
(2.34 x 106) (9.35 x 106)
The relative stabilities of the recombinant
wild-type haFGF, the single Ser mutants and the
multiple Ser mutants were determined. Mitogenic
~.3~(~~3
-71-
activites were measured following 0, 1 and 8 day
incubations in serum-free DME solutions, normally
used for serial sample dilutions, that were
C02-buffered to pH 7.3 at 37°C containing 1 mg/ml
human serum albumin. Mitogen samples were stored at
512 ng/ml, with or without 500 erg heparin/ml,
equivalent to the 10-fold concentrates from which the
highest concentration point in the assay is diluted.
Each sample was stored and assayed either in the
presence or absence of heparin. The relative
stabilities, following scaling to set each day 0
activity to 100%, are shown in Figure 2 as a function
of storage time. In Fig. 2: 1 corresponds to
wild-type; 1 corresponds to haFGF (Ser 16);
corresponds to haFGF (Ser (83); ~ corresponds to
haFGF (Ser (117); 0 corresponds to haFGF (Ser
16,83); ~ corresponds to haFGF (Ser 16,117); D
corresponds to haFGF (Ser 83,117); and o
corresponds to haFGF (Ser 16,83,117).
The loss of activity of wild-type haFGF and
the mutants in the presence of heparin closely fits
an exponential decay, see Fig. 2A. The activities of
all the mutants except Ser (16) are more stable than
the wild-type mitogen. The most stable mutants, in
descending order of stability are Ser 16,83,117), Ser
(117) Ser (16,83), Ser (83,117), Ser (16,83) and Ser
(83), Ser (16). The stability of Ser (83) was only
slightly higher than the wild-type. The various
forms of aFGF were less stable in the absence of
heparin and with the apparent exception of Ser
(16,83,117), the decay appeared not to be a simple
exponential of the time period.
- 72 -
A sample of the expression vector
pKK-haFGF(Ser 117) designated A48-lal containing the
gene capable of expressing the serine 117 mutant in
E. coli DH5 was deposited in the American Type
Culture Collection, 12301 Parklawn Drive, Rockville,
Maryland 20852 USA, on September 30, 1987 under the
Budapest Treaty and has been assigned ATCC number
67522.
15
25
