Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/50—Fibroblast growth factor [FGF]
  • C07K14/501—Fibroblast growth factor [FGF] acidic FGF [aFGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/50—Fibroblast growth factor [FGF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• FGFs fibroblast growth factors
• the FGFs are mitogenic and chemotactic for a variety of cells of epithelial, mesenchymal, and neural origins.
• FGFs are angiogenic, that is they are able to stimulate the formation of blood vessels.
• Members of the FGF family include acidic FGF, basic FGF, KGF, Int-2, HST, FGF-5, and FGF-6.
• Acidic FGF (aFGF) and basic FGF (bFGF) are considered to be two "original" members of the FGF family. Both aFGF and bFGF are believed to be derived from the same ancestral gene, with both molecules having approximately 55% sequence identity in addition to the same intron/exon structure. Acidic FGF and bFGF are also known to bind to the same receptor, although the existence of specific aFGF and bFGF receptors has not been ruled out. Several molecular weight forms of aFGF and bFGF are found in different tissues. However,
• KGF exhibits potent mitogenic activity for a variety of cells and it binds to cell surface receptors on Balb/MK keratinocytes to which aFGF and bFGF may also bind (Bottaro,et al (1990), J. Biol . Chem. , 265: 12767- 12770. However, KGF is distinct from the known FGFs (e.g., aFGF and bFGF) in that it is not mitogenic for fibroblasts or endothelial cells. Rubin et al, (1989), Proc. Natl . Acad . Sci . USA, ___: 802-806. KGF also has different receptors on NIH/3T3 fibroblasts from the receptors for aFGF and bFGF which fail to interact with KGF. Bottaro et al .
• a shared distinguishing feature of aFGF and bFGF is the propensity of these factors to bind tightly to heparin.
• the affinity of aFGF for heparin appears to be weaker than for bFGF, with aFGF having an anionic isoelectric point (Thomas et al (1984), Proc. Nat . Acad. Sci . USA, £2:6409-6413.
• the unique heparin binding property of aFGF and bFGF has greatly facilitated purification of these factors.
• heparin-like molecules can play a direct role in FGF function, including the activation, or potentiation, of aFGFs (Uhllrich et al (1986) , Biochem. Biophys . Res . Com . , 112:1205-1213.
• FGF proteins are believed to be effective in promoting the healing of tissue subjected to trauma.
• the unique angiogenic property of FGFs makes these factors especially valuable in the healing of deep wounds.
• the bFGF native proteins have been alleged to be useful in the treatment of myocardial infarction (U.S. Patents No. 4,296,100 and 4,378,347).
• human bFGF has been found to increase neuronal survival and neurite extension in fetal rat hippocampal neurons, suggesting that this factor may also be useful in the treatment of degenerative neurological disorders, such as Alzheimer's disease and Parkinson's disease (Wallicke et al (1986), Proc . Natl . Acad. Sci . USA, i: 3012-3016) .
• a major stumbling block to the effective use of aFGF in therapeutic applications appears to be related to its significantly lower biological activity, as compared with bFGF.
• studies with heparin suggest that the observed difference in potency between aFGF and bFGF can be substantially diminished by using heparin to boost the activity of aFGF to a level comparable to that of bFGF, the use of heparin in pharmaceutical preparations may not always be desirable.
• heparin a highly sulfated glycosaminoglycan of heterogeneous structure, is known to be an anticoagulant which functions by accelerating the rate at which antithrombin III inactivates the proteases of homeostasis (Jacques (1980), Pharmacol Rev, 11:99-166). It is not known whether it might be deleterious to use heparin in a pharmaceutical preparation for the treatment of deep wounds, where some degree of coagulation may be desired to achieve proper healing.
• the present invention provides novel analogs of proteins in the FGF family.
• One such analog is an aFGF analog that is more stable and exhibits greater biological activity in the absence of heparin than naturally occurring aFGF.
• Another such analog is a KGF analog that has enhanced thermal stability as compared to naturally occurring KGF. Enhanced stability is achieved by substituting at least one amino acid having higher loop-forming potential for an amino acid residue of lower loop-forming potential in or about the loop- forming sequence Asn-His-Tyr-Asn-Thr-Tyr of the naturally occurring protein. In the case of aFGF, this loop-forming sequence occurs in the area of about amino acids 92 to 96. In the case of KGF, this loop-forming region occurs in the area of about amino acids 115-119.
• a preferred analog of the present invention incorporates the substitution of an amino acid having higher loop- forming potential for the histidine residue in the loop- forming sequence.
• FIG. 1 shows the nucleic acid and amino acid sequences of recombinant bovine [Ala47-Gly93] aFGF.
• FIG. 2 show the amino acid sequence of recombinant human [Gly 93 ] aFGF.
• FIG. 3 demonstrates the elution profiles for bovine [Ala47] and [Ala47,Gly93] aFGF analogs using hydrophobic interaction chromatography.
• FIGS. 4A and 4B show the circular dichroic spectra for bovine [Ala47] and [Ala47,Gly93] a FGF analogs.
• FIG. 6 is a graph showing a plot of the log of the concentration of bovine [Ala47] a nd [Ala47-Gly93] aFGF analogs and human [Ser70-Ser88] bFGF versus the percentage of maximal stimulation.
• FIG. 7 is a graph showing the loss of activity over time of bovine [Ala47] and [Ala47,Gly93] aFGF analogs in the absence of heparin as compared with human [Ser70,ser88] bFGF.
• FIG. 8 shows the structure of the bovine [ la47.Gly93] aFGF analog of the present invention, as determined by X-ray crystallography.
• FIG. 9 shows the nucleic acid and amino acid sequences of naturally occurring KGF.
• FIG. 10 shows the nucleic acid and amino acid sequences of recombinant [Glyll6] KGF. Detailed Description of the Invention
• Novel analogs of the FGF family are provided in accordance with the present invention. These analogs exhibit improved stability, as compared with the corresponding naturally occurring protein. In the case of the aFGF analog of the present invention, the analog exhibits enhanced stability and biological activity in the absence of heparin. In the case of the KGF analog of the present invention, the analog exhibits enhanced thermal stability.
• the analogs of the present invention have at least one different amino acid residue from the corresponding naturally occurring protein in or about the loop-forming sequence Asn-His-Tyr-Asn-Thr-Tyr found in the naturally occurring form.
• the loop forming sequence occurs in the area of about amino acid residues 92 to 96 (based on the numbering of the known amino acid sequence for bovine aFGF, as shown in Fig. 1) .
• the loop forming sequence occurs in the area of about amino acid residues 115-119, as shown in Figs. 9 and 10.
• the different amino acid(s) is selected for its higher loop-forming potential in order to stabilize this area of the analog.
• Amino acids having relatively high loop-forming potential include glycine, proline, tyrosine, aspartic acid, asparagine, and serine [Leszcynski et al .
• the analogs of the present invention may also optionally include an amino acid substitution for non-conserved cysteine residues (e . g.
• the analogs of the present invention which are expressed from E. coli host cells may include an initial methionine amino acid residue (i.e., at position -1, as shown in Fig. 1) .
• one or more of the terminal amino acid residues may be deleted from the DNA sequence, as is known to those skilled in the art, while substantially retaining the enhanced biological activity of the corresponding naturally occurring protein.
• DNA sequences coding for all or part of the analogs of the present invention are also provided. Such sequences preferably may include the incorporation of codons "preferred" for expression by selected E. coli host strains ("E. coli expression codons") , the provision of sites of cleavage by restriction endonuclease enzymes, and/or the provision of additional initial, terminal, or intermediate DNA sequences which facilitate construction of readily expressed vectors.
• E. coli expression codons selected E. coli host strains
• These novel DNA sequences include sequences useful in securing the expression of the analogs of the present invention in both eucaryotic and procaryotic host cells, such as E. coli .
• the DNA sequences of the present invention may comprise the DNA sequence set forth in Fig. 1, wherein at least one codon encoding an amino acid residue in the area of about amino acids 92 to 96 is replaced by a codon encoding a different amino acid residue having a higher loop-forming potential (hereinafter "aFGF analog sequence(s) " or “analog sequence(s) ”) , as well as a DNA sequence which hybridizes to one of the analog sequences or to fragments thereof, and, a DNA sequence which, but for the degeneracy of the genetic code, would hybridize to one of the analog sequences.
• aFGF analog sequence(s) or “analog sequence(s)
• the DNA sequences of the present invention may comprise the DNA sequence set forth in Fig. 10, wherein at least one codon encoding an amino acid residue in the area of about amino acids 115- 119 is replaced by a codon encoding a different amino acid residue having a higher loop-forming potential (hereinafter "KGF analog sequence(s) " or “analog sequence(s) ”) , as well as a DNA sequence which hybridizes to one of the analog sequences or to fragments thereof, and, a DNA sequence which, but for the degeneracy of the genetic code, would hybridize to one of the analog sequences.
• KGF analog sequence(s) or “analog sequence(s)
• the analogs of the present invention can be encoded, expressed, and purified by any one of a number of recombinant technology methods known to those skilled in the art.
• the preferred production method will vary depending upon many factors and considerations, including the cost and availability of materials and other economic considerations. The optimum production procedure for a given situation will be apparent to those skilled in the art through minimal experimentation.
• the analogs of the present invention can be expressed at particularly high levels using E. coli host cells, with the resulting expression product being subsequently purified to near homogeneity using procedures known in the art.
• a typical purification procedure involves first solubilizing the inclusion bodies containing the analogs, followed by ion exchange chromatography, then refolding of the protein, and finally, hydrophobic interaction chromatography.
• the analogs of the present invention exhibit a surprising degree of enhanced stability. Unlike naturally occurring aFGF, the aFGF analogs of the present invention demonstrate enhanced stability and biological activity in the absence of heparin. While it is known that more stable bFGF analogs can be obtained through the substitution of serine or other neutral amino acids in place of certain cysteine residues (for example, as disclosed in published PCT Patent Application No. 88/04189), substitution for the non- conserved cysteine residue at position 47 of naturally occurring bovine aFGF alone is not believed to be significant in enhancing the biological activity and/or stability of an aFGF analog.
• bovine [Ala 47 ,Gly93] a pcF analog of the present invention unlike the bovine [Ala 47 ] analog, exhibited enhanced stability, maintaining its original mitogenic activity in the absence of heparin over the course of 250 hours, while the bovine [Ala 47 ] analog rapidly lost activity.
• X-ray crystallography The X-ray crystallographic data obtained from examination of these crystals supports the suggestion from the hydrophobic interaction chromatography data that residue 93 is exposed to solvent, i.e., that the glycine for histidine substitution at position 93 makes the molecule less hydrophilic.
• Detailed examination of the bovine [Ala 47 ,Gly 3] aFGF analog sequence around residue 93 revealed a clustering of approximately 8 amino acids with high loop-forming potentials in the region from about the glutamic acid residue at position 90 to about the tyrosine residue at position 97.
• the KGF analogs of the present invention also demonstrate that the corresponding region in KGF is a solvent-exposed loop that may be involved in receptor binding.
• the LGlyll6]KGF analog of the present invention was found to exhibit an altered, decreased mitogenic activity compared to naturally occurring KGF, as set forth in the examples which follow.
• the [Glyll ⁇ ] KGF analog was also found to have 5-7°C higher thermal stability relative to naturally occurring KGF.
• amino acids are (in descending order of loop-forming potential) glycine, proline or tyrosine, aspartic acid or asparagine, serine, cysteine, glutamic acid, threonine, lysine, cystine, glutamine, arginine, phenylalanine, and tryptophan. Substitution of any of these residues for the histidine residue in the loop- forming sequence of the naturally occurring protein could be expected to result in an analog of the present invention having an enhanced stability.
• the present invention also contemplates the substitution of an amino acid having high loop-forming potential for other amino acid residues within the amino acid 92 to 96 region of naturally occurring aFGF (i.e., amino acids 92 and 94-96) and the amino acid 115 to 119 region of naturally occurring aFGF (i.e., amino acids 92 and 115-117-119) .
• the aFGF analogs of the present invention include, for example, aFGF analogs having the threonine residue at position 96 of naturally occurring aFGF replaced with glycine, proline or tyrosine, aspartic acid or asparagine, serine, or glutamic acid, in order of preference, although minimal enhancement of stability and/or biological activity would be expected with the substitution of glutamic acid for threonine, due to the similarity of loop-forming potential of these two amino acids.
• amino acid residues at positions 92, 94, 95, and 97 (asparagine, tyrosine, asparagine, and tyrosine, respectively) of naturally occurring aFGF have sufficiently high loop-forming potential such that minimal benefits are envisioned to arise from substitution for these particular residues.
• analogs of the present invention are seen to encompass analogs of both human and animal (e.g., bovine) origin, as well as all forms of a protein having the following loop-forming amino acid sequence:
• both the human and bovine forms of aFGF are known, and have been identified as having the identical amino acid sequence (shown above) at positions 92 to 96. Moreover, there is approximately 92% sequence identity between human and bovine aFGF, and a 97% "similarity" (i.e., 5% of the total 8% changes between the two aFGF forms are "conservative") . Both the human and bovine forms of naturally occurring aFGF exhibit substantially the same in vitro mitogenic activity.
• novel biologically active aFGF analogs of the present invention are particularly well suited for use in pharmaceutical formulations for the treatment by physicians and/or veterinarians of many types of wounds of mammalian species.
• the KGF analogs of the present invention because of their enhanced thermal stability, may also be well suited for use in pharmaceutical preparations.
• the amount of biologically active analog used in such treatments will, of course, depend upon the severity of the wound being treated, the route of administration chosen, and the specific activity or purity of the analog, and will be determined by the attending physician or veterinarian.
• the term "analog therapeutically effective" amount refers to the amount of analog determined to produce a therapeutic response in a mammal. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art.
• the analogs of the present invention may be administered by any route appropriate to the wound or condition being treated.
• Conditions which may be beneficially treated with therapeutic application(s) of the analog of the present invention include but are not limited to, the healing of surface wounds, bone healing, angiogenesis, nerve regeneration, and organ generation and/or regeneration.
• the formulations of the present invention comprise a therapeutically effective amount of analog together with one or more pharmaceutically acceptable carriers therefore and, optionally, other therapeutic ingredients.
• the carrier(s) must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not be deleterious to the recipient thereof.
• the formulations may be conveniently presented in unit dosage form and may be prepared by any of the methods well-known in the art. All methods include the step ⁇ f bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the analog with liquid carriers or finely divided solid carriers or both.
• a [Ala47.Gly93] a FGF analog and a [Glyll6] KGF analog were made using site-directed mutagenesis. It will be appreciated, however, that these and other analogs of the present invention can be made by other methods, including chemical synthesis.
• a bovine sequence was used in the case of the [Ala47-Gly93] aFGF analog. Specifically, the [Ala47-Gly93] aFGF analog was prepared as follows:
• a bovine aFGF analog according to the present invention was prepared and examined in the following examples.
• This analog bovine [Ala 7 ,Gly93] aFGF, was constructed to contain both a desired amino acid substitution (glycine for histidine at position 93) in the residue 92 to 96 loop-forming sequence of the aFGF molecule and an additional amino acid substitution of alanine for the non-conserved cysteine residue at position 47, as shown in Fig. 1.
• a bovine [Ala 47 ] aFGF analog having only the amino acid substitution of alanine for cysteine, was also prepared for use as a control for the desired bovine [Ala 47 ,Gly93] aFGF analog.
• a synthetic gene coding for the [Ala47-Gly93] analog of bovine aFGF was assembled in two sections from a total of 28 component oligonucleotides.
• the amino acid sequence of Gimenez-Gallego et al . (1985), Science, 230:1385-1388 was used as the basis for this gene, with codon choices selected to optimize expression of the analog in E. coli (Gimenez-Gallego et al . (1985), supra) .
• Section I was assembled from 16 oligonucleotides to yield a 287 nucleotide fragment which could be inserted into a plasmid vector at Xba I and Xho I restriction endonuclease sites.
• Section II was assembled from 12 oligonucleotides to give a 170 nucleotide fragment bounded by Xho I and Bam HI compatible ends. The two sections were inserted into the expression plasmid pCFM1156, which had been previously digested with Xba I and Bam HI in a 3- component ligation, yielding the complete aFGF gene under the control of the lambda pL promoter.
• the plasmid pCFM1156 is prepared from the known plasmid pCFM836.
• the preparation of plasmid pCFM836 is described in U.S. Patent No. 4,710,473/ the relevant portions of the specification, particularly examples 1 to 7, are hereby incorporated by reference.
• the resulting plasmid is then digested with Cla I and Kpn I and the excised DNA fragment is replaced with a DNA oligonucleotide of the following sequence: 5'-CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC-3' 3'-TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC-5'
• E. coli cells transformed with this plasmid were grown in a 16-liter fermentation vessel (as described in Fox et al (1988), J. Biol . Chem . , 20:18452-18458.
• the gene coding for the bovine [Gly93,Ala 47 ] aFGF was converted to the [Ala 47 ] form using oligo site- directed mutagenesis.
• the aFGF gene was first transferred into the phage vector Ml3n.pl8, and single- stranded DNA to serve as a template for the mutagenesis reaction was prepared. Approximately 0.5 ⁇ g of this DNA was mixed with 5 picomoles each of the mutagenic primer
• the annealed template-primer was mixed with ATP, a dNTP mixture, DNA polymerase I large fragment, and T4 DNA ligase, then incubated at 15°C for 4 hours. Aliquots of this reaction mixture were added to competent E. coli JM101 cells and plated in 0.7% L-agar. The resulting plaques were replicated onto nitrocellulose filters and the filters were hybridized with 32p-iabeled mutagenic primer. DNA prepared from phage which hybridized was sequenced to verify successful completion of the desired mutagenesis event. The resultant gene was then transferred back to the pCFM1156 vector for expression of the recombinant protein.
• Both the bovine [Gly 3,Ala 47 ] and [Ala 4 7] aFGF analogs were purified from the insoluble fraction obtained from centrifugation of mechanically lysed E. coli cells expressing the recombinant protein.
• the pellet fraction was solubilized in 8 M urea, 0.1 M glycine, pH 2.5, and centrifuged to remove insoluble materials.
• the supernatant was loaded onto an S- Sepharose* (Pharmacia, Uppsala, Sweden) column equilibrated with 6 M urea, 10 mM glycine, pH 3.0, and washed with 6 M urea, 20 mM sodium citrate, pH 6.5.
• Proteins which bound to the column were eluted with a linear 0 to 0.5 M sodium chloride gradient in 20 mM sodium citrate, pH 6.5.
• the fractions containing the aFGF were pooled, diluted 20-fold with 20 mM sodium citrate, 0.1 M ammonium sulfate, and centrifuged to remove any precipitate.
• the supernatant was mixed with one volume of 20 mM sodium citrate, 2 M ammonium sulfate, and loaded onto a phenyl-Sepharose* column equilibrated with 20 mM sodium citrate, 1 M ammonium sulfate, pH 6.5.
• the bound proteins were eluted from the column with a linear descending gradient (1 M to 0 M) of ammonium sulfate.
• the aFGF-containing fractions were pooled and dialyzed against 20 mM sodium citrate, pH 6.5. This product was essentially homogeneous, as demonstrated by the fact that no other bands in Coomassie blue appeared in the SDS gel, as shown in Fig. 4.
• Hydrophobic interaction chromatography was performed at room temperature using a phenyl-Superose* column on a Pharmacia FPLC system.
• the sample in 2 M ammonium sulfate, 20 mM sodium citrate, pH 6.5, was loaded onto the column which had been equilibrated with 2 M ammonium sulfate. After a 2 M ammonium sulfate wash, the remaining protein was eluted with an ammonium sulfate gradient descending from 2 M to 0 M, followed by a final wash with 20 mM sodium citrate, pH 6.5.
• Fig. 3 presents the elution profiles for the bovine [Ala 47 ] and [Ala 7 ,Gly93] a FGF analogs.
• the [Ala 47 ] aFGF showed a major peak eluting at 0.25 M ammonium sulfate, while the [Ala 47 ,Gly93] aFGF analog showed a single peak at 0.13 M ammonium sulfate, suggesting that both proteins exist primarily in a single distinct conformation.
• Circular dichroic spectra were determined at room temperature on a Jasco Model J-500C spectropolarimeter (Jasco, Tokyo, Japan) equipped with an Oki If 800 Model 30 computer (Oki, Tokyo, Japan) . Measurements were carried out at a band width of 1 nm using cuvettes of
• the thermal transition of proteins was determined on a Response II spectrophotometer (Gilford, Medfield, Massachusetts) equipped with thermal programming and a thermal cuvette holder. Samples were heated at an increment of 0.1°C/min or 0.5°C/min and their absorbance monitored at 287 nm. Protein concentrations were determined spectrophotometrically using an extinction coefficient of 0.98 for bFGF and 1.04 for both bovine aFGF analogs at 280 nm for 0.1% protein.
• FTIR Fourier-transform infrared
• Second derivative spectra were calculated (as described in Susi et al (1988), Biochem. Biophys . Res . Comm . , 115:391-397) .
• a 9-point smoothing function was applied to the water vapor-subtracted spectra.
• the frequencies of the component bands in this region are related to secondary structure content.
• the spectra show strong bands at 1630 and 1685 cirri which are indicative of a significant amount of ⁇ -structures in the two proteins.
• a strong band near 1647 cm ⁇ l is indicative of the presence of irregular or disordered structures.
• the weaker peaks near 1666 and 1673 cm-l arise from turn structures.
• a small peak is present near 1651 cm ⁇ l in the spectra of both proteins.
• Amide I' components near this frequency are typically assigned to ⁇ -helices.
• Heparin-Sepharose* (Pharmacia) was packed into a 1 x 8 cm column and equilibrated with 10 mM Tris-HCl, pH 7.2. The column was loaded, washed with 10 mM Tris-HCl, pH 7.2 and eluted with a linear gradient from 0 to 2.8 M sodium chloride in the same buffer at a flow rate of 0.5 ml/min using a Pharmacia FPLC system.
• Acidic and basic FGF are distinguished by their avid binding to heparin and heparin-like molecules. Both the bovine [Ala 47 ,Gly93] and [Ala47] aFGF analogs showed a single peak eluting at 1.54 M sodium chloride in 10 mM Tris-HCl, pH 7.2.
• NIH 3T3 cells were obtained from ATCC. The cells were grown in DME supplemented with 10% calf serum,
• cells were pulsed for 1 hour with 1 ml of the above media containing 2-10 ⁇ Ci of tritiated thymidine at 37°C. After the pulse, cells were washed once with media, then 250 mM sucrose, 10 mM sodium phosphate, 1 mM EDTA, pH 8 was added and the plates incubated at 37°C for 10 minutes to release the cells. Cells were harvested on a Skatron harvester (Skatron, Inc., Sterling, Virginia) . Filters were dried, placed in scintillation fluid, and counted in a Beckman scintillation counter (Beckman Instruments, Inc., Fullerton, California).
• the mitogenic activity of the bovine [Ala47,Gly93] a nd [Ala 47 ] aFGF analogs on NIH 3T3 cells was examined as shown in Fig. 6.
• the [Ala 47 ] aFGF analog produced a dose dependent stimulation of 3H-thymidine uptake in the range of 1 to 100 ng/ml, with half-maximal stimulation of 25 ng/ml.
• the [Ala 47 ,Gly93] aFGF analog was able to produce the same mitogenic effect at a much lower protein concentration, the half-maximal dose being about 1 ng/ml.
• Recombinant bFGF was 4-5 times more potent than the [Ala 47 ,Gly93] aFGF, with a half-maximal dose of 220 pg/ml.
• aFGF analog remaining more potent.
• the activities were enhanced such that the dose response of all three molecules were nearly identical, with a half-maximal dose of 90 pg/ml.
• Crystals of bovine [Ala 47 ,Gly93] aFGF analog were grown by vapor diffusion against 0.2 M NH4SO4, 2 M NaCl, 0.099 M sodium citrate, and 0.02 M sodium potassium phosphate, pH 5.6.
• the protein droplet contained equal volumes of the reservoir solution and a 10 mg/ml protein solution.
• Intensity data were collected with a Siemens (Madison, Wisconsin) multiwire area detector mounted on an 18 kw rotating anode generator. The Siemens suite of processing programs was used for data reduction.
• mir phases were calculated to 3 A resolution from two derivatives, with a figure of merit of 0.68. After solvent flattening, regions corresponding to two independent aFGF molecules in the asymmetric unit were identified. The general non- crystallographic symmetry relationships between these molecules were determined from rotation function, real- space translation function, and density correlation studies. A molecular envelope was defined around an averaged aFGF molecule with a modified B.C. Wang algorithm. The phases were iteratively refined by molecular averaging and solvent flattening.
• the coding sequence for naturally occurring KGF was obtained using RNA isolated from human fibroblast cells (cell line AG1523) as a starting material from which to make cDNA for KGF using standard techniques known in the art.
• the KGF cDNA was then used as a template in polymerase chain reactions (PCR) to amplify the KGF gene. Due to the presence of an internal Ndel site in the KGF gene, the PCR D ⁇ A was made as two fragments that were then linked together at a unique BS ⁇ II site. Oligonucleotides 238-21 and 238-24 (shown below) were used to make a D ⁇ A product that was subsequently cut with BsmI and BamHI and then isolated to yield a 188 base pair fragment of KGF.
• Oligonucleotides 238-22 and 238-24 were used to make a D ⁇ A product that was subsequently cut with Ndel and BsmI to yield a 311 base pair fragment of KGF.
• the two D ⁇ A fragments, when ligated together create the gene for naturally occurring KGF, shown in Fig. 9.
• oligonucleotides corresponding to KGF DNA regions 5*- and 3'- of the site-directed mutational change could be used to provide the outside primers for the overlapping mutagenesis PCR.
• An EcoRI to Ba/nHI DNA fragment containing the [Glyll ⁇ ] KGF analog coding sequence was then ligated into the previously described expression plasmid pCFM1156 already containing the KGF gene, so as to replace the corresponding region of the KGF gene with a region of the coding sequence containing the necessary changes to encode the [Glyll ⁇ ] KGF analog (Fig. 10) .
• the ligation DNA was then transformed into an FM5 (ATCC# 53911) host and colonies were isolated that contained the pCFM1156 [Glyll ⁇ ] KGF analog plasmid.
• the FM5/pCFM1156 KGF [GlyH ⁇ ] KGF analog strain was then fermented and cell paste harvested using standard fermentation techniques.
• Oligo 238-21 5'-ACAACGCGTGCAATGACATGACTCCA-3'
• Oligo 238-22 5'-ACACATATGTGCAATGACATGACTCCA-3'
• Oligo 238-24 5'-ACAGGATCCTATTAAGTTATTGCCATAGGAA-3'
• Oligo 315-17 5'-GGAAAACGGTTACAACACATATGCA-3'
• Oligo 315-18 5'-GTGTTGTAACCGTTTTCCAGAATTAG-3'
• the cells containing the [Glyl 16 ] KGF analog from the fermentation described above were first broken by suspending 665 g of the E. coli cell paste in ca . 4 L 20 mM sodium phosphate, pH 6.8, 0.2 M NaCl and then passing the suspension 3 times through a Gaulin Homogenizer at 9,000 psi. The suspension was then centrifuged in a Beckman JA-10 rotor (Beckman Instruments, Fullerton, California) at 10,000 rpm, for 30 minutes at 4°C.
• Ion exchange chromatography was performed by applying supernatant from the centrifuged suspension to an S-Sepharose* Fast Flow (Pharmacia, Uppsala, Sweden) column (5 x 23 cm., 450 ml total volume) equilibrated with 20 mM sodium phosphate, pH 7.5, 0.2 M NaCl, at a flow rate of 25 ml/min. The column was then washed with 2 L of 20 mM sodium phosphate, pH 7.5, 0.4 M NaCl, and the [Glyll6] KGF analog eluted with a linear gradient of 0.4 M to 0.6 M NaCl in 20 mM sodium phosphate, pH 7.5. Total gradient volume was 7 L (about 16 times column volume) . Fractions containing KGF were pooled and concentrated about 22-fold over a YM*-10 membrane (Amicon) in a 400 ml stirred cell.
• Size exclusion chromatography was performed by applying half of the volume of the concentrated KGF (total volume of 80 ml) obtained from ion exchange chromatography to a Sephadex ⁇ G-75
• a second ion exchange chromatography procedure was then performed by first pooling SEC fractions corresponding to the monomeric form of [GlyH6] KGF analog, then diluting the pooled fractions with five volumes of 20 mM sodium phosphate, pH 6.8, 0.2 M NaCl and applying the diluted fractions to an S-Sepharose* Fast Flow (Pharmacia) column (5 x 23 cm., 450 ml total volume) equilibrated with 20 mM sodium phosphate, pH 6.8, 0.4 M NaCl. This column was then washed with about 1.5 L of 20 mM sodium phosphate, pH 6.8, 0.4 M NaCl.
• the purified [Gly 16 ] KGF analog was eluted with a linear gradient of 0.4 M to 0.6 M NaCl in 20 mM sodium phosphate, pH 6.8. Total gradient volume was 10 L (about 22 times column volume) . Samples containing the [Glyll6] KGF analog, as ascertained by SDS-PAGE, were pooled and the KGF content was determined by UV absorption.
• the pD values were determined by adding 0.45 to the pH reading from a glass electrode pH meter, according to Covington et al . (1968), Anal. Chem. , U2:700-706. The final protein concentration was 30 mg/ml. Protein solutions were placed in IR cells with CaF2 windows and 100 ⁇ M Teflon spacers.
• the melting temperature, T m , for naturally occurring KGF was estimated to be 60°C, while the T for the [Glyll ⁇ ] KGF analog was estimated to be 65°C, 5°C higher than that of naturally occurring KGF, indicating that the [GlyllG] KGF analog has a higher relative thermal stability than naturally occurring KGF.
• MOLECULE TYPE cDNA
• SEQUENCE DESCRIPTION SEQ ID NO:1 :
• AACCTAAGCT TCTGTACTGC TCTAACGGCG GTTACTTCCT GCGCATTCTC CCGGATGGCA 120
• MOLECULE TYPE protein
• Lys Ala lie Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140
• MOLECULE TYPE cDNA
• SEQUENCE DESCRIPTION SEQ ID NO: 4 :
• MOLECULE TYPE protein
• MOLECULE TYPE cDNA
• cDNA SEQUENCE DESCRIPTION: SEQ ID NO: 13: ACAACGCGTG CAATGACATG ACTCCA 26
• MOLECULE TYPE cDNA
• cDNA SEQUENCE DESCRIPTION: SEQ ID NO: 14: ACACATATGT GCAATGACAT GACTCCA 27

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