U.S. patent application number 10/280864 was filed with the patent office on 2003-03-20 for method of producing biologically active human acidic fibroblast growth factor and its use in promoting angiogenesis.
Invention is credited to Chernykh, Svitlana I., Kordyum, Vitaliy A., Slavchenko, Iryna Yu., Stegmann, Thomas J., Vozianov, Oleksandr F..
Application Number | 20030054492 10/280864 |
Document ID | / |
Family ID | 22844736 |
Filed Date | 2003-03-20 |
United States Patent
Application |
20030054492 |
Kind Code |
A1 |
Stegmann, Thomas J. ; et
al. |
March 20, 2003 |
Method of producing biologically active human acidic fibroblast
growth factor and its use in promoting angiogenesis
Abstract
The gene of human acidic fibroblast growth factor 155 (haFGF
155) has been obtained by chemical synthesis. The nucleotide
sequence of haFGF 155 gene has been deduced on the basis of haFGF
155 amino acid sequence as described in the literature. The amino
acid sequence, of the synthesized haFGF 155 does not differ from
those described in the literature. The nucleotide sequence of haFGF
gene differs from those described previously. For chemical
synthesis of haFGF 155 gene, codons were used which are the ones
most often used by E. coli in highly expressed E. coli proteins. A
plasmid with haFGF 155 (phaFGF 155) gene was obtained and was used
to transform E. coli. Production of haFGF 154 protein was achieved
by cultivation of the producer strain under conditions which slow
down the lytic development of lambda phage. The haFGF 154 protein
accumulated in culture medium in a soluble condition as a result of
the producer strain cells lysis by the lambda phage. The haFGF 154
protein constituted 20% of the soluble protein accumulated in the
culture medium and its biological activity was demonstrated by its
ability to generate new vessels (angiogenesis). The initiator
methionine residue at position 1 of the FGF 155 protein was
completely removed during protein synthesis resulting in an FGF 154
amino acid product. The use of the phage-dependent method to
produce other forms of the haFGF protein is also disclosed.
Inventors: |
Stegmann, Thomas J.;
(Petersberg, DE) ; Kordyum, Vitaliy A.; (Kiev-053,
UA) ; Slavchenko, Iryna Yu.; (Kiev-140, UA) ;
Chernykh, Svitlana I.; (Kiev-127, UA) ; Vozianov,
Oleksandr F.; (Kiev-025, UA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
22844736 |
Appl. No.: |
10/280864 |
Filed: |
October 24, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10280864 |
Oct 24, 2002 |
|
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09929945 |
Aug 15, 2001 |
|
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60225406 |
Aug 15, 2000 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/501 20130101;
A61P 9/00 20180101; C12N 15/70 20130101 |
Class at
Publication: |
435/69.1 ;
530/350; 536/23.5; 435/320.1 |
International
Class: |
C07H 021/04; C12N
015/00; C12N 015/63; C12N 015/74; C07K 014/00; C12P 021/06; C12N
015/09; C12N 015/70; C07K 001/00; C07K 017/00 |
Claims
What is claimed is:
1. A human acidic fibroblast growth factor protein having the
sequence as set forth in SEQ ID NO: 8.
Description
RELATED APPLICATIONS
[0001] The present application is a divisional application of U.S.
application Ser. No. 09/925,945, filed Aug. 15, 2001 which claims
priority to provisional patent application serial No. 60/225,406,
entitled, "A Method of Producing Biologically Active Human Acidic
Fibroblast Growth Factor and Its Use in Promoting Angiogenesis,"
filed on Aug. 15, 2000.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The field of the invention relates to methods of producing a
recombinant fibroblast growth factor protein and its use in
promoting angiogenesis.
[0004] 2. Description of the Related Art
[0005] Fibroblast growth factors (FGF) are nine structurally
related polypeptides, which are potent regulators of cell
proliferation, differentiation and normal development. They also
take part in pathological processes of tumorogenesis and metastasis
(Galzie, et al. Biochem. Cell Biol. (1997) 75: 669-685). They are
potent mitogens and differentiation factors for a broad range of
mesoderm and neuroectoderm derived cells, including endothelial
cells.
[0006] The heparin proteoglycans, heparin or heparin sulfate, bind
several FGF molecules together as a complex which are presented to
the FGF receptors. FGF proteins bind to their receptors resulting
in the activation of protein tyrosine kinases. The phosphorylation
of these tyrosine kinases initiates multiple signals including the
transcription of new mRNA's.
[0007] Two fibroblast growth factors, basic and acidic, are
described as potent inducers of angiogenesis (Friesel et al. (1995)
FASEB J. 9: 919-925). Both basic and acidic factors have been
implicated in the control of blood vessel formation and their
involvement in normal and pathological angiogenesis (Slavin, J.
(1995) Cell Biology International 19(5): 431-444). These factors
have been purified, their amino acid sequences have been determined
and their CDNA has been cloned and sequenced.
[0008] Acidic Fibroblast Growth Factor (aFGF) has been described
under various names including embryonic kidney-derived angiogenesis
factor I, astroglial growth factor I, endothelial cell growth
factor (ECGF), retina-derived growth factor, heparin-binding growth
factor class 1, endothelial growth factor, eye-derived growth
factor II, prostatropin, and glial maturation factor
(Gospodarowicz, et al. (1987) Journal of Cellular Physiology
supplement 5: 15-26). Cloning, nucleotide sequence and chromosome
localization have been described (Jaye et al. (1986) Science 233:
541-545).
[0009] The aFGF gene is situated on chromosome 5. It has a single
copy and encodes three exons separated by two introns. A 4.8 kb
mRNA translates synthesis of a form of aFGF with 155 amino acids.
However, the N-terminal methionine residue is removed in vivo to
give a 154 amino acid form. This 154 amino acid form of the aFGF is
processed into two forms which are 140 and 134 amino acids. The
aFGF protein is an anionic mitogen of molecular weight
15,000-17,000 D.
[0010] The aFGF protein has been found in brain, retina, bone
matrix and osteosarcoma. Only forms with 140 and 134 amino acids
have been obtained from tissues. It has been suggested that the
truncated aFGF forms are an artifact created by specific proteases
during aFGF extraction and isolation (Gospodarowicz, et al. (1987)
Journal of Cellular Physiology supplement 5: 15-26; Jaye et
al.(1987) The Journal of Biological Chemistry 262
(34):16612-16617).
[0011] It has been suggested that heparin potentiates the
biological activity of the aFGF protein (Thornton et al. (1983)
Science 222 (4624): 623-625). Heparin binding to aFGF has been
observed (Maciag et al. (1984) Science 225 (4665): 932-935). This
heparin-binding characteristic has been used as an efficient
affinity chromatography method for the purification of aFGF
protein. Heparin potentiates the biological activity of aFGF and
the enhanced activity of the aFGF-heparin complex varies from
several to one hundred fold (Lobb, et al. (1986) Anal. Biochem.
154: 1-14).
SUMMARY OF THE INVENTION
[0012] In one embodiment, the present invention relates to a method
for producing a biologically active human acidic fibroblast growth
factor protein, including the steps of:
[0013] transforming a strain of E. coli with a plasmid having at
least one copy of an expressible gene encoding a biologically
active human acidic fibroblast growth factor protein, operably
linked to a promoter;
[0014] infecting the transformed bacterial host cell with a
bacteriophage .lambda. capable of mediating delayed lysis; and
[0015] cultivating the E. coli host cell under a culture condition
that induces lytic growth of said cell without lysis until a
desired level of production of said protein is reached, wherein
said protein is produced as a soluble, biologically-active human
acidic fibroblast growth factor protein.
[0016] In a preferred embodiment, the bacteriophage .lambda. has a
temperature-sensitive mutation. In a more preferred embodiment, the
temperature-sensitive mutation is cI.sub.857.
[0017] In a preferred embodiment, the E. coli host cells are grown
at a temperature which prevents lytic growth of the bacteriophage
.lambda. prior to the cultivating step.
[0018] In a preferred embodiment, the bacteriophage .lambda. has a
mutation in at least one gene capable of mediating delayed lysis.
In a more preferred embodiment the at least one gene capable of
mediating delayed lysis is selected from the group consisting of N,
Q and R.
[0019] In a preferred embodiment, the strain of E. coli produces a
suppressor for the repair of amber-mutations.
[0020] In an alternate embodiment, the strain of E. coli lacks a
suppressor for the repair of amber-mutations.
[0021] In a preferred embodiment, the infecting bacteriophage
.lambda. is provided at a multiplicity of infection in a range of
about 1 to about 100. In a more preferred embodiment, the infecting
bacteriophage .lambda. is provided at a multiplicity of infection
in a range of about 10 to about 25.
[0022] In a preferred embodiment, the bacteriophage-mediated
delayed lysis of the strain of E. coli is delayed at higher
multiplicities of infection relative to lower multiplicities of
infection.
[0023] In a preferred embodiment, the biologically active human
acidic fibroblast growth factor protein contains 154 amino acids.
In a more preferred embodiment, the human acidic fibroblast growth
factor protein has the sequence as set forth in SEQ ID NO: 8.
[0024] In a preferred embodiment, the promoter is a T7 polymerase
promoter and the E. coli strain is capable of expressing the gene
for T7 RNA polymerase. In a more preferred embodiment, the gene for
T7 RNA polymerase gene is under the control of an inducible
promoter. In an even more preferred embodiment, the inducible
promoter is a lac UV 5 promoter.
[0025] In an alternate embodiment, the biologically active human
acidic fibroblast growth factor protein contains 146 amino
acids.
[0026] In another embodiment, the biologically active human acidic
fibroblast growth factor protein contains 140 amino acids.
[0027] In another embodiment of the invention, the biologically
active human acidic fibroblast growth factor protein contains 134
amino acids.
[0028] In a preferred embodiment, a method of producing a
biologically active human acidic fibroblast growth factor protein
is provided which comprises:
[0029] a) growing a first strain of E. coli cells, which harbor a
strain of bacteriophage .lambda., wherein the bacteriophage
.lambda. has a temperature-sensitive mutation,
[0030] b) adjusting the temperature to provide for lysis of the
first strain of E. coli cells and release of the bacteriophage
.lambda.,
[0031] c) providing a second strain of E. coli cells which have
been transformed with a plasmid having at least one copy of an
expressible gene encoding said biologically active human acidic
fibroblast growth factor protein, said expressible gene being
operably linked to a T7 polymerase promoter under the control of an
inducible promoter, wherein the second strain of E. coli cells may
be induced to express the gene for T7 RNA polymerase by addition of
an inducer;
[0032] d) infecting the second strain of E.coli cells with the
bacteriophage .lambda. released from the first strain of E. coli
cells; and
[0033] e) incubating the infected second strain of E. coli cells in
a culture medium containing the inducer, such that protein is
produced and released into the culture medium upon lysis of the
second strain of E. coli cells, wherein said protein is produced as
a soluble, biologically-active protein at a concentration greater
than 100 microgram/ml.
[0034] Another aspect of the invention encompasses a chemically
synthesized nucleic acid having the sequence set forth in SEQ ID
NO: 1.
[0035] For purposes of summarizing the invention and the advantages
achieved over the prior art, certain objects and advantages of the
invention have been described above. Of course, it is to be
understood that not necessarily all such objects or advantages may
be achieved in accordance with any particular embodiment of the
invention. Thus, for example, those skilled in the art will
recognize that the invention may be embodied or carried out in a
manner that achieves or optimizes one advantage or group of
advantages as taught herein without necessarily achieving other
objects or advantages as may be taught or suggested herein.
[0036] Further aspects, features and advantages of this invention
will become apparent from the detailed description of the preferred
embodiments which follow.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] These and other feature of this invention will now be
described with reference to the drawings of preferred embodiments
which are intended to illustrate and not to limit the
invention.
[0038] FIG. 1 shows the chemically synthesized nucleotide sequence
for human acidic fibroblast growth factor (155 amino acids) (SEQ ID
NO: 1) which has been modified by substitution of naturally
occurring codons with codons found in highly expressed E. coli
proteins and the translated amino acid sequence (SEQ ID NO: 2).
[0039] FIG. 2 shows the modifications made in the chemically
synthesized haFGF 155 codons. FGF fr HUMECGFB is the sequence
obtained from GenBank (at NCBI) (SEQ ID NO: 3). HaFGF 155 is the
chemically synthesized sequence of the present invention (SEQ ID
NO: 1).
[0040] FIG. 3 shows the pET24-155@rev construct which contains the
chemically synthesized haFGF 155 gene (SEQ ID NO: 1).
[0041] FIG. 4 shows purification of the culture medium containing
recombinant haFGF 154 (SEQ ID NO: 8). In the electrophoregram: lane
1, crude media containing recombinant haFGF 154 (225 mg
FGF-1/liter); lane 2, Heparin-Sepharose column purified recombinant
haFGF 154; lane 3, purification of haFGF 154 by HPLC C-18 column.
The unlabelled lane at the far left contains molecular weight
markers.
[0042] FIG. 5 shows the pET24-134@rev construct which contains the
chemically synthesized haFGF 134 gene (SEQ ID NO: 4).
[0043] FIG. 6 shows the chemically synthesized nucleotide sequence
for human acidic fibroblast growth factor (134 amino acids) (SEQ ID
NO: 4) which has been modified by substitution of naturally
occurring codons with codons found in highly expressed E. coli
proteins and the translated amino acid sequence (SEQ ID NO: 5).
[0044] FIG. 7 shows the pET24-140@rev construct which contains the
chemically synthesized haFGF 140 gene (SEQ ID NO: 6).
[0045] FIG. 8 shows the chemically synthesized nucleotide sequence
for human acidic fibroblast growth factor (140 amino acids) (SEQ ID
NO: 6) which has been modified by substitution of naturally
occurring codons with codons found in highly expressed E. coli
proteins and the translated amino acid sequence (SEQ ID NO: 7).
[0046] FIG. 9 shows a 12.5% SDS polyacrylamide gel containing
proteins produced by the phage-dependent method described herein:
lane 1: molecular weight standards, 2 .mu.g each standard; lane 2:
40 .mu.l of culture media containing the recombinant FGF 134
protein; lane 3: 40 .mu.l of culture media containing the
recombinant FGF 140 protein; lane 4: 40 .mu.l of culture media
containing recombinant interferon .alpha.2B; lane 5: 40 .mu.l of
culture media containing recombinant FGF 154 protein; lane 6: 40
.mu.l of culture media containing recombinant human growth hormone;
lane 7: 40 .mu.l of culture media containing recombinant methionine
aminopeptidase; lane 8: 40 .mu.l of culture media containing
.beta.-galactosidase of E. coli.
[0047] FIG. 10 shows a 12.5% SDS polyacrylamide gel containing
recombinant proteins purified according to the presently claimed
invention: lane 1: molecular weight standards; lane 2: 5 .mu.g of
purified FGF 134 protein; lane 3: 5 .mu.g of purified FGF 140
protein; lane 4: 5 .mu.g of purified FGF 146 protein; lane 5: 5
.mu.g of purified interferon .alpha.2B protein; lane 6: 5 .mu.g of
purified FGF 154 protein; lane 7: 5 .mu.g of purified methionine
amino peptidase protein; and lane 8: molecular weight
standards.
[0048] FIG. 11. Chicken embryo CAM blood vessels on the 14.sup.th
day of development after FGF treatment. Formation of chicken egg
CAM new blood vessels on the 4.sup.th day after application of the
154 amino acid form of the haFGF protein. Magnification 3.times..
FIG. 11A shows the effect of 1 .mu.gm of the 154 amino acid form of
the haFGF protein. The vessels under application are mainly small
and show radial growth. FIG. 11B shows the control sample.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0049] While the described embodiment represents the preferred
embodiment of the present invention, it is to be understood that
modifications will occur to those skilled in the art without
departing from the spirit of the invention. The scope of the
invention is therefore to be determined solely by the appended
claims.
[0050] The haFGF155 gene encodes a protein containing 155 amino
acid residues (SEQ ID NOS: 1 & 2). The first amino acid of the
haFGF 155 sequence is the initiator methionine residue, which under
normal situations would be removed during protein synthesis
resulting in an FGF protein of 154 amino acids (SEQ ID NO: 8).
However, it has only been possible to isolate two shorter aFGF
forms from tissue samples. The two isolated forms contain 140 and
134 amino acid residues. The aFGF form containing 140 amino acids
is considered complete, while the aFGF form containing 134 amino
acids is considered to be truncated. It has not been possible to
extract the aFGF form containing 155 or 154 amino acids from tissue
samples. It is not known whether the shorter isoforms occur as a
normal function of cell processing or as an artefact produced
during the isolation procedure by specific proteases in the process
of aFGF extraction. Western Blot analysis of the protein produced
from the isolated DNA recombinant molecules for the three aFGF
forms showed high expression of the 140 and 134 forms and a low
expression level of the 154 form.
[0051] In a preferred embodiment of the present invention, the gene
for human acidic fibroblast growth factor encodes the 154 amino
acid form of the aFGF protein and is chemically synthesized (SEQ ID
NO: 1). The nucleotide sequence of the haFGF 155 gene has been
deduced on the basis of the previously described haFGF 155 amino
acid sequence (SEQ ID NO: 2). The amino acid sequence of the
synthesized haFGF155 gene does not differ from those previously
described such as the translated sequence of the human aFGF
nucleotide sequence of SEQ ID NO: 3 obtained from GenBank. However,
the preferred nucleotide sequence of haFGF gene differs from those
previously described. In a preferred embodiment of the present
invention, the haFGF 155 gene has been chemically synthesized using
the codons which are most often used by E. coli for intensively
synthesized bacterial proteins. Preferred codon usage tables for E
coli are well known and available. See, for example,
http://pshche.uthct.edu/shaun/Sbla- ck/codonuse.html. Chemical
synthesis of polynucleotides was carried out using well known
methodology (Edge et al. (1983) Nucleic Acids Research 11 (18):
6419-6435).
[0052] Alternatively, other well known forms of the haFGF gene
could be used by those skilled in the art in the practice of the
present invention including isolated DNA from animal tissues
encoding other forms of the haFGF protein known to those skilled in
the art including the 154, the 146, the 140 and 134 isoforms and
any variants, derivatives, analogs or fragments thererof. The human
aFGF proteins may be used in methods to stimulate angiogenesis.
Human aFGF produced by the practice of the claimed invention may
also be used in a composition with a suitable pharmaceutical
carrier. Such carriers include, but are not limited to, saline,
buffered saline, water, dextrose and combinations thereof. In a
preferred embodiment, a fibringlue such as Tissucal.TM. (Baxter
International, Duarte, Calif.) is used as carrier.
[0053] FIG. 1 shows the complete nucleotide sequence of the haFGF
155 gene, as synthesized by the present inventors (SEQ ID NO: 1). A
sequence for human acidic fibroblast growth factor from GenBank
(SEQ ID NO:3) was compared to the chemically synthesized sequence
of FIG. 1. The comparison is shown in FIG. 2. There are
distinctions in 80 codons.
[0054] Expression and cloning vectors typically contain a promoter
that is recognized by the host organism and is operably linked to
the haFGF nucleic acid. Promoters are untranslated sequences
located upstream (5') to the start codon of a structural gene
(generally within 100-1000 base pairs) that control the
transcription and translation of particular nucleic acid sequences,
such as the haFGF nucleic acid sequence, to which they are operably
linked. Such promoters typically fall into two classes, inducible
and constitutive. Inducible promoters are promoters that initiate
increased levels of transcription from DNA under their control in
response to some change in culture conditions, e.g., the presence
or absence of a nutrient or a change in temperature. At this time a
large number of promoters recognized by prokaryotic host cells are
known. These promoters are operably linked to haFGF-encoding DNA by
removing the promoter from the source DNA by restriction enzyme
digestion and inserting the isolated promoter sequence into the
vector.
[0055] Promoters known to those skilled in the art include
.beta.-lactamase and lactose promoter systems (Chang et al. (1978)
Nature 275: 615; Goeddel et al. Nature (1979) 281: 544), alkaline
phosphatase, and a tryptophan (trp) promoter system (Goeddel (1980)
Nucleic Acids Research 8: 4057; Ep36,776). However, other known
bacterial promoters are suitable. A most preferred promoter is the
T7 promoter system. One skilled in the art would know how to ligate
them to haFGF DNA using suitable linkers or adaptors to provide
appropriate restriction sites. Promoters may also be used in tandem
to achieve higher levels of expression.
[0056] Any number of prokaryote host cells are suitable for
expressing the haFGF gene cloned into the vectors described herein.
Preferred prokaryotic hosts include eubacteria such as
Gram-negative or Gram-positive organisms, for example,
Enterbacteriaceae such as Escherichia. A most preferred prokaryote
host is E. coli.
[0057] Transformation means introducing DNA into an organism so
that the DNA is capable of replication, either as an
extrachromosomal element or by integration into the chromosome.
Transformation of prokaryotic cells is performed using techniques
well known to those skilled in the art such as treatment with
CaCl.sub.2 or electroporation.
[0058] An important advantage of infecting producer cells with a
bacteriophage is that the phage causes a profound rearrangement of
all macromolecular synthesis in the bacterial host cells. By
turning off transcription of bacterial genes, phages may increase
the copying of the targeted gene, and consequently, increase the
output of desired product.
[0059] In one embodiment of the present super-production system,
phage .lambda. with amber-mutations that delay bacterial lysis
(e.g., Q.sup.- and R.sup.- mutations) are provided in a strain of
E. coli, designated Su.degree., which lacks the suppressor
responsible for correcting amber-mutations in phage .lambda.. In
order to obtain a non-suppressing (Su.degree.) strain of E. coli,
Su.degree. clones are selected from the wild-type Su.sup.+
population. Preferably, a selection marker is inserted into the
phage DNA, e.g., tetracycline or ampicillin resistance.
[0060] Selection of non-suppressing (Su.degree.) strains of E.
coli, for example, E. coli K 802 was carried out with phage
.lambda. cI.sub.857 N.sub.am7N.sub.am53 bla tet (hereinafter
.lambda. bla N'). Strain E. coli C600 (.lambda. bla N') served as
source of the phage. This phage was obtained by insertion of
plasmid pCV 11 (bla tet) at EcoRI site into single-site (EcoRI)
vector carrying ts-mutation in repressor gene (cI.sub.857). Then
two amber-mutations were introduced into the phage N gene by
recombination in vivo.
[0061] Clones were tested for non-lysogenicity with phage .lambda.
clear. In addition to phage .lambda. bla N', phage .lambda.
cI.sub.857 Q.sub.am117 R.sub.am54 was used to check for
suppressor.
[0062] As is known, phage .lambda. N' mutant is not able to lyse
the host cells and is present in cells in the form of extremely
unstable plasmids. If the host cells contain suppressor, the
amber-mutation is phenotypically corrected, the N protein is
synthesized and the phage can develop lytically. This difference in
the viability of Su+ and Su.degree. cells, infected by .lambda. N',
is used as a basis for selection of spontaneously appearing
Su.degree. revertants from the E. coli Su.sup.+ cell population.
Phage .lambda. with an inserted plasmid that introduced the
ampicillin and tetracycline resistance markers into cells was used
to prevent the nonlysing Su.degree. cells from masking the search
for mutants. The phage also contains ts-mutation in the repressor
gene that permits lytic development of such phage resulting in cell
lysis.
[0063] If the medium supplemented with ampicillin and tetracycline
is inoculated with Su.sup.+ culture after its infection with phage
.lambda. bla N' with subsequent growth at 43.degree. C., single
suppressor-free cells containing phage .lambda. bla N' in the form
of plasmids must develop on plates. Curing the cells from the
phage, we must obtain Su.degree. derivatives of the parent
cultures. The method can be subdivided into several stages.
[0064] 1. Infection of Culture With Phage .lambda. bla N'
[0065] The culture E. coli Su+was grown on the M9 medium with
maltose at 37.degree. C. under intense agitation to a density of
1-2.times.10.sup.8 cells/ml. The cells were infected with phage
.lambda. bla N' at a multiplicity of 5-10 particles per cell and
incubated for 20 min at 20.degree. C. Under given conditions, the
infection efficiency is about 100%, in addition to the bulk of
Su.sup.+ cells, the phage also infects single Su.degree. cells.
[0066] 2. Selection of Suppressor-Free Cells Containing Marker
Phage
[0067] After infection, cells were plated out on agar medium
supplemented with 12 y/ml tetracycline and 20 y/ml ampicillin and
grown at 43.degree. C. In 24 h, single colonies developed, which
were replated on agar medium with antibiotics and grown at
37.degree. C.
[0068] 3. Curing of the Selected Clones from Phage .lambda. bla
N'
[0069] Since phage .lambda. N' in the E. coli Su.degree. cells is
in the form of extremely unstable plasmids, in order to cure from
the phage the selected clones were plated on selective agar medium
without antibiotics and grown at 37.degree. C. The number of cells
that had lost the phage in the first passage on the medium without
antibiotics amounted to 12-35%. The selection of such cells was
carried out by monitoring the loss of antibiotic resistance and the
acquisition of sensitivity to phage .lambda. clear.
[0070] 4. Testing of Cells for Repressor
[0071] The ability of phage .lambda. with amber-mutations to form
plaques on lawns of cured clones was checked. Isogenic
suppressor-free derivatives of the parent E. coli Su.sup.+ strains
are clones, on which phage .lambda. bla N' did not form plaques,
phage .lambda. cI.sub.857 Q.sub.am117 R.sub.am54 produced
1-3.times.10.sup.5 PFU/ml, and phage .lambda. cI.sub.857 without
mutations in genes Q and R produced 1.times.10.sup.10 PFU/ml.
[0072] Using this method, we obtained Su.degree. revertants of E.
coli K 802 Su.sup.+. Based on the cell number at the moment of
infection and the number of Su.degree. revertants among them, the
frequency of occurrence of suppressor-free cells was
3.times.10.sup.-7.
[0073] In a preferred embodiment, the gene of interest is cloned
into pET-24a(+) under the control of the T7 promoter. Preferred
genes include, but are not limited to, genes encoding human aFGF
134 amino acid form, human aFGF 140 amino acid form, and human aFGF
146 amino acid form and human aFGF 155 amino acid form. In an
alternate embodiment, the gene of interest may be cloned into both
a bacterial plasmid and the .lambda. phage under the control of
appropriate promoters. In a most preferred embodiment, chemically
synthesized haFGF 155 gene (SEQ ID NO: 1) is cloned into pET-24a(+)
under the control of the T7 promoter.
[0074] The T7 promoter is recognized only by T7 RNA polymerase and
is not recognized by the RNA polymerase of E.coli. The obtained
plasmid with an haFGF gene was transformed into E. coli BL21(DE3).
This strain contains the T7 RNA polymerase gene. The T7 RNA
polymerase gene is under the control of the inducible lac Uv5
promoter in order to induce T7 RNA polymerase synthesis only when
necessary as this protein is toxic for the E. coli cell. The
induction of the lac promoter is carried out by adding IPTG to the
nutrient medium. In order to obtain a haFGF protein, the producer
strain, containing the recombinant plasmid with the haFGF gene, is
cultured under conditions of intensive aeration to a cell density
of 5.times.10.sup.7-5.times.10.sup.9 cells in 1 ml at a temperature
of 20-40.degree. C. Then it is infected by lambda phage with the
ts-mutation cI repressor gene with a multiplicity from 0.1 to 100
phage bodies per cell and incubation is continued at 20-37.degree.
C. for 2-14 hours. Simultaneously with the phage, IPTG at a
concentration of 1 mM is introduced.
[0075] Production of the haFGF proteins was achieved by cultivation
of the producer strain under conditions which slow down the lytic
development of the lambda phage Such conditions include lowered
temperature of cultivation and use of amber mutations in late
lambda phage genes such as Q and R genes.
[0076] The haFGF proteins accumulated in the culture medium as a
soluble proteins as a result of the producer strain cells lysis by
lambda phage. The output of each haFGF protein generally
constituted 20% of the soluble proteins accumulated in the culture
medium. Debris was removed from the culture medium by
centrifugation. The haFGF can then be purified from contaminant
soluble proteins and polypeptides with the following procedures,
which are exemplary of suitable purification procedures: by
fractionation on an ion-exchange column; ethanol precipitation,
reverse phase HPLC; chromatography on silica; immunoaffinity;
SDS-PAGE; ammonium sulfate precipitation; and gel filtration. In
one embodiment, the haFGF recombinant protein was purified using a
C18 HPLC column. In another embodiment, the haFGF recombinant
proteins were applied to heparin sepharose in order to obtain
purified haFGF. The purified haFGF was then subjected to automated
amino-terminal sequence analysis for 15 cycles. This analysis
indicated that all the initiator methionine at position number 1 of
FGF155 had been removed during synthesis resulting in the
production of an FGF molecule containing 154 amino acids. The amino
acids detected in cycles 2-14 of the above analysis were identical
to positions 2-14 of FGF155.
[0077] Biological activity of the purified haFGF recombinant
proteins was demonstrated by the ability to generate new vessels
(angiogenesis). The assay involved the study of haFGF influence on
the formation of new blood vessels using the model of chicken
embryonic chorio-allantoic membrane (CAM).
[0078] A more detailed description of the present invention is
provided below. While the described embodiment represents the
preferred embodiment of the present invention, it is to be
understood that modifications will occur to those skilled in the
art without departing from the spirit of the invention. The scope
of the invention is therefore to be determined solely by the
appended claims.
EXAMPLE 1
Production of Human aFGF 154 by Phage-Dependent Method
[0079] Cultures of Escherichia coli BL21(DE3) (NOVAGEN) were
transformed by plasmid pET24-155 @rev (FIG. 3), which contains one
copy of the chemically synthesized haFGF 155 gene encoding human
acidic fibroblast growth factor (155 amino acids) (SEQ ID NO: 1).
Cultures of BL21(DE3) contain a single copy of the gene for T7 RNA
polymerase under the control of the inducible lac UV5 promoter in
the bacterial genome (Studier et al. (1986) J. Mol. Biol. 189:
113-130). Into the plasmid pET-24a(+) (NOVAGEN) was inserted the
chemically synthesized haFGF 155 gene (SEQ ID NO: 1) under the
control of the T7 promoter to produce plasmid pET24-155 @rev.
Expression of the haFGF 155 gene begins only after the appearance
of T7 polymerase in the cells which is mediated through the
induction of the lac UV5 promoter by IPTG.
[0080] Cultures of E. coli BL21(DE3) with pET24-155 @rev were grown
with shaking at 37.degree. C. in LB medium, containing 50 .mu.g/ml
kanamycin, to a density of 2.times.10.sup.8 cells/ml. Then the
cells were infected with phage .lambda. cI.sub.857 Q.sub.am117
R.sub.am54 at a multiplicity of about 10 phage bodies per 1
bacterial cell and cultivated with shaking at 21.degree. C. for
about 14 hour. Simultaneously with phage, 1 mM IPTG was introduced
into the medium.
[0081] Phage .lambda. cI.sub.857 Q.sub.am117 R.sub.am54 was
prepared from lysogenic cultures of E. coli RLMI, which were grown
in LB medium at 30.degree. C. with intensive aeration to a density
of approximately 1.times.10.sup.8 cells/ml. The lysogenic culture
was warmed to 43.degree. C. and incubated for 20 minutes to
inactivate cI repressor. The temperature was then decreased to
37.degree. C. and after 60-70 minutes the bacterial cells underwent
lysis, with phages being formed at 1-2.times.10.sup.10 PFU/ml.
[0082] After incubation with the phage-infected cells for 14 hours,
debris was removed from the culture medium by centrifugation. The
culture medium, containing the haFGF 154 protein was applied to a
heparin sepharose column to obtain pure haFGF 154.
[0083] The culture medium containing the haFGF 154 was analyzed by
SDS-polyacrylamide gel electrophoresis under denaturing conditions
and stained with Coomassie Blue. An electrophoregram of the culture
medium, containing haFGF 154 protein is compared to purified haFGF
protein in FIG. 4. Lane 1 shows crude media containing recombinant
haFGF 154 (225 mg FGF-1/liter). Lane 2 shows Heparin-Sepharose
column purified recombinant haFGF 154. Lane 3 shows purification of
haFGF 154 by HPLC C-18 column. The unlabelled lane at the far left
contains molecular weight markers. The overall purification yield
was about 65%. Bioactivity was measured by two different assays, a
3T3 cell proliferation assay and a rat hind limb angiogenesis assay
(not shown). The bioactivity was equipotent with FGF-1 obtained
from Sigma-Chem. An assay using chicken embryo chorio-allantoic
membrane is shown in Example 7, below.
[0084] The production of haFGF 154 protein in phage-infected
cultures was about 20% of the total cellular protein. The molecular
weight of haFGF 154 was 17,908 Daltons as determined by
densitometer Image Master VDS (data not shown). N-terminal sequence
analysis of FGF 154 indicated an alanine residue at the first
position, with no initiator methionine detected.
EXAMPLE 2
Production of Human aFGF 134 Amino Acid form by Phage-Dependent
Method
[0085] Cultures of Escherichia coli BL21(DE3) (NOVAGEN) were
transformed by plasmid pET24-134 @rev (FIG. 5), which contains one
copy of the chemically synthesized gene encoding human aFGF 134
amino acid form (FIG. 6; SEQ ID NO: 4). The translated amino acid
sequence is shown in SEQ ID NO: 5. Cultures of BL21 (DE3) contain a
single copy of the gene for T7 RNA polymerase under the control of
the inducible lac UV5 promoter in the bacterial genome (Studier et
al. (1986) J. Mol. Biol. 189: 113-130). Into the plasmid pET-24a(+)
(NOVAGEN) was inserted the human aFGF 134 amino acid form gene
under the control of the T7 promoter. Expression of the human aFGF
134 amino acid form gene begins only after the appearance of T7
polymerase in the cells which is mediated through the induction of
the lac UV5 promoter by IPTG.
[0086] Cultures of E. coli BL21(DE3) with pET24-134 @rev were grown
with shaking at 37.degree. C. in LB medium, containing 50 .mu.g/ml
kanamycin, to a density of 2.times.10.sup.8 cells/ml. Then the
cells were infected with phage .lambda. cI.sub.857 Q.sub.am117
R.sub.am54 at a multiplicity of about 10 phage bodies per 1
bacterial cell and cultivated with shaking at 21.degree. C. for
about 14 hour. Simultaneously with phage, 1 mM IPTG was introduced
into the medium.
[0087] Phage .lambda. cI.sub.857 Q.sub.am17 R.sub.am54 was prepared
from lysogenic cultures of E. coli RLMI, which were grown in LB
medium at 30.degree. C. with intensive aeration to a density of
approximately 1.times.10.sup.8 cells/ml. The lysogenic culture was
warmed to 43.degree. C. and incubated for 20 minutes to inactivate
cI repressor. The temperature was then decreased to 37.degree. C.
and after 60-70 minutes the bacterial cells underwent lysis, with
phages being formed at 1-2.times.10.sup.10 PFU/ml.
[0088] After incubation with the phage-infected cells for 14 hours,
debris was removed from the culture medium by centrifugation. The
culture medium containing the haFGF 134 amino acid form was applied
to a heparin sepharose column to obtain pure human aFGF 134 amino
acid form.
EXAMPLE 3
Production of Human aFGF 140 Amino Acid form by Phage-Dependent
Method
[0089] Cultures of Escherichia coli BL21(DE3) (NOVAGEN) were
transformed by plasmid pET24-140 @rev (FIG. 7), which contains one
copy of the chemically synthesized gene encoding human aFGF 140
amino acid form (FIG. 8; SEQ ID NO: 6). The corresponding protein
is shown as SEQ ID NO: 7. Cultures of BL21(DE3) contain a single
copy of the gene for T7 RNA polymerase under the control of the
inducible lac Uv5 promoter in the bacterial genome (Studier et al.
(1986) J. Mol. Biol. 189: 113-130). Into the plasmid pET-24a(+)
(NOVAGEN) was inserted the human aFGF 140 amino acid form gene
under the control of the T7 promoter. Expression of the human aFGF
140 amino acid form gene begins only after the appearance of T7
polymerase in the cells which is mediated through the induction of
the lac UV5 promoter by IPTG.
[0090] Cultures of E. coli BL21(DE3) with pET24-140 @rev were grown
with shaking at 37.degree. C. in LB medium, containing 50 .mu.g/ml
kanamycin, to a density of 2.times.10.sup.8 cells/ml. Then the
cells were infected with phage .lambda. cI.sub.857 Q.sub.am117
R.sub.am54 at a multiplicity of about 10 phage bodies per 1
bacterial cell and cultivated with shaking at 21.degree. C. for
about 14 hour. Simultaneously with phage, 1 mM IPTG was introduced
into the medium.
[0091] Phage .lambda. cI.sub.857 Q.sub.am117 R.sub.am54 was
prepared from lysogenic cultures of E. coli RLMI, which were grown
in LB medium at 30.degree. C. with intensive aeration to a density
of approximately 1.times.10.sup.8 cells/ml. The lysogenic culture
was warmed to 43.degree. C. and incubated for 20 minutes to
inactivate cI repressor. The temperature was then decreased to
37.degree. C. and after 60-70 minutes the bacterial cells underwent
lysis, with phages being formed at 1-2.times.10.sup.10 PFU/ml.
[0092] After incubation with the phage-infected cells for 14 hours,
debris was removed from the culture medium by centrifugation. The
culture medium containing the haFGF 140 amino acid form was applied
to a heparin sepharose column to obtain pure human aFGF 140 amino
acid form.
[0093] Human aFGF 140 produced by the method disclosed above had
biological activity based upon the chick membrane assay (Example
6).
EXAMPLE 4
Production of Human aFGF 146 Amino Acid Form by Phage-Dependent
Method
[0094] Cultures of Escherichia coli BL21(DE3) (NOVAGEN) were
transformed by plasmid pET24-146 @rev, which contains one copy of
the chemically synthesized gene encoding human aFGF 146 amino acid
form. Cultures of BL21(DE3) contain a single copy of the gene for
T7 RNA polymerase under the control of the inducible lac Uv5
promoter in the bacterial genome (Studier et al. (1986) J. Mol.
Biol. 189: 113-130). Into the plasmid pET-24a(+) (NOVAGEN) was
inserted the human aFGF 146 amino acid form gene under the control
of the T7 promoter. Expression of the human aFGF 146 amino acid
form gene begins only after the appearance of T7 polymerase in the
cells which is mediated through the induction of the lac UW5
promoter by IPTG.
[0095] Cultures of E. coli BL21(DE3) with pET24-146 @rev were grown
with shaking at 37.degree. C. in LB medium, containing 50 .mu.g/ml
kanamycin, to a density of 2.times.10.sup.8 cells/ml. Then the
cells were infected with phage .lambda. cI.sub.857 Q.sub.am117
R.sub.am54 at a multiplicity of about 10 phage bodies per 1
bacterial cell and cultivated with shaking at 21.degree. C. for
about 14 hour. Simultaneously with phage, 1 mM IPTG was introduced
into the medium.
[0096] Phage .lambda. cI.sub.857 Q.sub.am117 R.sub.am54 was
prepared from lysogenic cultures of E. coli RLMI, which were grown
in LB medium at 30.degree. C. with intensive aeration to a density
of approximately 1.times.10.sup.8 cells/ml. The lysogenic culture
was warmed to 43.degree. C. and incubated for 20 minutes to
inactivate cI repressor. The temperature was then decreased to
37.degree. C. and after 60-70 minutes the bacterial cells underwent
lysis, with phages being formed at 1-2.times.10.sup.10 PFU/ml.
[0097] After incubation with the phage-infected cells for 14 hours,
debris was removed from the culture medium by centrifugation. The
culture medium, containing the haFGF 146 amino acid protein, was
applied to a heparin sepharose column to obtain pure human aFGF 146
amino acid form.
[0098] Human aFGF 146 produced by the method disclosed above had
biological activity based upon the chick membrane assay (Example
6).
EXAMPLE 5
Purification of haFGF
[0099] The culture medium containing haFGF is diluted with one
volume of 0.04M KH.sub.2PO.sub.4 buffer, pH 7.0, and applied to a
heparin-sepharose column equilibrated with 0.02 M KH.sub.2PO.sub.4,
pH 7.0. The flow rate is adjusted to 80 ml/hour. After application
of the culture medium containing the haFGF protein, the column is
washed with 0.02M KH.sub.2PO.sub.4 buffer, pH 7.0. Next, the column
is washed with 0.02 M KH.sub.2PO.sub.4 buffer containing 0.6M NaCl,
pH 7.3. Elution is carried out using 0.02 M KH.sub.2PO.sub.4 buffer
with 1.5 M NaCl, pH 7.5. All steps are carried out at 4.degree.
C.
EXAMPLE 6
Gel Analysis of Recombinant Proteins Produced by the
Phage-Dependent Method
[0100] Culture media containing human aFGF 134 amino acid form,
human aFGF 140 amino acid form, and human aFGF 154 amino acid form
were analyzed by SDS-polyacrylamide gel electrophoresis under
denaturing conditions and stained with Coomassie Blue. An
electrophoregram of culture media, containing human aFGF 134 amino
acid form, human aFGF 140 amino acid form, human and aFGF 146 amino
acid form was compared to molecular weight standards in FIG. 9.
Lane 2 shows 30 .mu.l of the culture medium containing human aFGF
134 amino acid form. Lane 3 shows 30 .mu.l of culture media
containing the recombinant FGF 140 protein. Lane 5 shows 30 .mu.l
of culture media containing recombinant FGF 154 protein. Lane 1
shows 2 .mu.g of each molecular weight standard (Amersham Pharmacia
Biotech). From the top, the molecular weight standards are: 94,000;
67,000; 43,000; 30,000; 20,100; and 14,400.
[0101] Quantitation of amounts of human aFGF 134 amino acid form,
human aFGF 140 amino acid form, and human aFGF 154 amino acid form
in a mixture was accomplished by scanning the stained protein bands
on a polyacrylamide gel with densitometer Image Master VDS
(Pharmacia Biotech). The production of the recombinant proteins in
phage-infected cultures was about 20% of the total cellular
protein.
[0102] An electrophoregram containing purified recombinant human
aFGF 134, haFGF 140, haFGF 146, and haFGF 154 protein was compared
to molecular weight standards (FIG. 10). Lane 2 shows 5 .mu.g of
the purified aFGF 134 protein. Lane 3 shows 5 .mu.g of the purified
human aFGF 140. Lane 4 shows 5 .mu.g of the purified human aFGF 146
amino acid form. The production of human aFGF 146 amino acid form
in phage-infected cultures was about 20% of the total cellular
protein. Lane 6 shows 5 .mu.g of haFGF 154 protein. Lanes 1 and 8
show 2 .mu.g of each molecular weight standard (Amersham Pharmacia
Biotech).
EXAMPLE 7
A Method of Studying FGF Influence on the Formation of New Blood
Vessels in the Chicken Embryo Chorio-Allantoic Membrane (CAM).
[0103] The method of studying angiogenesis on the model of chicken
embryos (Thomas et al. (1985) Proc. Natl. Acad. Sci, USA 82:
6409-6413) was adapted to determine the effects of the haFGF 154,
146, and 140 recombinant proteins on angiogenesis compared to pure
brain-derived acidic fibroblast growth factor. Pure brain-derived
acidic fibroblast growth factor is a potent angiogenic vascular
endothelial cell mitogen with sequence homology to interleukin.
[0104] The shells of three-day old chicken embryos were sterilized
with ethyl alcohol. The shell and under shell cover were removed
from the air chamber using forceps and the eggs were covered by the
bottom of a plastic 35 mm Petri dish. The embryos were incubated at
37.degree. C. for 5-6 days. At the end of this period, the embryos
were examined and the eggs with well-developed blood vessels of CAM
were selected for experimentation.
[0105] Filter paper disks with deposited gel containing FGF were
laid on the eggs CAM with the gel towards the blood vessels and
incubated in a thermostat at 37.degree. C. for another 3 days. The
gel was prepared in the following way: the tested quantity of FGF
was dissolved in 30 .mu.l of Eagle's medium (solution 1); then in
30 .mu.l of Eagle's medium, 10 .mu.g of heparin was dissolved and
2% of agarose added (solution 2). Then equal volumes of solution 1
and 2 were mixed and the obtained mixture was deposited in aliquots
by 60 .mu.l on 12 mm diameter filter paper disks.
[0106] On the 4.sup.th day, the filter paper disks were removed.
Rich cow milk (10% milkfat) was injected under CAM in a quantity of
about 1 ml or less. The result was a white background against which
the CAM vessels were easily observed.
[0107] The results of the experiment were recorded with a video
camera in conjunction with a computer. The formation of new CAM
vessel under the affect of FGF was evaluated by the following
parameters: the nature and direction of vessel growth, their
quantity and quality (large, medium, small), the presence or
absence of anastomosis, etc. These data were compared with the
control samples which had not been exposed to FGF. FIG. 11 shows
Chicken embryo blood vessels on the 14.sup.th day of development
after treatment with FGF154 produced by the phage-dependent
recombinant method described herein and purified on heparin
sepharose as described.
[0108] FIG. 11A demonstrates the correlation between application of
recombinant FGF154 protein and the formation of new blood vessels.
On the fourth day after application of 1 .mu.g of FGF154, vessels
are mainly small and show radial growth (FIG. 11A). Increasing the
amount of FGF154 to 3 .mu.g results in a corresponding increase in
the size of the blood vessels (not shown). Medium vessels are
observed with radial growth. A further increase to 4 .mu.g of
FGF154 applied (not shown) results in development of large, medium
and small blood vessels at 4 days after application. Untreated
control is shown in FIG. 11B.
[0109] It will be understood by those of skill in the art that
numerous and various modifications can be made without departing
from the spirit of the present invention. Therefore, it should be
clearly understood that the forms of the present invention are
illustrative only and are not intended to limit the scope of the
present invention.
Sequence CWU 1
1
8 1 630 DNA Artificial Sequence Chemically synthesized sequence for
human acidic Fibroblast Growth Factor (155 amino acids) using
preferred codons for E. coli 1 gcgtagagga tcgagatctc gatcccgcga
aattaatacg actcactata ggggaattgt 60 gagcggataa caattcccct
ctagaaataa ttttgtttaa ctttaagaag gagatataca 120 t atg gct gaa ggg
gaa atc acc acc ttt aca gcg tta acg gag aaa ttt 169 Met Ala Glu Gly
Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe 1 5 10 15 aac ctt
ccg ccc ggg aat tac aaa aaa ccc aag ctt ctt tac tgc agt 217 Asn Leu
Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser 20 25 30
aac gga gga cac ttc ctg cga att ctg cca gat ggc aca gta gat ggg 265
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly 35
40 45 act cgc gat cgc tcc gac cag cac att cag ctg caa ctc tcg gcc
gaa 313 Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala
Glu 50 55 60 agc gtt gga gag gtc tat atc aag tcg acg gag act ggc
cag tac ctt 361 Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly
Gln Tyr Leu 65 70 75 80 gcc atg gac acc gat ggg ctt ctg tat ggc tca
cag acg cct aac gaa 409 Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser
Gln Thr Pro Asn Glu 85 90 95 gaa tgc ttg ttt cta gaa aga cta gaa
gaa aac cat tac aac acg tac 457 Glu Cys Leu Phe Leu Glu Arg Leu Glu
Glu Asn His Tyr Asn Thr Tyr 100 105 110 ata tcg aaa aaa cat gca gag
aag aac tgg ttt gta ggc ctt aaa aaa 505 Ile Ser Lys Lys His Ala Glu
Lys Asn Trp Phe Val Gly Leu Lys Lys 115 120 125 aat ggt tcc tgt aag
cgt gga cca cgg act cac tat ggc caa aag gct 553 Asn Gly Ser Cys Lys
Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala 130 135 140 atc ttg ttc
ctg cca cta cca gtg agc tcc gac taa g gatccgaatt 600 Ile Leu Phe
Leu Pro Leu Pro Val Ser Ser Asp * 145 150 155 cgagctccgt cgacaagctt
gcggccgcac 630 2 155 PRT Homo sapiens 2 Met Ala Glu Gly Glu Ile Thr
Thr Phe Thr Ala Leu Thr Glu Lys Phe 1 5 10 15 Asn Leu Pro Pro Gly
Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser 20 25 30 Asn Gly Gly
His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly 35 40 45 Thr
Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu 50 55
60 Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu
65 70 75 80 Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro
Asn Glu 85 90 95 Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His
Tyr Asn Thr Tyr 100 105 110 Ile Ser Lys Lys His Ala Glu Lys Asn Trp
Phe Val Gly Leu Lys Lys 115 120 125 Asn Gly Ser Cys Lys Arg Gly Pro
Arg Thr His Tyr Gly Gln Lys Ala 130 135 140 Ile Leu Phe Leu Pro Leu
Pro Val Ser Ser Asp 145 150 155 3 468 DNA Homo sapiens 3 atggctgaag
gggaaatcac caccttcaca gccctgaccg agaagtttaa tctgcctcca 60
gggaattaca agaagcccaa actcctctac tgtagcaacg ggggccactt cctgaggatc
120 cttccggatg gcacagtgga tgggacaagg gacaggagcg accagcacat
tcagctgcag 180 ctcagtgcgg aaagcgtggg ggaggtgtat ataaagagta
ccgagactgg ccagtacttg 240 gccatggaca ccgacgggct tttatacggc
tcacagacac caaatgagga atgtttgttc 300 ctggaaaggc tggaggagaa
ccattacaac acctatatat ccaagaagca tgcagagaag 360 aattggtttg
ttggcctcaa gaagaatggg agctgcaaac gcggtcctcg gactcactat 420
ggccagaaag caatcttgtt tctccccctg ccagtctctt ctgattaa 468 4 630 DNA
Artificial Sequence Chemically synthesized sequence for human
acidic Fibroblast Growth Factor (134 amino acids) using preferred
codons for E. coli 4 gcgtagagga tcgagatctc gatcccgcga aattaatacg
actcactata ggggaattgt 60 gagcggataa caattcccct ctagaaataa
ttttgtttaa ctttaagaag gagatataca 120 t atg aat tac aaa aaa ccc aag
ctt ctt tac tgc agt aac gga gga cac 169 Met Asn Tyr Lys Lys Pro Lys
Leu Leu Tyr Cys Ser Asn Gly Gly His 1 5 10 15 ttc ctg cga att ctg
cca gat ggc aca gta gat ggg act cgc gat cgc 217 Phe Leu Arg Ile Leu
Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg 20 25 30 tcc gac cag
cac att cag ctg caa ctc tcg gcc gaa agc gtt gga gag 265 Ser Asp Gln
His Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu 35 40 45 gtc
tat atc aag tcg acg gag act ggc cag tac ctt gcc atg gac acc 313 Val
Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr 50 55
60 gat ggg ctt ctg tat ggc tca cag acg cct aac gaa gaa tgc ttg ttt
361 Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe
65 70 75 80 cta gaa aga cta gaa gaa aac cat tac aac acg tac ata tcg
aaa aaa 409 Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser
Lys Lys 85 90 95 cat gca gag aag aac tgg ttt gta ggc ctt aaa aaa
aat ggt tcc tgt 457 His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys
Asn Gly Ser Cys 100 105 110 aag cgt gga cca cgg act cac tat ggc caa
aag gct atc ttg ttc ctg 505 Lys Arg Gly Pro Arg Thr His Tyr Gly Gln
Lys Ala Ile Leu Phe Leu 115 120 125 cca cta cca gtg agc tcc gac
taaggatccg aattcgagct ccgtcgacaa 556 Pro Leu Pro Val Ser Ser Asp
130 135 gcttgcggcc gcactcgagc accaccacca ccaccactga gatccggctg
ctaacaaagc 616 ccgaaaggaa gctg 630 5 135 PRT Homo sapiens 5 Met Asn
Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His 1 5 10 15
Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg 20
25 30 Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly
Glu 35 40 45 Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala
Met Asp Thr 50 55 60 Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn
Glu Glu Cys Leu Phe 65 70 75 80 Leu Glu Arg Leu Glu Glu Asn His Tyr
Asn Thr Tyr Ile Ser Lys Lys 85 90 95 His Ala Glu Lys Asn Trp Phe
Val Gly Leu Lys Lys Asn Gly Ser Cys 100 105 110 Lys Arg Gly Pro Arg
Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu 115 120 125 Pro Leu Pro
Val Ser Ser Asp 130 135 6 630 DNA Artificial Sequence Chemically
synthesized sequence for human acidic Fibroblast Growth Factor (140
amino acids) using preferred codons for E. coli 6 gcgtagagga
tcgagatctc gatcccgcga aattaatacg actcactata ggggaattgt 60
gagcggataa caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatataca
120 t atg ttt aac ctt ccg ccc ggg aat tac aaa aaa ccc aag ctt ctt
tac 169 Met Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu
Tyr 1 5 10 15 tgc agt aac gga gga cac ttc ctg cga att ctg cca gat
ggc aca gta 217 Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp
Gly Thr Val 20 25 30 gat ggg act cgc gat cgc tcc gac cag cac att
cag ctg caa ctc tcg 265 Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile
Gln Leu Gln Leu Ser 35 40 45 gcc gaa agc gtt gga gag gtc tat atc
aag tcg acg gag act ggc cag 313 Ala Glu Ser Val Gly Glu Val Tyr Ile
Lys Ser Thr Glu Thr Gly Gln 50 55 60 tac ctt gcc atg gac acc gat
ggg ctt ctg tat ggc tca cag acg cct 361 Tyr Leu Ala Met Asp Thr Asp
Gly Leu Leu Tyr Gly Ser Gln Thr Pro 65 70 75 80 aac gaa gaa tgc ttg
ttt cta gaa aga cta gaa gaa aac cat tac aac 409 Asn Glu Glu Cys Leu
Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn 85 90 95 acg tac ata
tcg aaa aaa cat gca gag aag aac tgg ttt gta ggc ctt 457 Thr Tyr Ile
Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu 100 105 110 aaa
aaa aat ggt tcc tgt aag cgt gga cca cgg act cac tat ggc caa 505 Lys
Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln 115 120
125 aag gct atc ttg ttc ctg cca cta cca gtg agc tcc gac taaggatccg
554 Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140
aattcgagct ccgtcgacaa gcttgcggcc gcactcgagc accaccacca ccaccactga
614 gatccggctg ctaaca 630 7 141 PRT Homo sapiens 7 Met Phe Asn Leu
Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr 1 5 10 15 Cys Ser
Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val 20 25 30
Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser 35
40 45 Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly
Gln 50 55 60 Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser
Gln Thr Pro 65 70 75 80 Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu
Glu Asn His Tyr Asn 85 90 95 Thr Tyr Ile Ser Lys Lys His Ala Glu
Lys Asn Trp Phe Val Gly Leu 100 105 110 Lys Lys Asn Gly Ser Cys Lys
Arg Gly Pro Arg Thr His Tyr Gly Gln 115 120 125 Lys Ala Ile Leu Phe
Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 8 154 PRT Homo sapiens
8 Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe Asn 1
5 10 15 Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser
Asn 20 25 30 Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val
Asp Gly Thr 35 40 45 Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln
Leu Ser Ala Glu Ser 50 55 60 Val Gly Glu Val Tyr Ile Lys Ser Thr
Glu Thr Gly Gln Tyr Leu Ala 65 70 75 80 Met Asp Thr Asp Gly Leu Leu
Tyr Gly Ser Gln Thr Pro Asn Glu Glu 85 90 95 Cys Leu Phe Leu Glu
Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile 100 105 110 Ser Lys Lys
His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys Asn 115 120 125 Gly
Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala Ile 130 135
140 Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 145 150
* * * * *
References