DK175856B1 - New bovine and human acidic fibroblast growth factors - useful for stimulating DNA synthesis in responsive cells and esp. for wound heating - Google Patents

Abstract

Recombinant bovine acidic fibroblast growth factor (aFGF) is new. It pref. has a specified amino acid sequence. Microheterogenous forms of recombinant bovine aFGF are new. Nucleotide sequence coding for bovine aFGF is new. Expression plasmid comprising a nucleotide sequence as defined above is new. Host compatible with and contg. a plasmid as defined above is new. Recombinant human aFGF is new. Microheterogeneous forms, nucleotide sequence, expression plasmid and host, as defined for bovine aFGF, are also new for human aFGF.

Description

DK 175856 B1 i

Description of figure.

FIG. 1 schematically shows the pKK223-3 plasmid containing the gene for aFGF. BACKGROUND OF THE INVENTION.

Home-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 then shown that pituitary extracts also had potent mitogenic activity for fibroblasts, Armelin, Proc. Natl. Acad. Sci. USA 70: 2702-2706 (1973). Partial purification of both brain and pituitary fibroblast growth factor (FGF) revealed 10 mitogenic activity against a number of different types of adrenal cells, including vascular endothelial cells, Gospodarowicz et al., Natl. Cancer Inst. Mo nogr. 48: 109-130 (1978). It has been shown that FGF exists in two forms, acidic FGF (aFGF) and basic FGF (bFGF), and both forms have been identified in home remedies, Thomas and Gimenez-Gallego, TIBS 11:81-15 84 (1986) . Several cell types respond to stimulation with either purified aFGF or bFGF by synthesizing DNA and the portion, including primary fibroblasts, vascular cells and cornea endothelial cells, chondrocytes, osteoblasts, myoblasts, smooth muscle cells and glial cells, Esch et al., Proc.

Natl. Acad. Sci. USA 82: 6507-6511 (1985), Kuo et al., Fed. Proc. 44: 695 (1985).

Pure cattle home-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 fibroblast mitogenic activity of aFGF can also be used to promote wound healing, Thomas, U.S. Patent No. 4,444,760. The present invention provides a genetic construct and agents for expression that allow production of large amounts of pure aFGF that can be used. therapeutically.

Objects of the Invention.

The object of the present invention is to provide a nucleotide base sequence for both bovine aFGF and human aFGF from the amino acid sequences of the specific proteins. Another object is to produce genes encoding the specific aFGF as well as to incorporate the genes into appropriate cloning 35 hosts. A further object is to transform an appropriate host with each of the recombinant vectors and to induce expression of the specific aF-2GF genes. Another object is to isolate and purify biologically active bovine aF-GF and human aFGF. These and other purposes will be apparent from the following description.

Summary of the Invention.

Unique genes are constructed that encode the amino acid sequence of bovine acidic fibroblast growth factor (aFGF) and human aFGF. The bovine gene is derived from reverse translation of the aFGF amino acid sequence, while the human gene is derived by specific point mutations of the bovine gene. Each genius construct is inserted into an expression vector used to transform an appropriate host. The transformed host cells produce recombinant aFGF (r-aFGF), human or bovine, which is purified and has an activity equivalent to native protein.

15 Detailed description

Acidic fibroblast growth factors exist 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, which is a peptide produced from a single gene unit of 20 DNA, which DNA is structurally modified after translation. However, the structural modifications do not result in any significant changes in biological activity of the peptide. The modifications can take place either in vivo or during the isolation and purification process. In vivo modification results in, but is not limited to, proteolysis, glycolysis, phosphorylation or acetylation at the N-terminal end. Proteolysis may comprise exoproteolysis in which one or more terminal amino acids are sequentially cleaved, enzymatically to produce a microheterogeneous form having fewer amino acids than the original gene product. Endoproteolytic modification results from the action of endoproteases that cleave the peptide at specific locations within the amino acid sequence. Similar modifications may occur during the purification process, which also results in the production of microheterogeneous forms. The most common modification that occurs during purification is proteolysis, which is generally kept to a minimum using protea signal inhibitors. In most conditions, a mixture of microheterogeneous forms is present after purification of native aFGF. Native aFGF refers to aFGF isolated and purified from tissues or cells containing aFGF.

3 DK 175856 B1

The invention encompasses all microheterogeneous forms of mammalian acidic fibroblast growth factor. The preferred embodiments include bovine and human micro-heterogeneous forms of aFGF. The most preferred microheterogeneous forms. more for bovine aFGF comprises a 154 amino acid form, one with 140 amino acids nos acids and one with 134 amino acids. The form of 140 amino acids is shown in Table III and it is the most preferred of the above bovine species. The 154 amino acid form further comprises the following amino acids: Ala-Glu-Gly-Glu-Thr-Thr-Thr-Phe-Thr-Ala-Leu-Thr-Glu-Lys, wherein the carboxyl terminal Lys is attached to the amino-terminal Phe at. first position in the form of 140 amino acids. The form with the 134 amino acids is identical to the form with the 140 amino acids except that the first 6 amino acids from the amino terminal end have been removed. After isolation, the relative amounts of these microheterogeneous forms vary depending on the method used, but all preparations contain at least a portion of each form.

Human aFGF exhibits a similar microheterogeneity as bovine aFGF. The most preferred microheterogeneous forms of human aFGF include a form of 154 amino acids, 140 amino acids or 139 amino acids. The 140 amino acid human form differs from the 11 amino acid bovine form as shown in Table V. The 154 amino acid form contains the exact sequence of the 140 amino acid human form plus 14 additional amino acids associated with the 154 bovine form. amino acids on an exception near. The amino acid in the fifth position from the N-terminal end or position -10 as determined from the 140 amino acid Phe N-terminus in the human form and replaces threonine in the bovine form. The additional 14 amino acids in the human N-terminal sequence are:

Ala-Glu-Gly-Glu-Ile-Thr-Thr-Phe-Thr-Ala-Leu-Thr-Glu-Lys.

A third form of human aFGF contains 139 amino acids and is equivalent to the human form of 140 amino acids, where the amino terminal phenylalanine is removed. The amino-terminal asparagine residue may be deamidated to aspartic acid in the human form of aFGF of 139 amino acids. The 140 and 139 amino acid forms are the most preferred forms of the human microheterogeneous forms.

Mammalian r-aFGF produced by cloning the natural gene from either the genomic DNA or cDNA or by constructing a gene for one of the micro-heterogeneous forms of the protein based on the known amino acid sequence of these microheterogeneous forms of mammalian aFGF, including the human happened. Genome DNA is extracted from mammalian 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 a suitable 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 RNA is extracted from cells expressing aFGF by the method of Aviv and Leder, Proc. Natl. Acad. Sci.

69: 1408-1412 (1972). cDNA is prepared using reverse transcriptase and DNA polymerase using standard techniques as described by Maniatis et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1982). cDNA is analyzed and cloned into a suitable vector, usually pBR322, 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 hybridization with an oligonucleotide probe. The sequence of the oligonucleotide hybridization probe is based on the particular amino 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) discloses various procedures for screening genome and cDNA clones.

The preferred procedure for obtaining a mammalian aFGF gene is to synthesize the gene. The gene can be synthesized based on the amino acid sequence of a microheterogeneous form of aFGF obtained from any mammal, including the human. The preferred method is to use the bovine amino acid sequence of aFGF and the chemical point mutant base sequence to produce the genes for other species. The amino acid sequences of bovine and human aFGF are 5. disclosed in U.S. Patent Application No. 868,473, filed May 30, 1986, which is a continuation-in-part of U.S. Patent Application No. 774,359 filed September 12, 1985, which is again a continuation-in-part. of U.S. Patent Application No. 685,923, filed December 24, 1984, and which has now declined.

10

The synthetic genes are based on the particular bovine amino acid sequence previously 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). 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: 20 6 DK 175856 B1

TABLE I

5 '0 IS 20

Phe Asn Leu Pro leu 61 y Asn Tyr Lys Lys Pr <> Lys Uu L # u Tyr Cys Ser Asn G1y G1y

5 TTQ AAQ CTN CCN CTN 6GN AAQ TAQ AAP AAP CCN AAP C TN CTN TAO TGQ TCN AAQ GGN GGN

TTP TTP TTP TTP agq 25 30 35 40

Tyr Phe Leu Arg Ile leu Pro Asp Gly Thr Val Asp G1y Thr Lys Asp Arg Ser Asp Gin

io TAQ TT (> c ™ CGH ATG c ™ CCN GGN ACN G ™ ^ 06.1 ACN ^ CGN TCN ^ CAP

TTP AGP ATA TTP AGP AGQ

45 50 55 60

His Ile Gin Leu Gin Leu Cys AU Glu Ser lle Gly Glu Val Tyr Ile Lys Ser Thr Glu

CAQ ATO CAP CTN CAP CTN TGQ GCN GAP TCN ATQ GGN GAP GTN TAQ ATQ AAP TCN ACN GAP

^ 5 ATA TTP TTP AGQ ΑΤΑ ΑΤΑ AGO

65 70 75 BO

Thr Gly Gir, Phe Leu Ala With Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gin Thr Pro Asn

ACN GGN CAP TTQ CTN GCN ATG GAQ ACN GAQ GGN CTN CTN TAQ GGN TCN CAP ACN CCN AAQ

7TP TTP TTP AGO

20 85 90 95, 00

Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr lie Ser Lys

GAP GAP TGQ CTN TTQ CTN GAP CGN CTN GAP GAP AAQ CAQ TAQ AAQ ACN TAQ ATQ TCN AAP

TTP TTP AGP TTP ATA AGQ

25 105 1.0 1 15 120

Lys His Ala Glu Lys His Trp Phe Val Gly Leu Lys Lys Asn G1 y. 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

125 130 135 ho.

30 Pr® Arg Thr His Phe Gly Gin Lys Ala He 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

wherein Q - C or T,

P = A or G, and N = A, T, C or G

DK 175856 B1 7 ·

The nucleotide sequence of the invention encompasses the following properties, codons preferred by Escherichia coli and mammalian cells, where possible, elimination of sequences with multiple complementarities, incorporation of unique restriction sites through the gene, terminal restriction enzyme sticky ends to facilitate insertion of the gene, a centrally located unique restriction site to allow the gene to be cleaved in two halves, preferably an N-terminal methionine codon as the start for translation and tandem translation stop codons.

The following description and examples illustrate the present invention with respect to a particular nucleotide sequence for bovine aFGF, but the present invention may comprise any of the permutations purified in Table I. Table II contains the preferred nucleotide sequence: 175856 B1

TABLE II

TTCAATCTGCCACTGGGTAATTACAAAAAGCCAAAGCTTCTTTACTGCTCTAACG6TGGT 60 5 TACTTTCTCCGCATCCTGCCAGATGGTACCGTGGACGGCACCAAAGATCGTTCTGATCAA CATATTCAACTGCAGCTGTGCGCCGAATCTATCGGTGAAGTTTACATCAAATCTACCGAA 120 10 180 15 240 20 ACTGGTCAATTCCTTGCCATGGACACTGATGGCCTGCTGTACGGATCCCAGACCCCAAAC GAGGAGTGCCTTTTCCTGGAGCGCCTGGAGGAAAACCATTACAACACCTACATCTCTAAA AAGCATGCTGAGAAACATTGGTTCGTAGGCCTTAAGAAAAATGGCCGCTCTAAACTGGGC 300 25 360 30 420 35 9 CCTCGTACTCACTTTGGTCAAAAAGCTATCCTGTTCCTGCCACTGCCAGTGAGCTCTGAC DK 175856 B1

The gene is constructed with a leader moiety containing a single restriction 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 complementary self.

DNA enclosure allows a selection of base sequences which in turn allows the incorporation of unique restriction enzyme cleavage sites distributed within the gene. The preferred gene base sequence with localization of restriction enzyme cleavage step is shown in Table III: 10 DK 175856 B1

W * - <L · O O

«U o £ υ o

(Λ O Η <H * O <C> - I

si <c ir α u L * -15 O ^ 04 <* -

<(J O <3 O O

Qi k <1. U 13 * - *

M <►- «O X

<<2 o v> Η <§ «* ^ µ *>» «k d> 1 ^ K f- l71 Q ω

J <H- O U U

L · (J 19 k · L> O

je oLiL> »<* -> * υ (β · a iD o r · o o« »* - <O O O« 4 U * 3

On O O 3 O O

V »« i l · · φ k · <<o υ -i u α

r- 43 O> *. ISLAND ISLAND

<0 O μ- <r- 0430> O (3 o o rv u u o w ^ o O OO. CM ^ <m jc u o η γν w <* ">

Η <K C <0 O

> »Μ <d u» - <* - O O> C JC. o 0 o o o »- <k α μ- <α o o v · <μ ιΑ <^ <« o o <· o υ o ^ <μ * - »^ u n

k O u O Φ k <O

fi. M o O X <»- U

3 (3 0 * O O Z

0 µ <- O O

~ J O O <O O

Φ O O 3 M ^ r— µ · »<Φ Η <O0 * - -« * * - -i o o w

<λ O U O «" · Φ O U O

4- (D O <J ^ -C O i- <<U o ^ cl c \ j µ · <3 O O C <M *

01 H- <. »- <H

Y U 0 13 O O

Or µ- <> 1 ►- <4S »- <r- r—« 3 0

O.> - <43 O O

L U 0 U w k- <

>% <►- £ · O O

µ- µ- <µ- <µ- o> * h <03 ί µ · «Ν * µ- (30 O» - <> - o o o o o u

> »N * <k O O

«- 43 O jC O O

> 1 ooo µ- <♦ - mc OO l · µ- <t— m <r µ- φ oo u-ι «co <µ v> o µ <4- ^ U = L · <v * O <» - 0 »(J 43> S« - <uj (Λ μ <-j <μ-

Γ «* (J o Φ O O

£> »co - ► · <PC o µ <µ * <f ►— S i (jew oro- 2>» <µ-> ι <µ- Μ µ- µ- <µ- µ- «ς 3 · µ * <r— · μ <Οι μ »<Φ μ <-4 ο Ο> 4JS Ο 3» - <. ο <. μ- φ μ <μ f- «^ _Ι ϋ Ο μ Ο • Λ ο ο %>% μ- <> 1 <μ- C r— Ο Ο <μ »Ο 43 Ο Ο <μ- χ Φ Ο Ο k Ο Ο r- Ο μ- <ο_ ο υ i? μ * ο ^ μ »Ο μ ο ο ο k μ <χ <κ m οι t_> Ο · - * Ο <μ- ν> μ * <« **

Μ <µ- 3 <Η- C

_j <^ ο <3 ο χ.

k u Ο m ο ο> η γ ^. -C µ- ι— ο Ο <* ο ο / - »C * - * μ <νι υ Ο £ vi <μ >> Ο .ο w · <<<~ ο 1— <> * * - <3 Ο Ο <- r— Ο Ο ^ Φ «μ <μ Ο 19 υ ν otnoOs 3 43 Ο * - * C μι Ο Ο> φ μ <μ- <μ» ο.

_J U 0 ο ο ο W

Μ Ο <μ 3 ο ο U · μ μ rg ο ο «μ *« νι α. υ ο ο μ υ Ο ο.

3 ο ο c <μ- ο »μ- <μ- <ο ο <3 ο ο ο C μ- <Φ μ- <ΙΛ <μ- c— μ <<<μ * * - <►- # - φ ο ο i «μ- <-c. <- · <μα. μ- <χ ο 43 ** (3ο ο c <μ φ ►- <* τ ι— <μ- X Ο U Ο «kJ 43 μ Ο. μ <μ μ- α νι <ι- u μ- ο < 0 U Ο <w

«- ^ UJ

* 11 DK 175856 B1 «Λ <ft—> i <►- —i O ^ * - t so» - <

o (*) u W

«/> ^ <Σ» u d u u u <u o> «u o

r O U

19 19 Vi C · µ- <<3 · - • Λ «<►->» <> · —J · <f- • λ <3 o> *. <H- -i <»· 3 µ» << * O> - -tf -iu “« -.

o >> ro o o 3 - f— CS O «i« —O C g (Λ »- <* -« 0 ft- <

> C <J

ei u O.

£ ΓΓ 5 α ου ou O o ^ rk ►- <tt V «fr— <- <µ- X Vi O ft— * - · V» <fu r-> * «« ft— O ft -i <µ- <* Λ 3 OO li ~ O 19 We - '

p- N <H> Μ ΤΓ U O

O Π O U - v> - <>

«Ft— <w <H- OC

«- u o» - <o <O U «<ft— ti

* Η- <Λ u U W

· * -. <ft- A. · <µ- X U O «Λ ^ <«) m O U <^ • J. tfC »- µ- <OM W tf H O O. u o

O X <K V M tf K

J. - -J <C ft— - <O u

Φ O u O 41 O O

«1 ft— <« Λ I— <<- »7 oo O lu <3 wr * p— * - · <φ o we ms L · - * O ·« ft- ΙΛ O <»Λ ruoo O» - cm <3 u U> »Γ0 <t— <0 T7 ft— <I— * L- ft- * - <> ou« s. Looo <r ft— »-

£ .OO Ur— O O W

♦ - tt p- o. M o o

IH C U U 3 * - O O

L-J ** <»- Φ * - · H <r <-2 w h o 3 H V · O O O. <μ

>> <ft- L. O O

1 »- ►- <o O O

H «* * - <3 O O

(aj - <ft- 4> ft— <C

/ r z <- O o o o • r c .Li O 4l o o <** <c ft- jC µ- <r O <<5 H- CL · ft— <

^ 3 <I— 3 O O

r- O <»- Φ O µ- <

O € 0 0 0 O o O O

03 N O U O «^ U O

O 'p · <Ή D p · H <O O O P> M <>.

3 O o «ο» - tt

41 µ- -tf »- uHJ

—J U O H <o o CA U O µ M tf µ · C o O φ> s tf µ.

«« Oo «o« «tf ft— 3 O O X C tf ft-

P * T <H p- tf H

O O O O O o 3 O O> * ft- <

4 »H tf p— O

o o O <- * o o o φ O O O 4) ft— <JC ft— <-. C µ- tf CL * - tf w CL. ^ <

3 O µ tf M OOO

4> tf) µ <µ- <Q tf µ - * cm o o x ro o o V »O O L µ- <

X 13 O £ U O

υ η μ <μ- tf μ ι - »- 3 <n OOO) μ <vf— μ« tf μ μ 19 y _ »o O o <oo« - »3 OOO μ tf * - · <μ >> μ · Uo OO o (L ro o O · - «

O C O O O> * - O O <D

CO M tf µ fSJ p— µι O O Q.

tf tf µ - O O O <o tf µ 3. o o L O O 41 ft— <o. O O O o o 12 DK 175856 B1

The gene sequence of each strand of the double-stranded molecule is randomly divided into 8 nucleotide sequences. The oligonucleotides are constructed with overlapping ends to allow formation of the double-stranded DNA. The following table contains one of the several possible oligonucleotide arrangements used to prepare the bovine aFGF gene.

TABLE IV

10 01IG0-1 10 20 30 40 50 S8 5 'AATTCATGTT CAATCTGCCA CTGGGTAATT ACAAAAAGCC AAAGCTtCTT FACTGCTC 3' 15 OLIGO-2 10 20 30 40 45 5 'AGAAGCTTTG GCTTTTTGTA ATTACCCAGT GGCAGATTGGGGGAGGT TACT TEXT TO CTA. 30 40 50 60 65 5 'GTAAACTTCA CCGATAGATT CGGCGCACAG CTGCA6TTGA ATATGTTGAT CAGAACGATC TTTGG 3'

OlIGO-7 10 20 30 40 50 60 67 3 ° 5 'GAAGTTTACA TCAAATCTAC CGAAACTGGT CAATTCCTTG CCATGGACAC TGATGGCCTG CTGTACG 3 · 35 13 DK 175856 B1} TABLE IV (continued) 5 01IG0-8 10 20 30 40 50 60 62 ACGG GAGGTATATA GAGGTATTATAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAGTAG! 20 30 40 48 5 'CCATTACAAC ACCTACATCT CTAAAAAGCA TGCTGAGAAA CATTGGTT 3' 15 0LIG0-12 10 20 30 40 46 S 'GGCCTACGAA CCAATGTTTC TCAGCATGCT TTTTAGAGAT GTAGGT 3 * OLIGO-13 10 20 30 40G 20 OCT-14 10 20 39 40 50 55 ♦ 5 'GCTTTTTGAC CAAAGTGAGT AC6AGGGCCG AGTTTAGAGC GGCCATTTTT CTTAA 3' OLIGO-15 10 20 30 40 50 56 25 5 * 6TCAAAAAGC TATCCTGTTC CTGCCACTGC CAGTGAGC 01 'TCGACGATAT CTATTAGTCA GAGCTCACTG GCAGTGGCAG GAACAGGATA 3 '30

The oligonucleotides shown in Table IV are presented merely as an example of oligonucleotide subunits and should not be considered limited thereto. The composite base sequence showing overlap and arrangement of the oligonucleotides is shown in Table III.

35 14.

DK 175856 B1

The bovine gene is assembled in two steps: first the half corresponding to the N-terminal part of the protein, and then the second C-terminal half. Generally, the oligonucleotides are kinase with T4 polynucleotide kinase in the presence of either APT or 32P-labeled ATP. In the first reaction of each step, the oligonucleotides that make up one strand of the gene are kinase except for most 5 'oligonucleotide. In the second reaction, the oligonucleotides constituting the second strand are kinase, with the exception of most 5 'oligonucleotide. When kinased oligonucleotides are used, ca. 1 pmol of the 32P-labeled oligonucleotide for later identification of the products. Voltage equalization is performed in a suitable buffer, such as one containing, but not limited to, approx. 60 mM Tris, approx. pH 7.6, approx. 5 mM dithiothreitol (DTT), ca. 10 mM MgCfe and co. 30 μΜ ATP at approx. 90 ° C for approx.

4 minutes followed by a quick transfer to approx. 60 ° C and a rapid cooling to approx. 30 ° C. Ugering is carried out in a suitable buffer, such as one containing, but not limited to, approx. 60 mM Tris, approx. pH 7.6, approx. 10 mM DTT, approx. 10 mM MgCfe, ca. 30 μΜ ATP and approx. 0.03 units of T4 DNA ligase at ca. 20 ° C for approx. 1.5 hours.

The ligating oligonucleotides are purified by polyacrylamide gel electrophoresis followed by an ethanol precipitate. The oligonucleotides are redissolved in a buffer containing ca. 20 µΙ approx. 80% formamide, ca. 50 mM Tris-borate, ca. pH 8.3, approx. 1 mM ethylenediaminetetraacetic acid (EDTA), ca. 0.1% by weight / vol. xylenesanol and approx. 0.1% by weight / vol. bromophenol. Each sample is heated at approx. 90 ° C for approx. 3 minutes and electrophoresed in a ca. 10% urea-polyacrylamide gel at approx. 75 watts for approx. 5 hours. The 231 base N-terminal bands are removed, combined and eluted at ca. 4 ° C for approx. 0.5 M ammonium acetate containing approx. 1 mM EDTA at approx. pH 8. The 209 base C-terminal bands are treated in the same way.

The synthesized gene sequences encoding either the N-terminal or the C-terminal portion of aFGF are incorporated into the pBR322 plasmid. It is particularly desired and intended that the invention encompass the use of other plasmids into which the aFGF gene can be incorporated and which will allow expression of the aFGF gene. Voltage-equalized oligonucleotides, ca. 300 fmol 35 and approx. 100 fmol of the recovered 231 base pair N-terminal end are each ligated to ca. 100 µmol agarose gel purified approx. 3.9 kilobase (kb) EcoRI-BamHI

15 DK 175856 B1 pBR322 for the N-terminal end. The 209 bp C-terminal end is constructed similarly using BamHI-SalI pBR322. Ligation is performed in a buffer containing ca. 20 mM Tris, ca. pH 7.8, approx. 1 mM DTT, approx. 10 mM MgCl2, ca. 0.4 mM ATP, with approx. 1 unit of T4 DNA ligase for approx. 1 5 hours at approx. 20 ° C. Each half-ligated vector is used to transform competent bacterial cells, such as E. coli RR1 (Bethesda Research Laboratories, BRL), following the supplier's procedures. The transformed cells are selected for growth in ampicillin and screened for the presence of either the 231 base pair (bp) EcoRI-BamHI inserted or the 209 bp BamHI-SalI inserted by restriction analysis of minilysate plasmid preparations. The DNA sequence for clones containing an insert of an appropriate size is determined using Maxam and Gilbert, Proc. Natl. Acad. Sci. USA 74: 560-564 (1977) Chemical DNA Sequence Technique.

The final full length synthetic aFGF gene was cloned by cleavage of the N-terminal half-clone with restriction enzymes BamHI and SalI, treatment with alkaline phosphatase and ligation thereof to the gel-purified 209 bp BamHI-SalI inserted by the C-terminal half-clone. This ligated material was used to transform competent RR1 cells as before.

20

Expression of the synthetic aFGF gene is followed by a variety of promoter expression systems. It is desired and intended that the invention encompass the use of other promoter expression systems for 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. Night. Acad. Sci. USA 80: 21-25 (1983). Plasmid pKK 223-3 (Pharmacia), which contains the tac promoter and rmB rRNA transcription terminator, was modified to remove the pBR322-derived SalI restriction enzyme site. The rmB 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).

pKK223-3 plasmid DNA is cleaved with restriction enzymes to produce a 2.7 kb DNA fragment to generate clone pKK 2.7. The synthetic aFGF gene 35 is cleaved from its pBR322 vector and transferred to the pKK 2.7 plasmid after restriction by pKK 2.7 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 effective way to transfer the amino acid sequence from a mammalian species of aFGF to aFGF amino acid sequence of another species. The following description relates to the site-specific mutagenic transfer of bovine aFGF, 140 amino acid form to human aFGF, it being understood that the process can be used to transfer any mammalian aFGF to aFGF from another species. The only restriction on the transfer is that the amino acid 10 sequence of both aFGFs must be known. The following table lists the substituted amino acids and the location on the bovine aFGF amino acid map, Table III, where the substitutions are made: TABLE V__

Amino acid Substituted amino acids for_location_ human αFGF_ bovine αFGF_ 5_Pro_Leu_ 21_Jjis_Tyr__ 35_Arg_Lys _ 47_Ser_Cys_ 51_Val_Jle__ 64_Tyr_Phe _ 106_Asn ._His_ 116 _Ser_Ar_Ar_Ar_Ar

TABLE VI

OLIGO-1 5 'CTGCCACCGGGTAATTAC 3' OLIGO-2 5 5'CGGTGGTCACTTTCTCCG 3 'OLIGO-3 5' CGGCACCAGAGATCGTTC 3 'OLIGO-4 5' GCAGCTGTCCGCCGAATCTGTCGGTGAAG 3 'OLIGO-5 5' CT66TCAATACCTTGCCATGG 3 'OLIGO-6 10 5' GCTGAGAAAAATTGGTTC6 3 ' OLIGO-7 5 'GGCCGCGTTTACAGCTGCCA11 111CTTAAGG 3' OLIGO-8 5 'CGTACTCACTATGGCCAAAAAGCTATCC 3' 15

As with the bovine gene sequence, 8 oligonucleotides representing the human gene sequence are constructed by the same procedure as that used for the bovine oligonucleotides. Table VI contains one of several oligonucleotide arrangements used to produce the human aFGF 20 gene.

The cloned synthetic bovine gene for aFGF is transferred to a human synthetic gene for aFGF by a series of directed point mutations. Oligonucleotide-directed mutagenesis of the cloned gene allows modification of the base sequence of bovine aFGF so that the resulting amino acid sequence contains the substituted amino acids shown in Table V and is human aFGF. To prepare the human 139 amino acid microheterogeneous form of aFGF, the amino terminal phenylalanine is removed by a deletion in the bovine gene. A point mutation is performed to replace asparagine at the second position 30 with aspartic acid. Alternatively, the asparagine is deamidated to aspartic acid. The methods for performing these procedures are described below and known in the art. The oligonucleotide-directed mutagenesis is performed using standard procedures known in 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 performed by standardized oligonucleotide-directed mutagenesis are shown in the following Table Vil. The location of the base mutagenesis can be seen in Table III. The point mutations are shown only as an example of changes that will result in the human aFGF gene and should not be considered as limiting.

5

TABLE VII

Base Substituted base for corresponding localization_humant aFGF bovine aFGF_human amino acid 22_C_T_Pro 69_C_T _His 112_G_ A _Arg 148_C___G_Ser 159_G_A_Val_ 199_A_T_ Tyr_ 324_A C_ Asn_ 35_G C_ As__

The expression clones are grown at ca. 37 ° C in a suitable growth medium consisting of ca. 1% tryptone, approx. 0.5% yeast extract, approx. 0.5% NaCl, approx. lo 0.4% glucose and approx. 50 µg / ml ampicillin. When the optical density at 550 nm reaches approx. 0.5, isopropyl-β-D-thioglactopyranoside (IPTG) can be added to a final concentration of approx. 1 mM and growth is continued at approx. 37 ° C for approx. 3 hours. The cells from 1 liter of culture medium are harvested by centrifugation and redissolved in a disruption buffer containing ca. 10 mM sodium phosphate 15 at approx. pH 7.2, approx. 5 mM EDTA, ca. 10.6 pg / ml N-p-toluenesulfonyl-L-phenylalanine chloromethyl ketone (TPCK), approx. 34.3 pg / mlk pepstatin A, ca. 87 pg / ml phenylmethylsulfonyl fluoride (PMSF), ca. 15 pg / ml bovine pancreatic trypsin inhibitor (BPTI) and approx. 25.2 pg / ml leupeptin. Cells are either disrupted or frozen and stored at -70 ° C and disrupted immediately after thawing at ca.

19 DK 175856 B1 three passages through a French pressure cell at approx. 12,000 psi at approx. 4 ° C.

The supernatant is collected by centrifugation.

The recombinant aFGF is purified for homogeneity by a unique two-step chromatographic procedure using heparin / Sepharose® affinity chromatography combination followed by reverse-phase HPLC. The crude r-aFGF is placed on a heparin / Sepharose column in a diluted buffer such as ca. 10 mM phosphate or Tris, ca. pH 6-8 which is then washed with a low salt solution such as ca. 0.8 M NaCl until the absorbance at 10 280 nm drops to approx. background absorbance. r-aFGF is then eluted with a high salt buffer solution, such as ca. 10 mM sodium phosphate or Tris, ca. pH 6-8, containing approx. 1.5 M NaCl. The eluate is then purified by reverse-phase HPLC on a resin consisting of covalently bonded alkyl silane chains having alkyl groups of 3-18 carbon atoms, preferably 4 carbon atoms. r-aFGF is directly applied to the equilibrated HPLC column in a dilute acid such as ca. 10 mM trifluoroacetic acid, acetic acid or phosphoric acid and eluted with a linear gradient organic solvent such as acetonitrile or ethanol. Bovine brain-derived aFGF was previously described as binding to both heparin / Sepharose suits and to reverse-phase HPLC columns, cf. Maciag et al., Science 225: 932-935 (1984) and Thomas et al., Proc. Natl. Acad. Sci. USA 81: 357-361 (1984) as part of multi-stage purifications. In part, based on the relatively high amount of r-aFGF in bacterial lysates, these two steps alone appear to be sufficient to obtain homogeneously pure r-aFGF by ca. 16,000 daltons as determined by electrophoresis in 25 polyacrylamide gels. These two steps alone do not produce pure aFGF from the brain.

Mitogenic activity of the purified aFGF is determined by incorporation of 3 H-thymidine into DNA with cell line fibroblasts, preferably BALB / c 3T3 A31 (American Type Culture Collection). The recombinant aFGF shows a peak response at ca. 1 ng of protein or less per ml in the fibroblast stimulating sample.

Another embodiment of the invention is a method of promoting wound healing by application of the novel peptide, either with or without 35 heparin, preferably with heparin, ca. 1 to approx. 500 µg / cm2 to the wound area either 20 topically or subcutaneously in the wound in an amount of approx. 0.1 to 100 µg / cm 2 of surface for topical application.

For application, various pharmaceutical formulations are useful, such as ointments, pastes, solutions, gels, solid water-soluble polymers such as albumins, gelatins, hydroxypropyl cellulose, pluronics, tetronics or alginates wherein the active ingredient is incorporated in amounts of approx. 1 to approx. 100 µg / ml.

The ability of aFGF to stimulate healing 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 in mammals, including humans, by administering novel r-aFGF to patients in need of such treatment.

15

The invention is further explained by the following examples. EXAMPLE 1 Oligonucleotide Synthesis

Oligonucleotides were synthesized according to the technique described by Mat-teucci and Cauthers in J.A. Chem. Soc. 103: 3185-3191 (1981) and by Beaucage and Caruthers in Tetrahedron Letters 22: 1859-1862 (1981). The base sequences of the synthesized oligonucleotides are shown in Table IV.

EXAMPLE 2 Collection of the aFGF gene

The oligonucleotides of Example 1 were pooled as two separate units, the N-terminal half (231 bp) and the C-terminal half (209 bp). The halves were then combined to make the synthetic gene intact, see Table III. Initially, the oligonucleotides were kinased in the following reaction mixture: 70 mM Tris pH 7.6, 5 mM DTT, 10 mM MgCfe, 33 µM ATP, 0.3 units T4 polynucleotide per ml. microliters. The mixture was incubated for 1.5 hours at 35 ° C and then another 1 hour after supplementing the mixture with 0.2 units / liter kinase and ATP to give a concentration of 100 mM. For radiolabelling, the original mixture contained 37 nCi / pliter [γ-32P] -ATP.

The stress equalization and ligations were made in two separate reactions. In each reaction, 100 pmol of each of the 8 oligonucleotides was added. In one reaction, the oligonucleotides which form a strand of the C-terminal or N-terminal half of the gene were kinated with the exception of the 5 'oligonucleotide. In the second reaction, the oligonucleotides that make up the opposite strand were kinased, again with the exception of the 5 'oligonucleotide. Thus, in 10 each reaction, 3 oligonucleotides were kinased and 5 oligonucleotides were not kinased. When kinased oligonucleotides were used, 1 pmol of the 32 P-labeled oligonucleotide was also added for later identification. products. Each reaction contained 200 liters of 70 mM Tris pH 7.6, 5 mM DTT, 10 mM MgCl 2 and 30 µM ATP. The oligonucleotides were offset by heating to 90 ° C for 4 minutes, then the reaction was immediately transferred to 60 ° C and allowed to cool slowly to 30 ° C. Ligation was performed in 400 plits containing 60 mM Tris pH 7.6, 10 mM DTT, 10 mM MgCfe, 1 mM ATP and 0.03 units of T4 DNA ligase per ml. rubbing on incubation at 20 ° C for 1.5 hours.

Polyacrylamide gel electrophoresis was used to purify the ligated oligonucleotides. The ligated oligonucleotides were precipitated with ethanol, redissolved in 20 plots of 80% formamide, 50 mM Tris-borate pH 8.3, 1 mM EDTA, 0.1% w / v. xylenesanol and 0.1% by weight / vol. bromophenol. Each sample was heated at 90 ° C for 3 minutes and electrophoresed in 10% 25 urea polyacrylamide gel at 75 W for 5 hours. The oligonucleotide bands were made visible by exposing the gel to X-ray film.

The 231 basebands for each reaction for the N-terminal end were cut out of the gel, combined and eluted at 4 ° C in 1 ml of 0.5 M ammonium acetate, 1 30 mM EDTA pH 8. The eluted DNA was precipitated with ethanol and redissolved in ethanol. 30 plies 70 mM Tris pH 7.6, 5 mM DTT and 10 mM MgCl2. The 209 basebands for the C-terminal end were eluted in the same way.

The gel purified oligonucleotides were voltage equalized before transformation 35 by heating to 90 ° C for 4 minutes and slowly cooled to 20 ° C. Assuming a 5% yield from the original oligonucleotides, 300 DK 175856 B1 22 fmol and 100 fmol recovered, voltage-equalized 231 bp oligonucleotides were each ligated to 100 fmol agarose gel purified 3.9 kb EcoRI-BamHI pBR322 fragment Tr pH 7.8, 1 mM DTT, 10 mM MgCl 2, 0.4 mM ATP with 1 unit of T4 DNA ligase for 1 hour at 20 ° C. The equalized 209 5 bp oligonucleotides were ligated to the agarose-purified 3.9 kb Bam HI-SalI pBR322 fragment DNA under the same conditions as the 231 base pair fragments. The ligation reactions were diluted 1: 5 in H 2 O and 1 pleat dilution was used to transform 20 pl 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 bp EcoRI-BamHI or the 209 BamHI-SalI inserted by restriction analysis of minilysate plasmid preparations.

The DNA sequence of clones containing insert of appropriate size 15 was determined using the chemical DNA sequence technique of Ma-xam and Gilbert, Proc. Natl. Acad. Sci. USA 74: 560-564 (1977). Since none of the 231 bp clones had the correct sequence, a clone containing the correct sequence was prepared as follows. A clone with the correct sequence between KpnI and with the BamHI sites was cleaved with KpnI and with 20 SalI, which cleaved in the pBR322 vector. The 400 bp band was gel purified and ligated to the 3.8 kb KpnI-SalI band from another 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 that the desired sequence had been obtained.

25

Once a clone containing the correct 209 bp sequence was obtained, no further manipulation of these clones was required. The final full-length aFGF synthetic gene was cloned by cleavage of the N-terminal half-clone 30 with BamHI and SalI, treatment with alkaline phosphatase, and ligation to the gel-purified 209 bp BamHI-SalI insert of the C-terminal half-clone. This ligated material was used to transform competent RR1 cells as before.

EXAMPLE 3

Expression of the synthetic bovine aFGF gene

The intact aFGF gene of Example 2 was incorporated into a modified 5 pKK223-3 plasmid. The pKK223-3 plasmid (Pharmacia) contains the tac promoter, which is a hybrid between regions of the trp-prpm motor and the lac promoter, deBoer et al., proc. Natl. Acad. Sci. USA 80: 21-25 (1983). This plasmid also contains rmB rRNA transcription terminator, a strong terminator sequence that has been shown to allow expression from strong promoters, Gentz et al., Proc. Natl. Acad. Sci. USA 78: 4936-4940 (1981), Brosius,

Gene 27: 161-172 (1984). The pKK223-3 plasmid was modified to remove the pBR322-derived SalI restriction enzyme site. This was done by cleavage of pKK223-3 plasmid DNA with Ndel and Narl and recycling the 2.7 kb DNA fragment to form clone pKK2.7. The synthetic aFGF gene was then cleaved from its pBR322 vector and transferred to pKK2.7 after restriction of this expression vector by EcoRI and SalI. This construct places the initiating methionine for the synthetic gene 11 bases after the Shine-Dalgamo ri-bosom exchange site. The resulting recombinant shown in FIG. 1, was transformed into E. coli JM105 cells and also into E. coli DH5 cells.

20

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 10 µg / ml ampicillin.

When the optical density at 550 nm reached 0.5, IPTG was added to give 1 mM and the culture was continued at 37 ° C for 3 hours. The cells were harvested by centrifugation at 10,000 xg for 20 minutes and the cells from 1 liter culture were redissolved in 20 ml of 10 mM sodium phosphate pH 7.2 (heparin / sucrose buffer) 5 mM EDTA, 10 N 6 pg / day. ml TPCK, 34.3 pg / ml pepstatin A, 87 pg / ml PMSF, 15 pg / ml BPTI and 34.3 pg / ml leupeptin. The redissolved cells were quickly frozen in a dry ice / ethanol bath and stored overnight at -70 ° C.

EXAMPLE 4

Extraction and purification of recombinant aFGF 35 The frozen cells of Example 3 were thawed, an additional 87 µg / ml of PMSF was added and the preparation was passed through a French pressure cell at 12,000 psi three times at 4 ° C. The resulting lysate was centrifuged at 93,000 x g for 30 minutes to remove cell debris. The supernatant was removed, adjusted to pH 7.2 with 1 M NaOH and placed on a 1.6 x 10 cm Heparin / Sepharose column (Pharmacia) operated at 4 ° C with a flow rate of 20 ml. per. per hour while collecting 2 ml fractions. The pellet was redissolved in 5 ml of 10 mM sodium phosphate, 2 M NaCl, pH 7.2, re-centrifuged at 93,000 xg for 30 minutes, and the supernatant was diluted with 3 volume 10 mM sodium phosphate, pH 7.2, reset to pH 7.2 with 1 M NaOH, if necessary, and placed on the same heparin / sepharose column. After 10 application, the column was washed with 10 mM sodium phosphate, 0.8 M NaCl, pH 7.2 until the absorbance at 280 nm decreased to the background absorbance. Bound r-aFGF was eluted as a single peak with 10 mM sodium phosphate, 1.5 M NaCl, pH 7.2. The total fractions from the heparin / sepharose column were purified by reverse-phase HPLC using a 4.6 mm x 25 cm C4-15 column (Separations Group) as described by Thomas et al., Proc. Natl. Acad.

Sci. USA 81: 357-361 (1984). r-aFGF was eluted as a single main peak, which was dissolved from several less polluting peaks, suggesting that the protein was homogeneously pure. Polyacrylamide gel electrophoresis was used to confirm purity. The purified r-αFGF was electrophoresed following the technique of O'Farrell, J. Biol. Chem. 250: 4007-4021 (1975). Silver staining revealed a single band with a molecular mass of 16,000 daltons. Identity of the protein as aFGF was confirmed by both amino acid analysis and amino-terminal sequencing.

EXAMPLE 5

Biological activity of bovine recombinant aFGF

Biological activity of the purified r-aFGF of Example 4 was assessed using a fibroblast mitogenic sample as described by Thomas et al., J.

Biol. Chem. 225: 5517-5520 (1980). BALB / c 3T3 A31 fibroblasts (American Type Culture Collection) were plated at 2 x 10 4 cells per cell. 35 mm diameter well in a culture medium containing 10% heat inactivated calf serum and incubated in 7% CO 2 (pH 7.35 ± 0.05). The cells were fully stained by replacing the medium with 0.5% heat-inactivated calf serum 6 and 35 again 24 hours later. 55 hours after ironing, 50 µg of heparin, test samples and 1.1 µg of dexamethasone were added, at 70 hours each well was supplemented with 2 µCi [methyl-3-H] thymidine (20 Ci / mmol, New England Nuclear) and 3 µg of unlabeled thymidine (Sigma), and at 95 hours, the cells were processed to determine radiolabelling into DNA. Each dose response point was the average of three determinations. The results are shown in the following table:

TABLE VIII

Mitogenic response of BALB / c 3T3 Fibroblasts to bovine r-aFGF

Concentration CPM___r-aFGF (ng / ml) _r-aFGF_Brain aFGF_ 0.003 _ 268_ 231 0.01_ 498 329_ 0.031 1550_ 1017_ 0.1_ 7031_ 3684_ 0.316_9319 _ 11353_ 1,000 | 4718 [9050

The activity of the recombinant aFGF was equal to or slightly greater than the activity of brain-derived aFGF. The purified r-aFGF had a half-maximal stimulation of DNA synthesis at ca. 71 pg / ml, while purified home-derived aFGF had a half-maximum value at 126 pg / ml.

Example 6 Mutagenesis of bovine aFGF gene to the human aFGF gene

To facilitate the mutagenesis of the bovine aFGF gene, the synthetic gene of 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 Enzymology, 100: 468-500 (1983); Norris et al., Nucleic Acids Research, 11: 5103-5112, and Zoller and Smith, DNA, 3: 479-488. The bovine pKK-αFGF plasmid was cleaved with EcoRI and SalI, see Table III, and the resulting 440 bp 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 then dephosphorylated. in 100 pliter 10 mM Tris pH 8.0 26 B1 buffer with 100 units of bacterial alkaline phosphatase. A ligation was made using 50 ng of the treated vector DNA and 12 ng of aFGF gene fragment DNA in 10 pls 25 mM Tris pH 7.8, 10 mM MgCl 2, 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 H 2 O and 1 pl of solution was used to transform 20 pl 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 obtained. A phage clone containing the bovine aFGF was selected, M13mp19-aFGF.

! Eight oligonucleotides were designed to specify the human sequence and synthesized, see Table VI.

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.

The human oligomers 1,2, 3, 4, 6 and 8 were phosphorylated, 15 pmol of each were individually fused with 0.5 pmol of M13mp19-aFGF single-stranded phage DNA in 10 pliter 20 mM Tris pH 7.5.10 mM MgCl 2, 50 mM NaCl, 1 mM DTT for 10 minutes at 65 ° C followed by 10 minutes at 23 ° C. Sealed circular double-stranded molecules were then prepared in 20 plits of 20 mM 25 Tris pH 7.5, 10 mM MgCl 2, 25 mM NaCl, 5.5 mM DTT, 0.5 mM ATP, 0.25 mM dATP, 0.25 mM dCTP, 0.25 mM dCTP, 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 radiolabelled using 32P-ATP and polynucleotide kinase. Hybridization conditions were optimized for each probe to prevent the formation of hybrids containing single base changes. Single-stranded DNA was isolated from the phage clone containing human oligomer 5 mutant 4 mutations, and the above procedure was repeated using human oligomer 5 to form a clone containing both oligomer 4 and 5 mutations.

In the following procedures, the bovine-to-human sequence mutations in M13-5-based clones were combined into a pBR322-based clone. RF DNA was prepared from clones containing base changes specified by human oligomers 1, 2, 6 and 8. DNA from the human 1 mutant clone was cleaved with EcoRI, the ends dephosphorylated with bacterial alkaline phosphatase, and DNA was cleaved with HindIII. The human 2 mutant DNA was digested with Hindο HindIII, treated with phosphatase and then digested with BamHI. The human 6 mutant DNA was digested with BamHI, treated with phosphatase and then digested with Apal. Likewise, the human 8 mutant DNA was cleaved with Apal, the ends were phosphorylated and DNA was cleaved with SalI.

These four DNA preparations were electrophoresed through 2% agarose, and the 15 fragments of 45bp, 190bp, 135bp and 70bp of mutant DNA contained within the | the human 1, 2, 6 and 8 mutations were eluted from the gel. Ca. 60 fmol of each fragment was collectively ligated to ca. 60 fmol of a gel purified 3.7 kb EcoRI-SalI fragment from pBR322 in 5 pliter 25 mM Tris pH 7.8, 10 mM MgCfe, 1 mM DTT, 0.4 mM ATP with 1.5 units of T4 DNA ligase for 16 hours at 120 ° C.

The reaction mixture was diluted 1: 5 in H 2 O and 1 pl of dilution was used to transform 20 pl 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 radiolabelled probes prepared from each of the oligomers. The 140 bp Kpnl-BamHI DNA 25 fragment isolated from cleaved RF DNA of the human 3 mutant M13 clone was ligated to endonuclease cleavage products of the human 1-2-6-8 mutant DNA and transformed into DH5 competent cells to form a clone with the human 1-2-3-6-8 mutations. BamHI-PstI degradation fragments of the latter clone were ligated to BamHI-PstI degradation fragments of RF DNA 30 from the human 4-5 M13-based clone and the mixture from the ligation 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 35 (BRL). Competent DH5 cells were transformed with ligated DNA and the transformed cells were propagated on JM105 host cells to form a clone. The single stranded phage DNA of this clone was fused to the human 7 oligomer and an M13 clone containing all the desired mutations was obtained by following the procedure described above. RF DNA was prepared from this clone and digested with 5 EcoRI and SalI. The resulting 440 bp band was gel purified and ligated to the 2.7 kb EcoRI-SalI DNA fragment of pKK2.7 tac promoter expression vector. This DNA was used to transform competent DH5 cells to form the human pKK-aFGF expression clone used to produce the human form of aFGF. in Island

The human r-aFGF was purified by the same procedure as used for the bovine r-aFGF, see Example 4. The human r-aFGF was judged to be at least 99.75% pure based on the presence of an intense band on a silver-colored SDS electrophoresis. gel applied to 400 ng of purified human r-aFGF and 15 with an intensity of approx. 1 ng / band. The protocol is described in Example 4.

The pure recombinant human aFGF was tested for mitogenic activity using 3H-thymidine incorporation into confluent BALB / c 3T3 cells as described for the bovine recombinant protein of Example 5. As previously observed with human brain-derived aFGF tested on vascular endothelial cells. the recombinant human protein has a greater difference in heparin (50 pg / ml) activation than is the case for both brain-derived and recombinant bovine aFGF, Gimenez-Gallego et al., Biochem. Biophys. Res. Comm. 135: 541-548 (1986); The results are recombinant human aFGF on 25 BALB / c 3T3 cells are shown in the following table: 29 DK 175856 B1

TABLE IX

Mitogenic response of BALB / c 3T3 fibroblasts to human r-aFGF concentration

r-aFGF CPM

(picogram / ml) "_- heparin_ + heparin" 0 3574 991 1 4156 1336 3.16 4216 1802 10.0 4092 2617 31.6 4155 4824 100 4274 10489 316 6060 14584 1000 (1 ng) 6811 10547 3160 7910 12357 10000 8597 9143 31600 9700 9057 100000 11166 9277 1000000 15864 12425 * picogram - 10'12 grams In the presence of heparin, the half-maximal stimulation occurs at approx.

42 pg / ml. In the absence of heparin, the peak was not clearly reached even at the highest concentration but must be greater than ca. 50 ng / ml.

Claims (5)

1. A nucleotide sequence coding for recombinant human acidic fibroblast growth factor having the following amino acid sequence: 5 10 20 25 L • PheAsnLeuProProGlyAsnTyrLysLysProLysLeuLeuTyrCysSerAsnGlyGlyHisPhelieuArglle LeuProAspGlyThrValAspGlyThrArgAspArgSerAspGlnHisIleGlnLeuGlnLeuSerAlaGluSer 30 40 50 51 60-70 75 80 90 100 ValGlyGluValTyrIleLysSerThrGluThrGlyGlnTyri.euAlaMetAspThrAspGlyLeuLeuTyrGly SerGlnThrProAsnGluGluCysLeuPheLeuGluArgLeuGluGluAsnHisTyrAsnThrTyrlleSerLys 101110120125 10 LysHisAlaGluLysAsnTrpPheValGlyLeuLysLysAsnGlySerCysLysArgGlyProArgThrHisTyr 130140 GlyGlnLysAlalleLeuPheLeuProLeuProValSerSerAsp wherein in the phenylalanine at the first position is associated with a methionine, and wherein the base sequence is: I 15 60 80 2β 4Q AATTCATGTTCAAlCTGCCACCGGGTAAriACAAeAAGCCAAAGCTrCITT'ACTGCTCTAACGCTGGTCACTnCTCCGC GT ACAAGTI AGACGGT GGCCCAn AAIGTTITI CGCTTTCGAAGaAATGaCGAGATTGCCACCAGTGAAAGAGGCG too U0 140th 160 ATCCTGCCAGAIGGTACCGTGGACGGCACCAGAGATCGTTCTGATCAACATATTCAACTGCÅGCIGTCCGCCGAATCTGT · j TAGGACGGTCIACCATGGCACCIGCCGTGGTCTCTAGCAAGACTAGTTGTATAAGTTGGGATA 200 * 220 240 CGGTGAAGmACATCAAATCTACCGAAACTGGTCAAlACCTTGCCATGGACACTGATGGCCTGCTGTACGGATCCCAGA GCCACTTCAAAIGTAGTTTaGATGGCT.TTGACCAGTI AIGGAACGGTACCGGGGGGGACGG 280300320 CCCCAAACGAGGAGTGCCTTTTCCTGGAGCGCCTGGÅGGAAAACCA7TACAACACCTACATCICTAÅ4AAGCATGCiGAG GGGGTITGCTCCTCACGGAAAAGGACCT CGCGGACC1CCTTT1GGT aaIGT TGTGGATGl AGAGATTTTTCGT ACGACTC 340360380400 aaaaatiggttcgtaggccttmgaaaaatggcagctgtaaacgcggcccicgtactcactatggccaaaaagciatcct tttti * accaagcatccggaattctttttaccgtcgacatttgcgccgggagcatgagtgataccggiitttcgatagga 420,440 GTICCIGCCACTGCCAGTGAGCICIGACTAArAGATATCG CAAGGaCGGTGACGGTCACICGAGaCIGATTATCIATAGCaGCT. DK 175856 B1 in _ An expression plasmid wherein the nucleotide sequence of claim 1 is inserted. A host compatible with and containing the plasmid as claimed in claim 2. The plasmid according to claim 2, which is capable of expressing the gene for human acidic fibroplast growth factor. A process for purifying acidic fibroblast growth factor (aFGF) in pure form comprising the following steps: a. Partial purification of recombinant aFGF by a heparin-Sepharose affinity chromatography matrix and an acceptable eluent; followed by 15 b. final purification partially purified recombinant aFGF by reverse phase hplc (high performance liquid chromatography) using alkyl silane substrate and an acceptable eluent.