MXPA97002481A

MXPA97002481A - Method for the purification of growth factors of queratinoci

Info

Publication number: MXPA97002481A
Application number: MXPA/A/1997/002481A
Authority: MX
Inventors: W Hsu Eric; C Kenney William; Tressel Tim
Original assignee: Amgen Inc; W Hsu Eric; C Kenney William; Tressel Tim
Priority date: 1994-10-13
Filing date: 1997-04-04
Publication date: 1997-12-01

Abstract

The present invention relates to a method for purifying a keratinocyte growth factor (KGF), the method is characterized in that it comprises the following steps: a) obtaining a solution containing the KGF, b) loading the solution from step a) onto a cation exchange resin, c) eluting the KGF in an eluate solution of the cation exchange resin, d) passing the solution from step c) through either (i) a molecular weight exclusion matrix, or ( ii) a hydrophobic interaction chromatography matrix and e) recover the KGF, with the proviso that the method does not comprise a step of affinity chromatography to hepari

Description

METHOD FOR PURIFYING KERATINOCYTE GROWTH FACTORS FIELD OF THE INVENTION The present invention relates to the field of protein purification. Specifically, the present invention relates to the field of purification of keratinocyte growth factors.

BACKGROUND OF THE INVENTION Polypeptide growth factors are important mediators of intercellular communication (Rubin et al. (1989), Proc. Nati. Acad. Sci. USA, 82: 802-806). These molecules are generally released by a cell type and have an effect influencing the proliferation of other cell types. A family of growth factors is formed by fibroblast growth factors (FGF-fibroblast growth factors). At present, eight members of the FGF family are known, which show an affinity between their primary structures: the fibroblast growth factor REF: 2í »í» 06 basic, bFGF (Abraham et al. (1986), EMBO J., 5: 2523-2528); the acid fibroblast growth factor, aFGF (Jaye et al. (1986), Science, 233: 541-545); the product of the int-2 gene, int-2 (Dickson &Peters (1987), Nature, 326: 833); the hst / kFGF (Delli-Bovi et al. (1987), Cell, 5_0: 729-737 and Yoshida et al. (1987). Proc.

Nati Acad. Sci. USA, 8_4: 7305-7309); FGF 5 (Zhan et al. (1988), Mol. Cell Biol., 8 ^ 3487-3495); the FGF-6 (Marics et al. (1989), Oncogene, _4: 335-340); the keratinocyte growth factor (Finch et al (1989), Science, 2_4: 752-755; Rubin et al. (1989), Proc. Nati. Acad. Sci. USA, 8_6: 802-806; Ron et al. (1993), The Journal of Biological Chemistry, 268 (4): 2984-2988, and Yan et al (1991), In Vitro Cell, Dev. Biol., 27A: 437-438); and hisactofilin (Habazzettl et al. (1992), Nature, 359: 855-858). Among the FGF family of proteins, keratinocyte growth factor (KGF) is a unique effector for the proliferation of non-fibroblast epithelial cells (particularly keratinocytes) derived from skeletal tissues. The term "native KGF" refers to the natural human polypeptide (hKGF) or the recombinant polypeptide (rKGF) (with or without a signal sequence) as represented by the amino acid sequence presented in SEQ. FROM IDENT. NO: 2 or an allelic variant thereof. [Unless otherwise indicated, the amino acid numbering for the molecules described herein must correspond to that presented for the mature form of the native molecule (ie, minus the signal sequence), as represented by the amino acids 32 to 194 of the SEC. FROM IDENT. NO: 2.]. Native KGF can be isolated naturally. For example, hKGF can be isolated from a medium conditioned with an embryonic lung fibroblast cell line (Rubin et al (1989), supra.) To obtain a purified hKGF preparation, three chromatographic steps, called affinity chromatography, were used. heparin-Sepharose ™ (Pharmacia, Piscataway, NJ), HPLC gel filtration and reverse-phase HPLC: 10 liters of conditioned medium recovered approximately 6 mg of hKGF These chromatographic steps only recovered 0.8% of the total amount of hKGF detected by a mitogenic activity assay A broader example shows the use of another chromatographic step using heparin-Sepharose MR affinity chromatography and Mono-SMR ion exchange chromatography (Pharmacia, Piscataway, NJ) for the isolation of rKGF produced in bacteria (Ron et al. (1993), Journal of Biological Chemistry, 268: 2984-2988) The properties of kerati growth factors nocytes suggest a potential for the application of these as a drug to promote specific stimulation of epithelial cell growth. Accordingly, it would be desirable to develop a method or methods for obtaining relatively high levels of homogeneous keratinocyte growth factors in order to provide sufficient amounts of material for a biological evaluation understandable in vitro and in vivo and for a potential therapeutic application. The aim of the present invention is to provide a new method for the purification of keratinocyte growth factors.

BRIEF DESCRIPTION OF THE INVENTION The present invention is directed to a first method for purifying keratinocyte growth factor (KGF), this method comprises the following steps: a) obtaining a solution containing KGF; b) the bond of the KGF of the solution of point (a) with a cation exchange resin; c) the elution of KGF, in an eluate solution, from the cation exchange resin; d) passing the eluate solution from point (c) through a molecular weight exclusion matrix; and e) the recovery of KGF from the molecular weight exclusion matrix.

In addition, the invention is directed to a second method for purifying keratinocyte growth factor (KGF), which comprises the following steps: a) obtaining a solution containing KGF; b) the KGF link of the point solution (a) to a cation exchange resin; c) the elution of KGF, in an eluate solution, from the cation exchange resin; d) carrying out the hydrophobic interaction chromatography in the eluate solution of point (c); and e) the recovery of the KGF from the step of the hydrophobic interaction chromatography of point (d). In general, the step of cation exchange chromatography of the first method or the second method can be carried out with any suitable buffer (eg, phosphate buffer, sodium acetate or tris-HCL) preferably at a pH between 6.8-7.5. . Suitable columns for use in this step include carboxymethyl cellulose, carboxymethyl agarose and sulfated agarose, and cellulose columns (eg, S-Sepharose Fast Flow ™ resin columns, Mono-SMR resin columns, and resin columns). CM-cellulose ™, commercially distributed by Pharmacia, Piscataway, NJ). The flow velocity will vary depending on the size of the column. The gel filtration step of the first method can be carried out in any suitable buffer (eg, phosphate buffer) at a pH of preferably between 7.0 and 7.5. Suitable columns for use in this step include size exclusion columns based on agarose, acrylated, silica based, or polymer based (eg, Sephadex G-75MR resin columns and resin columns). of Superdex-75MR, commercially distributed by Pharmacia). In a particularly preferred embodiment of the second method, the free sulfhydryl groups can be oxidized before the passage of the hydrophobic interaction, which is discussed below. Any form of oxidation can be used. For example, the protein can be exposed to atmospheric oxygen for an adequate period of time. Alternatively, various oxidation processes may be used. Such a method is particularly suitable for keratinocyte growth factors wherein one or more cysteine residues, when compared to the native KGF molecule, are deleted or replaced. In this process the oxidizing agent (for example, cystamine dihydrochloride or other suitable oxidizing agent, for example, cystine, oxidized glutathione or divalent copper) can be added to the final concentration, adjusting the pH preferably between about 7 - 9.5, being more preferably a pH of 9.0 + 0.3 ° C when cystamine dihydrochloride is used, and maintaining the temperature preferably between about 10-30 ° C, for a suitable period of time. The second procedure can be used to oxidize native KGF and other keratinocyte growth factors with similar patterns of cysteine residues. In this procedure, the oxidation can be completed by the addition of an appropriate amount of an ionic strength modifier, (e.g., (NH4) 2S04), by adjusting the pH preferably to about 7.5-9.5, and maintaining the temperature of preference between approximately 23 + 5 ° C for an adequate period. The step of the hydrophobic interaction of the second method can be carried out using any suitable buffer (e.g., sodium phosphate) at a pH preferably at about 6.0-8.0, more preferably at about 7.0 and eluting with a gradient of decreasing linear (NH) 2S? 4 ranging from 2-0 M to the columns suitable for use in this step include substituted alkyl or phenyl resins (eg, a Butyl-650M Toyopearl ™ resin column, commercially available from Tosohaas, Inc., Montgomerville, PA and phenyl Sepharose ™ and phenyl Superose ™ resin columns, commercially available from Pharmacia).

The process of the present invention can be used to purify KGF. Thus, it is to be understood that the terms "keratinocyte growth factor" and "KGF" as used in this disclosure are intended to include, and to mean interchangeably unless otherwise indicated, native KGF and proteins. KGF analogs (or "muteins") characterized by a peptide sequence, substantially equal to the peptide sequence of native KGF and which retains some or all of the biological activity of native KGF, particularly the proliferation of non-fibroblastic epithelial cells (e.g. a large stimulation of the BALB / MK keratinocyte cells approximately 500 times greater than that of the NIH / 3T3 fibroblast cells, and a greater stimulation of the BALB / MK keratinocyte cells of approximately 50 times greater than that of the epithelial cells BS / 589 or the one presenting the epithelial cells CC1208, which was determined by the incorporation of and H-thymidine). The phrase "characterized by a peptide sequence, substantially equal to the peptide sequence of native KGF" means a peptide sequence which is encoded by a DNA sequence capable of hybridizing at nucleotides 201 to 684 of SEQ. FROM IDENT. N0: 1, preferably under stringent hybridization conditions. The determination of the position of the corresponding amino acid between two amino acid sequences can be determined by aligning two sequences to maximize the alignment of the residues including the displacement of the amino terminal end and / or the carboxy terminal end, introducing the required cuts and / or deleting the residues present as inserts in the candidate. Database studies, sequential analysis and manipulations can be performed using one of the well-known and routinely used sequential homology programs / identity scanning algorithms (eg, Pearson and Lipman (1988), Proc.

Nati Acad. Sci. U.S.A., 85: 2444-2448; Altschul et al. (1990), J. Mol. Biol., 215: 403-410; Lipman and Pearson (1985), Science, 222: 1435 or Devereux et al. (1984), Nuc. Acids Res., 12: 387-395). The stringent conditions, within the context of hybridization, will be conditions rigorously combined with salinity, temperature, organic solvents and other parameters typically controlled in hybridization reactions. The rigorously exemplary hybridization conditions are hybridization in 4 X SSC at 62-67 ° C, followed by a wash in 0.1 X SSC at 62-67 ° C for about one hour. Alternatively, the rigorously exemplary hybridization conditions are hybridization in 45-55% formamide, 4 X SSC at 40-45 ° C. [See, T. Maniatis et al., Molecular Cloning (A Laboratory Manual); Cold Spring Harbor Laboratory (1982), pgs. 387 to 389]. Accordingly, the proteins include allelic variations, or deletion (s), substitution (s) or insertion (s) of amino acids, including fragments, chimeric or hybrid molecules of native KGF. An example of KGF includes proteins that have residues corresponding to Cys1 and Cys15 of the SEC DE IDENT. NO: 2 replaced or deleted, resulting in a molecule that exhibits improved stability when compared to the original molecule (as is usually shown in U.S. Patent Application Serial No. 08 / 487,825, filed July 7, 1995 , of common property). Another example of KGF includes charge-change polypeptides wherein one or more amino acid residues 41-154 of the native KGF (preferably residues Arg41, Gln43, Lys55, Lys95, Lys1a, Asn137, Gln138, Lys139, Arg144, Lys147, Gln152 , Lys153 or Thr154) are deleted or substituted with a neutral residue or with a negatively charged residue selected for the protein to have an effect with a reduced positive charge (as is usually shown in U.S. Patent Application Serial No. 08/323, 337, common property, registered on October 13, 1994). An even larger example of KGF includes proteins generated by substitution of at least one amino acid having a higher loop-forming potential of at least one amino acid and with a loop-forming region of Asn115-His116-Tyr117- Asn118 ~ Thr119 of native KGF as usually shown in co-owned US Patent Application Serial No. 08 / 323,473, filed October 13, 1994). An even broader example includes proteins having one or more amino acid substitutions, deletions or additions of a region of 123-133 (amino acids 154-164 of SEQ ID NO: 2) of native KGF; these proteins can have an agonistic or antagonistic activity. The specifically described proteins include the following KGF molecules (referring to the residue found at that position in the mature protein (minus the signal sequence) fourth point in SEQ ID NO: 2, followed by that amino acid position in the parentheses and then either the substituted residue or "-" to designate the deletion): C (1,15) S,? N15-? N24,? N3 / C (15) S,? N3 / C (15) -, ? N8 / C (15) S,? N8 / C (15) -, C (1, 15) S / R (144) E, C (1,15) S / R (144) Q,? N23 / R (144) Q, C (1,15,40) S, C (1, 15, 102) S, C (1, 15,102,106) S,? N23 / (137) E,? N23 / K (139) E, ? N23 / K (139) Q,? N23 / R (144) A,? N23 / R (144) E,? N23 / R (144) L,? N23 / K (147) E,? N23 / K ( 147) Q,? N23 / K (153) E,? N23 / K (153) Q,? N23 / Q (152) E / K (153) E; R (144) Q and H (116) G. Those skilled in the art will also appreciate that a variety of vector-host systems can be used to express the sequence of the KGF coding protein. This includes, but is not limited to, eukaryotic cell systems such as systems of mammalian cells infected with viruses (eg, vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (for example, Baculovirus); to microorganisms such as yeast vectors containing yeast; or to prokaryotic cell systems such as bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA. The expression elements of these vectors vary in their length and specificity. Depending on the vector-host system used, any number of translation elements and suitable transcription can be used. Once the protein product of KGF expression has been isolated, purified and tested for its KGF activity (using methods known to the person skilled in the art), it can be formulated in a wide variety of pharmaceutical compositions. Typically, such compositions include a suitable carrier or excipient, chemically defined, for the therapeutic agent and, depending on the desired form of administration, also includes other ingredients. The composition may include aqueous carriers or be composed of solid phase formulations in which KGF is incorporated into non-aqueous carriers such as collagens, hyaluronic acid, and various polymers. The composition can be suitably formulated to be administered in a variety of ways including by injection, oral, topical, intranasal and pulmonary deliberation routes.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) and the amino acid sequence (SEQ ID NO: 2) of the native KGF (the nucleotides coding for the mature form of the native KGF are represented by bases 201 to 684 of the SECTION ID NO: 1 and the mature form of KGF is represented by amino acid residues 32 to 194 of SEQ ID NO: 2). Figures 2A, 2B and 2C show the maps of the plasmids pCFM1156, pCFM1656 and pCFM3102, respectively. Figure 3 shows the nucleotide sequence (SEQ ID NO: 3) and the amino acid sequence (SEQ ID NO: 4) of the RSH-KGF construct. Figure 4 shows the nucleotide sequence (SEQ ID NO: 5) and the amino acid sequence (SEQ ID NO: 6) of the construct contained in the KGF of the plasmid. Figure 5 shows the chemically synthesized OLIGOs (OLIGO # 6 through OLIGO # 11; ID SEQ ID NO: 12-17, respectively) used to replace the DNA sequence between the Kpnl site and the EcoRI site for the Kpnl site (from the amino acid positions 46 to 85 of SEQ ID NO: 6) in the construct containing the KGF of the plasmid to produce the construction in the KGF (dsd) of the plasmid. Figure 6 shows the chemically synthesized OLIGOs (OLIGO # 12 to OLIGO # 24; ID SEQ ID NO: 18-30, respectively) used to construct the KGF (optimized codon). Figure 7 shows the nucleotide sequence (SEQ ID NO: 31) and the amino acid sequence (SEQ ID NO: 32) of C (1,15) S, analogous to KGF having serine substitutions for cysteine at amino acid positions 1 and 15 of the native KGF. Figure 8 shows the nucleotide sequence (SEQ ID NO: 33) and the. amino acid sequence (SEQ ID NO: 34) of C (1, 15) S / R (144) E, analogous to KGF having serine substitutions for cysteine at amino acid positions 1 and 15 and a substitution of glutamic acid by arginine at the position of amino acid 144 of the native KGF. Figure 9 shows the nucleotide sequence (SEQ ID NO: 35) and the amino acid sequence (SEQ ID NO: 36) of C (1, 15) S / R (144) Q, analogous to KGF which has serine substitutions by cysteine at the positions of amino acids 1 and 15 and a substitution of glutamine by arginine at the position of amino acid 144 of the native KGF. Figure 10 shows the nucleotide sequence (SEQ ID NO: 37) and the amino acid sequence (SEQ ID NO: 38) of ΔN15, analogous to KGF having a deletion of the first 15 amino acids of the end N-terminal of the native KGF. Figure 11 shows the nucleotide sequence (SEQ ID NO: 39) and the amino acid sequence (SEQ ID NO: 40) of? N23, analogous to KGF having a deletion of the first 23 amino acids of the N-terminus of native KGF. Figure 12 shows the nucleotide sequence (SEQ ID NO: 41) and the amino acid sequence (SEQ ID NO: 42) of N23 / R (144) Q, analogous to KGF having a deletion of the first 23 amino acids of the N-terminal end and a substitution of glutamine by arginine at the position of amino acid 144 of the native KGF DESCRIPTION OF THE SPECIFIC MODALITIES Normal methods for many of the methods described in the following examples suitable for alternative procedures are widely provided in recognized molecular biology manuals such as, for example, Molecular Cloning, Second Edition, Sambrook et al., Cold Spring Harbor Laboratory Press ( 1987) and Current Protocols in Molecular Biology, Ausabel et al., Greene Publishing Associates / iley-Interscience, New York (1990).

Example 1: Preparation of DNA encoding KGF and KGF analogs.

Complete cloning of the human KGF gene (encoding a polypeptide with the native KGF sequence) was performed by both polymerase chain reaction (PCR) of the RNA from animal cells and by PCR from oligonucleotides ("OLIGOs") chemically synthesized (codon optimized of E. coli). Both methods are described below: PCR amplification using RNA isolated from cells known to produce the polypeptide was modified. Initially, the cell line from human fibroblast cells AG1523A (obtained from the Human Genetic Mutant Cell Culture Repository Institute for Medical Research, Camden, New Jersey) was disrupted with guanidium thiocyanate, followed by extraction (according to the method of Chomyzinski et al. , (1987), Anal. Biochem., 172: 156). Using a normal reverse transcriptase protocol for total RNA, the KGF cDNA was generated. PCR amplification (PCR # 1) of the KGF gene was carried out using the KGF cDNA as an annealed and the 0LIG0 # 1 and 0LIG0 # 2 of the primers, which immediately encode the 5 'DNA sequences and 3 'of the KGF gene [model 9600 Thermocycler (Perkin Elmer Cetus, Norwalk, CT); 28 cycles; each cycle consists of one minute at 94 ° C for the denaturation, two minutes at 60 ° C for the union of the single chains (anneling), and three minutes at 72 ° C for the elongation]. A small aliquot of the PCR # 1 product was then used as an annealing for a second KGF PCR (PCR # 2), an identical amplification to the cycle conditions described above with the exception of the annealing temperature which was 50 ° C. For the cloning of expression of the KGF gene, the PCR primers were incubated to create convenient restriction sites at both ends of the KGF gene. OLIGO # 3 and OLIGO # 4 were used to modify the KGF DNA product of PCR # 2 to include the Mlul and BamHI restriction sites at the 5 'and 3' ends of the gene respectively [PCR # 3; 30 cycles; each cycle consists of one minute at 94 ° C for denaturation, two minutes at 60 ° C for single chain binding, and three minutes at 72 ° C for elongation]. This DNA was subsequently cut with Mlul and Ba HI, extracted with phenol and precipitated with ethanol. Later it was resuspended and bound (using T4 ligase) in a plasmid pCFM1156 (Figure 2A) which contained an "RSH" signal sequence to make the RSH-KGF construct (Figure 3). The ligation products were transformed (according to the method of Hanahan (1983), J. Mol. Biol., 166: 557) into E. coli strain FM5 (ATCC: 53911) and plated onto LB + kanamycin at 28 ° C. Several transformants were selected and grown in small liquid cultures containing 20 μg / ml kanamycin. The plasmid RSH-KGF was isolated from the cells of each culture and the DNA was sequenced. Due to the internal Ndel site in the KGF gene, it was not possible to directly clone the native gene sequence into the desired expression vector with the Ndel and BamHI-bound restriction sites. This was completed as a three-step link. The plasmid RSH-KGF was cut at the unique restriction sites of Bsml and SstI, and the fragment of ~3 kbp of DNA (containing the 3 'end of the KGF gene) was isolated following an electrophoresis through an agarose gel. at 1%. PCR was carried out (PCR # 4) as described for PCR # 3 except that 0LIG0 # 5 was replaced by OLIGO # 3. The PCR DNA product was subsequently cut with Ndel and Bsml and a DNA fragment of 311 bp was isolated following an electrophoresis on a 4% agarose gel. The third fragment used in the ligation was a DNA fragment of 1.8 kbp of plasmid pCFM1156 cut with Ndel and SstI isolated following an electrophoresis through a 1% agarose gel. Following a ligation (with T4 ligase), a transformation, a selection with kanamycin and DNA sequencing as described above, a clone was obtained containing the construction of Figure 4, and the plasmid designated KGF. Due to an internal ribosomal binding site which produced truncated products, the DNA sequence between the KGF between the unique Kpnl and EcoRI sites was replaced with chemically synthesized OLIGOs (from 0LIG0 # 6 to OLIGO # ll) to minimize the use of the internal start site (Figure 5). 0LIG0 # 1 (IDENTIFICATION SECTION N0: 7): 5 '-CAATGACCTAGGAGTAACAATCAAC-3' OLIGO # 2 (SEQ ID NO: 8): 5 »-AAAACAAACATAAATGCACAAGTCCA-3 ' OLIGO # 3 (ID SECTION NO: 9): 5 '-ACAACGCGTGCAATGACATGACTCCA-3' 0LIG0 # 4 (SEQ ID NO: 10): 5 '-ACAGGATCCTATTAAGTTATTGCCATAGGAA-3' OLIGO # 5 (ID SECTION NO: 11): 5 '-ACACATATGTGCAATGACATGACTCCA-3 » OLIGO # 6 (SEQ ID NO: 12): 5 '-CTGCGTATCGACAAACGCGGCAAAGTCAAGGGCACCC-3' OLIGO # 7 (ID SECTION NO: 13): 5 '-AAGAGATGAAAAACAACTACAATATTATGGAAATCCGTACTGTT-3' OLIGO # 8 (SEQ ID NO: 14): 5 '-GCTGTTGGTATCGTTGCAATCAAAGGTGTTGAATCTG-3 » OLIGO # 9 (ID SECTION NO: 15): 5 '-TCTTGGGTGCCCTTGACTTTGCCGCGTTTGTCGATACGCAGGTAC-3' OLIGO # 10 (ID SECTION NO: 16): 5 '-ACAGCAACAGTACGGATTTCCATAATATTGTAGTTGTTTTTCATC-3' OLIGO # ll (SEQ ID NO: 17): 5 '-AATTCAGATTCAACACCTTTGATTGCAACGATACCA-3' The OLIGOs were phosphorylated with T4 polynucleotide kinase and subsequently denatured by heat. The single-chain OLIGOs (ss) were subsequently left at room temperature, thus allowing a slow descent of the temperature to form the DNA fragments. T4 ligase was subsequently used to covalently ligate both the shaved ends of the internal OLIGO and the fragment of the OLIGO ds to the plasmid KGF cut with Kpnl and EcoRI. The new plasmid was designated KGF (dsd). The KGF gene of the optimized codon of E. coli was completely constructed by PCR amplification of OLIGOs # 12 through # 24.

OLIGO # 12 (ID SECTION NO: 18): 5 '-AGTTTTGATCTAGAAGGAGG-3 * 0LIG0 # 13 (ID SECTION NO: 19): 5 '-TCAAAACTGGATCCTATTAA-3' OLIGO # 14 (SEQ ID NO:.. 20): 5 '-AGTTTTGATCTAGAAGGAGGAATAACATATGTGCAACGACATGAC- TCCGGAACAGATGGCTACCAACGTTAACTGCTCCAGCCCGGAACGT-3' 0LIG0 # 15 (SEQ ID N0:.. 21): 5 '-CACACCCGTAGCTACGACTACATGGAAGGTGGTGACATCCGTGTTC- GTCGTCTGTTCTGCCGTACCCAGTGGTACCTGCGTATCGACAAA-3' 0LIG0 # 16 (SEQ ID NO: 22): 5 '-CGTGGTAAAGTTAAAGGTACCCAGGAAATGAAAAACAACTA-CAACATCATGGAAATCCGTACTGTTGCTGTTGGTATCGTTGCAATCAAA-3' 0LIG0 # 17 (SEQ ID NO: 23): 5 '-GGTGTTGAATCTGAATTCTACCTGGCAATGAACAAAGAAGGTAAAC-TGTACGCAAAAAAAGAATGCAACGAAGACTGCAACTTCAAAGAA-3' OLIGO # 18 (SEQ ID NO: 24): 5 '-CTGATCCTGGAAAACCACTACAACACCTACGCATCTGCTAAATGGA-CCCACAACGGTGGTGAAATGTTCGTTGCTCTGAACCAGAAAGGT-3 • OLIGO # 19 (SEQ ID NO: 25): 5 '-ATCCCGGTTCGTGGTAAAAAAACCAAAAAAGAACAGAAAACCGCT-CACTTCCTGCCGATGGCAATCACTTAATAGGATCCAGTTTTGA-3' OLIGO # 20 (ID SECTION NO: 26): 5 '-TACGGGTGTGACGTTCCGGG-3' OLIGO # 21 (ID SECTION NO: 27): 5'-CTTTACCACGTTTGTCGATA-3 'OLIGO # 22 (ID SECTION NO: 28): 5' -ATTCAACACCTTTGATTGCA-3 ' OLIGO # 23 (ID SECTION NO: 29): 5 '-CCAGGATCAGTTCTTTGAAG-3' OLIGO # 24 (ID SECTION NO: 30): 5 '-GAACCGGGATACCTTTCTGG-3' The OLIGOs from # 12 to # 24 were designated in such a way that the complete DNA sequence coding for the native KGF was represented by the OLIGOs either from "Watson" or from "Crick" the chain amplification and by PCR to produce the desired double-stranded DNA sequence (Figure 6) [PCR # 5, Model 9600 thermal cycler (Perkin-Elmer Cetus); 21 cycles; each cycle consists of 31 seconds at 94 ° C for denaturation, 31 seconds at 50 ° C for the union of single chains and 31 seconds at 73 ° C for elongation; followed by 21 cycles the PCR ended with an elongation step of 7 minutes]. After PCR amplification, the DNA fragment was cut with Xbal and BamHI and the 521 bp fragment was ligated into the expression plasmid pCFM1156 cut with the same enzymes. PCR # 5 used 0LIG0 # 12 and 0LIG0 # 13 of the external primers (100 pmol / 100 μl rxn) and 1 μl / 100 μl rxn of a KGF annealing derivative by ligation (by ligase T4) of the 0LIG0s # 14 to # 19 (the 0LIG0s # 15 to the OLIGOs # 18 were phosphorylated with the T4 polynucleotide kinase) using OLIGOs # 20 to OLIGOs # 24 as binding-aid oligos (Jayaraman et al., (1992), Biotechniques, 12 ^: 392) for ligation. The final construction was designated as KGF (codon optimized). All KGF analogs described herein are composed in part of DNA sequences found in KGF (dsd) or KGF (optimized codon), or in a combination of the two. The sequences were again modified by insertion at convenient restriction sites of the DNA sequences for the amino acid analogs of the particular KGF using one or more of the techniques described above for the synthesis of DNA fragments. Any of the analogs can be generated completely by any technique of those described above. However, as part of the design of the general OLIGO, optimized codons of E. coli were used where it was appropriate, on the other hand, the presence of the optimized codons of E. coli did not increase significantly, either in part or in all, of any From the genes that were examined the production of the protein that can be obtained from cultured bacterial cells. Figures 7 to 12 given below by the constructions of the nucleotide and amino acid sequences of the particular KGF analog as a convenient example: C (1,15) S (Figure 7); C (1, 15) S / R (14) E (Figure 8); C (1, 15) S / R (144) Q (Figure 9); N15 (Figure 10); ? N23 (Figure 11) and? N23 / R (144) Q (Figure 12). All of the analogous constructs of KGF described herein were confirmed DNA sequences.

Example 2: Purification from E. coli In the cloning of the KGF analog genes, three different expression plasmids were used. These were the plasmids: pCFM1156 (ATCC 69702), pCFM 1656 (ATCC 69576), and pCFM3102 (Figures 2A, 2B and 2C, respectively). Plasmid p3102 can be obtained from plasmid pCFM1656 by marking a series of basic changes directed to sites with a PCR-mutagenesis of overlapping oligos. Starting with the BglII site (pCFM1656 bp from plasmid # 180) immediately 5 'to the plasmid replication promoter, PCoPs / and proceeding to the plasmid replication genes, the changes in the base pairs are as follows: PCFM1656 # of pb in the pCFM1656 bp changed in pCFM3102 # 204 T / AC / G # 428 A / TG / C # 509 G / CA / T # 617 - two bp insert G / C # 677 G / CT / A # 978 T / AC / G # 992 G / CA / T # 1002 A / TC / G # 1005 C / GT / A # 1026 A / TT / A # 1045 C / GT / A # 1176 G / CT / A # 1464 G / CT / A # 2026 G / C deletion of pb # 2186 C / G T / A # 2479 A / T T / A # 2498-2501 AGTG GTCA TCAC CAGT pCFM1656 # of pb pb in pCFM1656 pb changed in pCFM3102 # 2 641 - 2 647 TCCGAGC pb deletion AGGCTCG # 3441 G / C A / T # 3452 G / C A / T # 3649 A / T T / A # 4556 insert of pbs (SEQ ID NO: 43): 5 '-GAGCTCACTAGTGTCGACCTGCAG-3' (SEQ ID NO: 44): 5 • -CTCGAGTGATCACAGCTGGACGTC-3 ' As seen in the above, the plasmids pCFM1156, pCFM1656 and pCFM3102 are very similar to each other and contain many same restriction sites. The plasmids were chosen for convenience and the vector of the DNA components can be easily changed for the purpose of new constructions. The host used for the entire cloning was E. coli strain FM5 (ATCC: 53911) and the transformations were carried out (according to the method of Hanahan (1983), supra) or by electroelution with the Gene Pulse ™ transfection apparatus. (BioRad Laboraties, Inc., Hercules, CA), according to the manufacturers protocol. Initially, a fresh, small culture inoculum of the desired recombinant of the E. coli clone carrying the desired construction in one of the three pCFM vectors was started with the transfer of 0.1 ml of the concentrate of the appropriate frozen strain in glycerol to a 2L flask containing 500 ml of Luria's medium. The culture was stirred for 16 hours at 30 ° C. After this, the culture was transferred to a 15 1 thermenator containing 8 1 of sterilized half lot (Tsai, et al (1987), J. Industrial Microbiol., 2: 181-187). The fermentation of the fed batch begins with the feeding of the food medium # l (Tsai, et al. (1987), supra). When the OD600 (600 optical density) reaches 35, the expression of the desired KGF analog was induced by a rapid increase in culture temperature to 37 ° C to allow amplification of the plasmid. After two hours at 37 ° C, the culture temperature increased rapidly to 42 ° C to denature the Cl repressor and the addition of Feed 1 was discontinued in favor of Feed 2, the addition rate of which was started at 300 ml / hr. Feed 2 comprised 175 g / 1 of peptone-trypticase, 87.5 g / 1 of yeast extract, and 260 g / 1 of glucose. After one hour at 42 ° C, the culture temperature was reduced to 36 ° C, maintaining this temperature for another 6 hours. After this, the fermentation was stopped and the cells were collected by centrifugation in plastic bags placed in 1 1 centrifugation bottles. The cells were pelleted by centrifugation at 400 rpm for 60 minutes, after which the cells were removed. supernatants and the cell mass was frozen at -90 ° C. Following the expression of several KGF analogs in E. coli, native KGF, C (1,15) S, C (1, 15) S / R (144) E, C (1, 15) S / R (144) Q,? N15,? N23, and protein? N23 / R (144) Q were purified using the following procedure. The cell mass from a high cell density fermentation was suspended at 4 ° C in a 10-20% solution (weight per volume) of 0.2 M NaCl, 20 mM NaP0, with a pH of 7.5 using high shear mixing appropriate. The suspended cells were then lysed by passing the solution through a homogenizer (APV Gaulin, Inc., Everett, MA) three times. The flowing homogenate was cooled to 4-8 ° C using an appropriate heat exchanger. The waste was removed by centrifuging the lysate in a J-6BMR centrifuge (Beckman Instruments, Inc., Brea, CA) equipped with a JS 4.2 rotor at 4,200 rpm for 30-60 min. at 4 ° C. The supernatants were carefully decanted and placed on a column of S-Sepharose Fast Flow ™ resin (Pharmacia) equilibrated with 0.2 M NaCl., 20 mM NaP04, with a pH of 7.5 at 4 ° C. The column was then washed with five column volumes (2250 ml) of 0.4 M NaCl, 20 M NaP04, with a pH of 7.5 at 4 ° C. The desired protein was eluted by washing the column with 5 1 of 0.5 M NaCl, 20 mM NaP04, with a pH of 7.5. Fractions of 50 ml were collected again and the A28o of the effluent was continuously monitored. The fractions identified by A28o as carriers of the eluted material were subsequently analyzed by SDS-PAGE (electrophoresis in polyacrylamide gels) through 14% gels to confirm the presence of the desired polypeptide. Those carrier fractions of the proteins of interest were then put together, to which an equal volume of distilled water was added. The diluted sample was then placed on a column, previously prepared 450 ml (5 cm x 23 cm) of S-Sepharose Fast Flow equilibrated with 0.4 M NaCl, 20 mM NaP04, with a pH of 6.8 at 4 ° C. This column was washed with 2250 ml of 0.4 M NaCl, 20 mM NaP04, with a pH of 6.8 and the protein was eluted using a linear volume gradient of column 20 within a range of 0.4 M NaCl, 20 mM NaP0, with a pH of 6.8 up to NaCl 0. 6 M, NaP04 20 mM, with a pH of 6.8. Again fractions of 50 ml were collected under a constant A2so monitoring of the effluent. Those carrier fractions of the protein (determined by SDS-PAGE 14%) were pooled again, and subsequently concentrated through a YM-10 membrane (molecular weight cut-off of 10,000) of an active cell volume of 350 cc (Amicon, Inc. Mayberry, MA) at a volume of 30-40 mi. After this, the concentrate was placed on a column, previously generated 1,300 ml (4.4 cm x 85 cm), of Superdex-75MR resin (Pharmacia) equilibrated in a column buffer composed of lx PBS (Dulbecco's Phosphate Buffered Saline, "D-PBS", free of calcium and magnesium) or in 0.15 M NaCl, 20 mM NaP0, with a pH of 7.0. After allowing the samples to run in the column, the protein was eluted from the mixed filtrate of the gel using a column buffer. After this, fractions of 10 ml were recovered and those fractions containing the analogue (determined by SDS-PAGE 14%) were pooled. Typically, the concentration of the protein was approximately 5-10 mg / ml in the obtained obtained. All previous procedures were performed at 4-8 ° C unless otherwise indicated. An alternative purification procedure was used to purify the native KGF, C (1,15) S and? N23. The procedure involves the following steps and, unless otherwise specified, all procedures, solutions and materials were performed at 23 + 5 ° C. To conclude the production phase of the bacterial fermentation, the cell culture was cooled to 4 minutes. -8 ° C and cells were collected by centrifugation or similar processes. Under the bases of the expected yield of the protein per unit weight of the cell mass and the amount of purified protein required, an appropriate amount of cell mass, by weight, was suspended in a mild buffer solution 0.2 M NaCl, NaP04. mM, at a pH of 7.5, weighing about five times what the cell mass weighs when resuspended. The cells were dispersed to a homogeneous solution using a shear mixer. The temperature of the dispersion of the cell mass was maintained at 4-8 ° C during homogenization.

Subsequently, the cells were used by pressure, for example by passing the dispersed cell mass twice through a cell homogenizer of appropriate size. The homogenate was stored cooled to 5 + 3 ° C. To clarify the cell lysate, a previously prepared deep filtrate container (Cuno, Inc., Meriden, CT) equipped with a filter having an appropriate amount of filtrate surface area, equilibrated with an appropriate volume of 0.2 M NaCl was used. , 20 mM NaP04, with a pH of 7.5. The equilibrium and clarification were carried out at a temperature of 5 + 3 ° C. Before clarification, an appropriate amount of an appropriate filter was used to pre-coat the filter and be thoroughly mixed with the cell lysate, after which the cell lysate was clarified by passing the solution through the filtration apparatus. The filter was washed with 0.2 M NaCl, 20 mM NaP04, with a pH of 7.5. The filtrate and any subsequent washing were collected in a cooling container of appropriate capacity, which was stored at less than 10 ° C. Following clarification, the lysate was subsequently passed through a previously prepared column of SP-Sepharose Fast Flow which contained at least 1 ml of resin per 2 g of the cell mass. The SP-Sepharose Fast Flow column was equilibrated with 0.2 M NaCl, 20 mM NaP04, at a pH of 7.5 cold (5 + 3 ° C). The temperature of the column was maintained at least at 10 ° C. The clarified lysate (5 + 3 ° C) was then placed on an ion exchange column with an absorbance at 280 nm (A2so) of the eluent which was continuously monitored. After having placed the sample, the column was washed with 0.2 M NaCl, 20 mM NaP04, at a pH of 7.5, cold, followed by a wash with 0.3 M NaCl, 20 mM NaP0, pH 7.5 at 23 + 5 ° C. To elute the desired protein, a linear gradient of 0.2-1 M NaCl, 20 mM NaP0, at a pH of 7.5 was used. The obtained product was collected in several fractions under the A2so reference of the eluate. Following the elution, these fractions were brought together and the volume was noted. To oxidize the free sulfhydryl groups, an oxidation step was carried out. For proteins having altered cysteine standards, when compared to native KGF, an oxidizing agent (eg, cystamine dihydrochloride or some other suitable oxidizing agent, eg, cystine, oxidized glutathione or divalent copper) was added to the final concentration of 1-20 mM and adjusted to 7-9.5, with a pH of 9.0 + 0.3 when cystamine dihydrochloride was used. Oxidation was carried out at 10-30 ° C for an appropriate period. For the native KGF protein, the oxidation was completed by adding an appropriate amount of (NH4) 2S04 such as (NH4) 2S04 1-2 M, adjusting the pH to 7.5-9.5, and maintaining the temperature at 23 + 5 ° C for a appropriate period. After oxidation, the pH of the solution was adjusted between 6.5 and 9.5. In case of need, the (NH4) 2S04 solid was added to the solution at a final concentration of 2 M. To remove the truncated particles, the solution was passed through appropriate clarification filters. The product of the filtered oxidation was subjected to a hydrophobic interaction chromatography (HIC). The base of the HIC was a Butyl-650M Toyopearl ™ resin (Tosohaas, Inc., Montgomeryville, PA). The carrier solution of the protein was deposited on the column, which was previously equilibrated with 2 M (NH4) 2 SO4, 0.15 M NaCl, 20 mM NaP04, with a pH of 7.0. After sample placement, the column was washed with 2 M (NH4) 2 SO4, 0.15 M NaCl, 20 M NaP04, with a pH of 7.0. Subsequently, the protein was eluted using a linear gradient of (NH4) 2S04 of 2-0 M, made in 0.15 M NaCl, 20 mM NaP04, with a pH of 7.0. When the desired protein began to elute, as indicated by the increase in A28o of the eluate, the fractions were collected. The aliquots of each fraction were then analyzed by SDS-PAGE. Those fractions that contained the desired protein were subsequently put together, completely mixed, and the volume of the obtained was determined, as well as the concentration of the protein in it. The eluate bearing the HIC protein obtained was concentrated and the elution buffer changed. Typically, the proteins were concentrated at 5.0-10.0 g / ml. The ultrafiltration was performed using an ultrafiltration system equipped with a Pellicon ™ tape system (Millipore, Inc., Bedford, MA) with an appropriately sized cutting membrane. After concentration, the sample was diafiltered with an appropriate buffer. The product retained in the concentration step was again diafiltered with 0.15 M NaCl, 20 mM NaP04, with a pH of 7.0 until the conductivity of the retained product was with 5% of the conductivity of the 0.15 M NaCl solution, NaP04 20 mM, with a pH 7.0.

In addition, to remove the precipitates and the bacterial endotoxin that could be present, the carrier sample of the diafiltered, concentrated protein was passed through a PosidyneMB filter of 0.1 μm (Pall, Inc., Cortland, NY). After determining the concentration of the protein in the solution and based on the desired concentration of the final loose product, the solution was diluted with 0.15 M NaCl, 20 mM sodium phosphate, with a pH of 7.0, until reaching the desired final concentration . A final aseptic filtration through a 0.22 μm filter was performed subsequently as the final loose product was transferred to a pyrogen-free container (at about 5 ° C) for a wider formulation.

Example 3: Purification of a Mammalian Cell Culture.

This example describes the expression, isolation and characterization of two forms of recombinant, biologically active KGF (rKGF) produced in a mammalian expression system. The human KGF gene was isolated by PCR amplification of a cDNA made from fibroblastic, dermal, normal human cells (Clonetec, Inc., Palo Alto, CA). Following the manufacture of the cDNA by reverse transcriptase, it was used to amplify the KGF gene. OLIGO # 25 and OLIGO # 26 were used to amplify the cDNA gene and OLIGOs # 27 and OLIGO # 28 were used to place the HindIII restriction sites and BglII at the ends of the fragment by a second PCR amplification as they were placed in Figure 1.

OLIGO # 25 (ID SECTION NO: 45): '-CAATCTACAATTCACGA-3 OLIGO # 26 (ID SECTION NO: 46) -TTAAGTTATTGCCATAGG-3 » OLIGO # 27 (ID SECTION NO: 47): '-AACAAAGCTTCTACAATTCACAGATAGGA-3' OLIGO # 28 (SEQ ID NO: 8): '-AACAAGATCTTAAGTTATTGCCATAGG-3 * Following the cloning and confirmation of the DNA sequence, the DNA of the KGF gene was then used. The amplification was carried out using two primers: OLIGO # 29 (ID SECTION NO: 49): '-CGGTCTAGACCACCATGCACAAATGGATACTGACATGG-3' OLIGO # 30 (ID SECTION NO: 50): '-GCCGTCGACCTATTAAGTTATTGCCATAGGAAG-3' The sense primer relative to the ATG start codon, OLIGO # 29, includes an Xbal site and a Kozak consensus translation sequence (5'-CCACC-3 *). The antisense primer, OLIGO # 30, includes a SalI cloning site and an additional termination codon. After 18 cycles of PCR amplification (30 sec of denaturation at 94 ° C, 40 sec of gluing the single strands at 55 ° C, and 40 sec of elongation at 72 ° C), the product was digested with Xbal and Sali and ligated with a DNA digested in a manner similar to plasmid pDSRa2 (according to the methods of Bourdrel et al. (1993), Protein Exp. &Purif., 4: 130-140 and Lu et al. (1992), Arch. Biochem Biophys., 298: 150-158). This gives rise to the KGF / pDSRa2 plasmid in which the human KGF gene is placed between the SV40 early promoter sequence and the a-FSH polyadenylation sequence. Two clones were harvested and analysis of the DNA sequence confirmed the construction of the desired vector. Then two micrograms of the KGF / pDSRa2 DNA were linearized with Pvul. Chinese hamster or guinea pig (CHO) ovary cells were seeded a day earlier at a density of 0.8 x 106 cells / 60 mm in the culture dish, where they were transfected with the treated DNA using a normal method of phosphate precipitation of calcium (Bourdrel et al., supra). Two weeks later, individual colonies were harvested and transferred to 24-well plates. The conditioned medium was considered serum-free when the cells reached confluence and the aliquots of these were analyzed by means of a (Western blotting) "protein immunodetection method by transfer to nitrocellulose membranes" using a polyclonal rabbit antiserum reactive against the KGF of human expressed in E. coli. Protein immunodetections were performed by running the samples through SDS-PAGE (electrophoresis in polyacrylamide gels) to 12.5% (P / V), followed by electrotransfer of the proteins for one hour at 400 A to the nitrocellulose membranes using a semi-dry transfer apparatus (Hoefer Scientific Instruments, San Francisco, CA). A solution of 20 mM Tris, 150 M glycine and 20% methanol was used as a transfer carrier. The blockade of the nitrocellulose membranes was carried out by incubating them with normal 10% goat serum in PBS. The rabbit anti-serum obtained against the KGF derived from E. Coli was used as the primary antibody. To be used, it was diluted 1 / 10,000 in 1% normal goat serum in PBS and incubated for 12 hours at room temperature with the blocked nitrocellulose membranes, after which the excess antibody was removed with three 30-ml washes. min in PBS. The nitrocellulose membranes were then incubated in 100 ml of 1% normal goat serum in PBS carrying the goat anti-rabbit IgG biotinylated with Vectastain ™ (secondary antibody, Vector Labs, Burlingame, CA) for 30 minutes at room temperature. After three 10-minute washes in PBS, a 30-min incubation at room temperature was carried out in 100 ml of a 1% normal goat serum solution containing biotinylated peroxidase and streptavidin, prepared according to the instructions of manufacturers (Vector Labs). After the three washes in PBS, the KGF cross-reactivity material was visualized by incubation in a mixture of 60 μl of 30% H202 (? / V) in 100 ml of PBS and 50 mg of 4-chloronaphthol in 20 ml. of methanol. The reaction was stopped at 10 min by rinsing with water. Analysis of protein immunodetections showed that KGF-specific antibody was associated with three different protein bands, two of which were closely related to nearby molecular weights of approximately 25-29 kDa and one of them with an estimated molecular weight of close to 17 kDa, when compared to the expected molecular weight of approximately 18.8 kDa of the mature protein of 163 amino acids. Additionally, several clones with a high level of expression, secrete more than 2.0 mg of rKGF per liter, judging from the Western analysis, were selected and amplified in rotating bottles (according to the method of Lu et al., Supra) for generate large volumes of serum-free conditioned medium for the purification of KGF by cation exchange chromatography and gel filtration, as described below. The 3 1 KGF was purified from a serum free conditioned medium by directly applying it to a cation exchange column (5 x 24 cm) made with 450 ml of a sulfoethyl column of SP-Sepharose Fast Flow (Pharmacia) pre-equilibrated with 20 mM sodium phosphate, pH 7.5. After washing with five volumes of the 20 M sodium phosphate column, 0.2 M NaCl, with a pH of 7.5, the rKGF was eluted using a linear volume gradient of column 20 of 0.2 to 1.0 M NaCl in sodium phosphate. M, with a pH of 7.5. Fractions of 50 ml were collected with continuous monitoring of the A28o- KGF protein was detected by analyzing the aliquots of each fraction by SDS-PAGE. SDS-PAGE was performed in an electrophoresis system (Novex, San Diego, CA) using pre-made gels of Tris-glycine pre-made at 14% (according to the method of Laemmli (1970), Nature, 227: 6801-685). The samples were mixed with a non-reducing SDS sample buffer, without heating prior to placement. The proteins were detected either by staining with Coomassi blue or by staining with silver. Two late peaks were seen containing protein bands corresponding to 25-29 kDa and 17 kDa bands detected by Western blot. Fractions containing each of these peaks were separately concentrated to a volume of less than 1.0 ml and subjected to gel filtration. Gel filtrations used Superdex-75MR resin columns (HR 10/30, Pharmacia) pre-equilibrated with PBS, with a pH of 7.2, and calibrated with the following known molecular weight standards (BioRad, San Francisco, CA): thyroglobulin (670 kDa), gammaglobulin (158 kDa), ovalbumin (44 kDa ), myoglobin (17 kDa) and vitamin B-12 (1.4 kDa). These purification steps gave an approximately 2000-fold purification of rKGF, which specifically includes 17 kDa and 30 kDa material, as estimated by silver staining. In the case of high molecular weight material, the rKGF that eluted with the largest symmetric peak was called KGF-a. With the SDS-PAGE analysis of the smaller amount of this material, 3 μg / lane versus 6 μg / lane, two bands with 1 or 2 kDa difference in molecular weight were revealed. In this case, the material with the lowest molecular weight was designated KGF-b, the gel filtration showed that the preparation of the protein had the expected mobility. For both KGF-a and KGF-b, the total yield after purification was approximately 30-40%. The amino acid sequences of KGF-a and KGF-b were also analyzed. These analyzes were carried out in an automatic sequencer (Model 477A or 470A, Applied Biosystems, Inc., Foster City, CA) equipped with an amino acid analyzer of PTH, Model 120A online and with the data collection system Model 900A (according to the method of Lu et al. (1991), J. Biol. Chem., 266: 8102-8107). The analysis of the Edman sequence of KGF-a showed a sequence of the N-terminal end of Xi-N-D-M-T-P-E-Q-M-A-T-N-V-X2-X3-S- (SEQ ID NO: 51). A minor sequence starting from the third amino acid of the N-terminal end, aspartic acid, was present in 1.6% of the total sequence protein Xi, X2 and X3 where they were not assigned due to the absence of phenylthiohydantoinyl amino acid signals ( PTH). It is very interesting that the sequential analysis of KFG-b at the N-terminus revealed an amino acid sequence of SYDYMEGGDIRV- (SEQ ID NO: 52), which indicates that this is a truncated form of the N-terminus of KGF that has been proteolytically cut in the peptide bond Arg23-Ser24. To further characterize KGF-a and Purified KGF-b, the protein was treated with glycosidases (neuraminidase, O-glycanase, and / or N-glycanase), using known techniques (Sasaki et al (1987), J. Biol. Chem., 262: 12059-12076; Takeuchi et al., (1988), J. Biol. Chem., 263: 3657-3663; Zsebo et al., (1990), Cell, 63: 195-201). These data indicate that KGF-a contains both O-linked and N-linked carbohydrates, although the lower molecular weight KGF-a form probably contains only N-linked sugars. Treatment with glycosidases does not cause molecular weight reduction for KGF-b, indicating that the molecule is not glycosylated.

Example 4: Biological activity Each KGF analog was diluted and examined for its biological activity by measuring the incorporation of [3 H] -thymidine from Balb / MK cells (according to the method of Rubin et al., (1989), supra). As a first step the samples were diluted in a medium for bioanalysis composed of Eagle's MEM not provided by the distributor, 50% of F12 not provided by the distributor, 5 μg / ml of transferrin, 5 ng / ml of sodium selenite, 0.0005 % HSA, and 0.005% Tween 20. The KGF samples were added to 96-well plates (Falcon primer 96-well plates) covered with Balb / MK cells. The incorporation of [3H] -thymidine during DNA synthesis was measured and the concentration of native KGF was determined by comparison with the normal curve of the native KGF. All analogs analyzed showed mitogenic activity. The interaction with the KGF receptor was analyzed using membrane preparations of the isolated KGF receptor, prepared from the Balb / MK mouse epidermal keratinocytes (according to the procedures described by Massague (19932), J. Biol. Chem. , 258: 13614-13620). Specifically, several forms of KGF were diluted with 50 mM Tris-HCl, with a pH of 7.5, with 0.2% of bovine serum albumin, such that the concentrations were within the range of 0.8 ng to 100 ng per 50 μl. These were incubated individually with the membrane preparation (75 ng / ml) and with the KGF (1.5 ng) of E. coli labeled with 125I. The binding to the receptor and the competition experiments were performed at 4 ° C for 16 h., After which the samples were taken, centrifuged, and washed twice with the diluent buffer above to eliminate the labeled KGF that did not He joined or joined specifically. The samples were counted to know the remaining radioactivity. Competency curves for receptor binding were constructed between the KGF and labeled KGF samples graphically representing the percent non-competition against the concentrations of each KGF sample. The non-compete experiments of a radio-receptor assay indicated that KGF derived from E. coli, KGF-a, and KGF-b have a very similar receptor binding activity. Although the present invention has been described above, both generally and in terms of the preferred embodiments, it is clear that other variations and modifications will occur to those skilled in the art in view of the foregoing.ST GENERAL INFORMATION: (i) ASPIRANTE: Amgen Inc. (ii) TITLE OF THE INVENTION: Method for the Purification of Keratipacite Growth Factors (iii) NUMBER OF SEQUENCES: 52 (iv) ADDRESS OF CORRESPONDENCE: (A) RECIPIENT: Amgen Inc. (B) STREET: 1840 DeHavilland Drive (C) CITY: Thousand Oaks (D) STATE: California (E) COUNTRY: US A. (F) C.P. 91320-1789 (v) COMPUTER LEGIBLE FORM: (A) MIDDLE TYPE: Soft disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: PatentIn Relay # 1.0, Version # 1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US 08 / 487,830 (B) FILLING DATE: (C) CLASSIFICATION: unknown (2) INFORMATION FOR SEC. FROM IDENT. NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 862 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: l: C? TCTAC? A TTC? CAGATA GGAA3AGGTC? ATGACCTAG GAGTAACAAT CAACTCAAGA 60 TTCATTTTC? TTATGTTATT CATGAACACC CGGAGCACTA CACTATAATG CACAAATGGA 120 T? CTG? CATG GATCCTGCCA ACTTTGCTCT ACAGATCATG CTTTCACATT ATCTGTCTAG_130_TGGGTACTAT ATCTTTAGCT TGCAATGACA TGACTCCAGA GCAAATGGCT ACAAATGTGA '240 ACTGTTCCAG CCCTGAGCGA CACACAAGAA GTTATCATTA CATGGAAGGA GGGGATATAA 300 GAGTGAGAAG ACTCT CTGT CG? ACACAGT GGTACCTGAG GATCGATAAA AGAGGCAAAG 360 TAA.V '? GGAC CC? AGAGATG AAGAATAATT ACAATATCAT GGAAATCAGG ACAGTGGCAG 420 TTGGAATTGT GGCAATCAAA GGGGTGC? AA GTGAATTCTA TCTTGCAATG AACAAGGAAG 480 G? A? ACTCT? TGCAAAG ?? A GAATGCA? TG AAGATTGTA? CTTCAAAGAA CTAATTCTGG 540 / - ??? CC? TT? C? CAC? TAT GCATCAGCTA AATGGACACA CAACGGAGGG G? AATGTTTG 600 TTGCCTTAAA, TCAAAAGGGG ATTCCTGT? A GAGGAAAAAA AACGAAGAAA GAACAAAAAA 660 CAGCCCACTT TCTTCCT? TG GCA? TAACTT A? TTGCATAT GGTATATAAA GAACCCAGTT 720? GC? GC-G? G? TT7CTT A AGTGGACTGT TTTCTTTCTT CTCAAAATTT TCTTTCCTTT 730? T 7? G A GC GCA? • • • • • • • * *. ACT AA AAA_1Y; A., CA d * 4 O < JZ r \\ TTTVr GTTTGTTT7 AG 852 (2) INFORMATION FOR SEC. FROM IDENT. N0: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 194 amino acids (B) TYPE: amino acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 2: Mee His Lys Trp He Leu Thr Trp He Leu Pro Thr Leu Leu Tyr Arg 1 5 10 15 Be Cys Phe His He He Cys Leu Val Gly Thr He Ser Leu Cys Wing 20 25 30 Asn Asp Mee Thr Pro Glu Gln Mee Wing Thr Asn Val Asn Cys Ser Ser 35 40 45 Pro Glu Arg Kis Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp He 50 55 60 Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg He Asp 65 70 75 80 Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn 85 90 95 He Met Glu He Arg Thr Val Wing Val Gly He Val Wing He Lys Gly 100 105 110 Val Glu Ser Glu Phe Tyr Leu Wing Mee Asn Lys Glu Gly Lys Leu Tyr 115 120 125 Wing Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu He Leu 130 135 140 Glu Asn His Tyr Asn Thr Tyr Wing Ser Wing Lys Trp Thr His Asn Gly 145 150 155 160 Gly Glu cC Phe Val Wing Leu Asn Gln Lys Gly He Pro Val Arg Gly 165 170 175 Lys Lys Thr Lys Lys Glu Gln Lys Thr Wing His Phe Leu Pro Met Wing 180 185 190 He Thr (2) INFORMATION FOR SEC. FROM IDENT. NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 595 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (iii) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 3: ? TCGATTTGA TTCT? CA? GG AGGAATA? CA TATGAAAAAG CGCGCACGTG CTATCGCCAT 60 TGCTGTGGCT CTGGCAGGTT TCGCAACTAG TGCACACGCG TGCAATGACA TGACTCC? GA 120 GCAAATGGCT ACAAATGTGA ACTGTTCCAG CCCTGAGCGA CACACAAGAA GTTATGATTA 180.

CATGGAAGGA GssGATATAA GAGTGAG? AG ACTCTTCTGT CGAACACAGT GGTACCTGAG 240 GATCGATAAA AGAGGCAAAG TAAAAGGGAC CCAAGACATG AAGAATAATT ACAATATCAT 300 G AAATCAGG ACAGTGGCAG TTGGAATTGT GGCAATCAAA GGGGTGGAAA GTGAATTCTA 360 TCTTGCAATG AACAAGGAAG GAAAACTCTA TGCAAAGAAA GAATGCAATG AAGATTGTAA 420CTAATTCTGG AAAACCATTA CAACACATAT GCATCAGCTA AATGGACACA 480 CAACGGAGGG G? AATGTTTG TTGCCTTAAA TCAAAAGGGG ATTCCTGTAA GAGGAAAAAA 540 ? ACGAAGAA? GAACAAAAAA CAGCCCACTT TCTTCCTATG GCAATAACTT? ATAG_595_(2) INFORMATION FOR SEC. FROM IDENT. NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH: 186 amino acids B) TYPE: amino acid C) POLARITY: unknown D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: protein (iii) DESCRIPTION OF THE SEQUENCE: SEC - DE IDENT. DO NOT: : Mee Lys Lys Arg Ala? Rg Ala He Ala Ala Ala Ala Ala Ala Ala Gly 1 5 10, '15 Phe Ala Thr Ser Ala Ala Ala Cys Asn Asp Met Thr Pro Glu Gln Met 20 25 30 Wing Thr Asn Val Asn Cys Ser Ser Pro Glu Arg His Thr Arg Ser Tyr 35 40 45 Asp Tyr Met Glu Gly Gly Asp He Arg Val Arg Arg Leu Phe Cys Arg 50 55 60 Thr Gla Trp Tyr Leu Arg He Asp Lys Arg Gly Lys Val Lys Gly Thr 65 70 75 80 Gln Glu Met Lys Asn Asn Tyr Asn He Met Glu He Arg Thr Val Wing 85 90 95 Val Gly He Val Ala He Lys Gly Val Glu Ser Glu Phe Tyr Leu Ala 100 105 110 Mee Asp Lys Glu Gly Lys Leu Tyr Ala Lys Lys Glu Cys Asn Glu Asp 115 120 125 Cys Asn Phe Lys Glu Leu He Leu Glu Asn His Tyr Asn Thr Tyr Ala 130 135 140 Be Ala Lys Trp Thr His Asn Gly Gly Glu Mee Phe Val Ala Leu Asn 145 150 155 160 Gln Lys Gly He Pro Val Arg Gly Lys Lys Thr Lys Lys Glu Gln Lys 165 170 175 Thr Ala His Phe Leu Pro Mee Wing He Thr 180 185 (2) INFORMATION FOR SECOND IDENT. NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 499 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEC. FROM IDENT. NO: 5: TATGTGCAAT GACATGACTC CAGAGCAAAT GGCTACAAAT GTGAACTGTT CCAGCCCTGA 60 GCGACACACA AGAAGTTATG ATTAC? TGGA AGGAGGGGAT ATAAGAGTGA GAAGACTCTT 120 CTGTCGAACA CAGTGGTACC TGAGGATCGA TAAAAGAGGC AAAGTAAAAG GGACCCAAGA 180 GATGAAGAA? ATTAC? ATA TCATGGAAAT CAGGACAGTG GCAGTTGGAA TTGTGGCAAT 240 CAAAGGGGTG GAA / V? TGAAT TCTATCTTGC AATGAACA? G GAAGGAAAAC TCTATGCAAA 300 GAAAG? ATGC AATGAAGATT GTAACTTCAA AGAACTAATT CTGGAAAACC ATTACAACAC 360 ATATGCATCA GCTAAATGGA C? C? CAACGG AGGGGAAATG TTTGTTGCCT TAAATCAAAA 420 GGGGATTCCT GTAAGAGGAA AAAAAACGAA GAAAGAACAA AAAACAGCCC ACTTTCTTCC 480 TATGGCAATA ACTTAATAG '499 (2) INFORMATION FOR SEC. FROM IDENT. NO: ß: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 164 amino acids (B) TYPE: amino acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO 6: Mee Cys Asn Asp Mee Thr Pro Glu Gln Mee Wing Thr Asn Val Asn Cys 1 5 10 15 Ser Ser Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Mee Glu Gly Gly 20 25 30 Asp He Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg 35 40 45 He Asp Lys Arg Gly Lys Val Lys Gly Thr Gln Glu MeC Lys Asn Asn 50 55 60 Tyr Asn He Ket Glu He Arg Thr Val Wing Val Gly He Val Wing He 65 70 75 80 Lys Gly Val Glu Ser Glu Phe Tyr Leu Ala Mee Asn Lys Glu Gly Lys 85 90 95 Leu Tyr Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu 100 105 110 He Leu Glu Asn Kis Tyr? Sn Thr Tyr Ala Ser Wing Lys Trp Thr His 115 120 125 Asn Gly Gly Glu Met Phe Val Wing Leu Asn Gln Lys Gly He Pro Val 130 135 140 Arg Gly Lys Lys Thr Lys Lys Glu Gln Lys Thr Wing His Phe Leu Pro 145 150 155 160 Mee Ala He Thr (2) INFORMATION FOR SEC. FROM IDENT. NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 7: CAATGACCTA GGAGTAACAA TCAAC 25 (2) INFORMATION FOR SEC. FROM IDENT. NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: c DNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 8: AAAACAAACA TAAATGCACA AGTCCA 26 (2) INFORMATION FOR SEC. FROM IDENT. NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (iii) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 9: ACAACGCGTG CAATGACATG ACTCCA 26 (2) PAPA INFORMATION SEC. FROM IDENT. NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 10: ACAGGATCCT ATTAAGTTAT TGCCATAGGA AT 31 (2) INFORMATION FOR IDENTIFYING SECTION: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: ll: ACACATATGT GCAATGACAT GACTCCA 27 (2) INFORMATION FOR SEC. FROM IDENT. N0: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 37 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (li) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 12: CTGCGTATCG ACAAACGCGG CAAAGTCAAG GGCACCC 37 (2) INFORMATION FOR SEC. FROM IDENT. NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEC DE ID £ NT. NO: 13: AAGAGATGAA AAACAACTAC AATATTATGG AAATCCGTAC TGTT 44 (2) INFORMATION FOR SEC. FROM IDENT. N0: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: Cadn (iii) ) DESCRIPTION OF THE SEQUENCE: s? C ^ m 1O? M, N0: 14: GCTGTTGGTA TCGTTGCAAT CAAAGGTGTT GAATCTG 37 (2) INFORMATION FOR ID £ NT > N0: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: ^ DE IDENT > NQ: 15: TCTTGGGTGC CCTTGACTTT GCCGCGTTTG TCGATACGCA GGTAC 45 (2) INFORMATION FOR SEC. FROM IDENT. NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 16: ACAGCAACAG TACGGATTTC CATAATATTG TAGTTGTTTT TCATC 45 (2) PAPA INFORMATION SEC. FROM IDENT. NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 17: AATTCAGATT CAACACCTTT GATTGCAACG ATACCA 36 (2) INFORMATION FOR SEC. FROM IDENT. NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: Cadn (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 18: AGTTTTGATC TAGAAGGAGG 20 (2) INFORMATION FOR SEC. FROM IDENT. NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 19: TCAAAACTGG ATCCTATTAA 20 (2) INFORMATION FOR SEC. FROM IDENT. NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 91 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (i) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 20: AGTTTTGATC TAGAAGGAGG AATAACATAT GTCCAACGAC ATGACTC 47 CGG AACAGATGGC TACCAACGTT AACTGCTCC GCCCGGAACG T 91 (2) INFORMATION FOR SEC. FROM IDENT. NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 90 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 21: CACACCCGTA GCTACGACTA CATGGAAGGT GGTGACATCC GTGTTCG 47 TCG TCTGTTCTGC CGTACCCAGT GGTACCTGCG TATCGACAAA 90 (2) INFORMATION FOR SEC. FROM IDENT. NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 90 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 22: CGTGGTAAAG TTAAAGGTAC CCAGGAAATG AAAAACAACT ACAACAT 47 CAT GGAAATCCGT ACTGTTGCTG TTGGTATCGT TGCAATCAAA 90 (2) INFORMATION FOR SEC. FROM IDENT. NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 90 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 23: GGTGTTGAAT CTGAATTCTA CCTGGCAATG AACAAAGAAG GTAAACT 47 GTA CGCAAAAAAA GAATGCAACG AAGACTGCAA CTTCAAAGAA 90 (2) INFORMATION FOR SEC. FROM IDENT. NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 90 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 24: CTGATCCTGG AAAACCACTA CAACACCTAC GCATCTGCTA AATGGAC 47 CCA CAACGGTGGT GAAATGTTCG TTGCTCTGAA CCAGAAAGGT 90 (2) INFORMATION FOR SEC. FROM IDENT. NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 88 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 25: ATCCCGGTTC GTGGTAAAAA AACCAAAAAA GAACAGAAAA CCGGTCA 47 CTT GCAATCACTT AATAGGATCC AGTTTTGA 88 (2) PARÍ INFORMATION SEC. FROM IDENT. NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC > FROM IDENT. NO: 26: TACGGGTGTG ACGTTCCGGG 20 (2) INFORMATION FOR SEC. FROM IDENT. NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 27: CTTTACCACG TTTGTCGATA 20 (2) INFORMATION FOR. SEC. FROM IDENT. NO: 28: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 28: ATTCAACACC TTTGATTGCA 20 (2) INFORMATION FOR SEC. FROM IDENT. NO: 29: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 29 CCAGGATCAG TTCTTTGAAG 20 (2) INFORMATION FOR SEC. FROM IDENT. NO: 30: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT_ ^^ GAACCGGGAT ACCTTTCTGG 20 (2) INFORMATION FOR SEC. FROM IDENT. NO: 31: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 495 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 31: ATGTCTAATG ATATGACTCC GGAACAGATG GCTACCAACG TTAACTCCTC CTCCCCGGAA 60 CGTCACACGC GTTCCTACGA CTAC? TGGAA GGTGGTGACA TCCGCGTACG TCGTCTGTTC 120 TGCCGTACCC AGTGGTACCT GCGTATCGAC AAACGCGGCA AAGTCAAGGG CACCCAAGAG 180 ATGAAAAACA ACTACAATAT TATGGAAATC CGTACTGTTG CTGTTGGTAT CGTTGCAATC 240 AAAGCTGTVG AATCTG? ATT CTACCTGGCA ATG? ACAAAG AAGGTAAACT GTACGCAAAA 300 A? AGAATGCA ACGAAG? CTG CA? CTTCAAA GAACTGATCC TGGAAAACCA CTACAACACC 360 T? CGCATCTG CTAAATGGAC CCACAACGGT GGTGAAATGT TCGTTGCTCT GAACCAGAAA 420 GGTATCCCGG TTCGTGGTAA A? AAACCAAA AAAGAACAGA AAACCGCTCA CTTCCTGCCG 480 ? TGGCAATCA CTTAA 495 (2) INFORMATION FOR SEC. FROM IDENT. NO: 32: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 164 amino acids (B) TYPE: amino acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 32: Met Ser Asn Asp Met Thr Pro Glu Gln Met Wing Thr Asn Val Asn Ser 1 5 10 15 Ser Ser Pro Glu? Rg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly 20 25 30? Sp He Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu? Rg 35 40 45 He Asp Lys? Rg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn 50 55 60 Tyr A = n He Met Glu lie Arg Thr Val Wing Val Gly He Val Wing He 65 70 75 80 Lys Gly Val Glu 'Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys 85 90 95 Leu Tyr Ala Lys Lys Gl u Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu 100 105 110 He Leu Glu Asn His Tyr Asn Thr Tyr Ala Be Ala Lys Trp Thr His 115 120 125 Asn Gly Gla Met Met Phe Val Ala Leu Asn Gln Lys Gly He Pro Val 130 135 140 Arg Gly Lys Lys Thr Lys Lys Glu Gln Lys Thr Ala His Phe Leu Pro 145 15 C 155 160 Met Ala He Thr (2) INFORMATION FOR SEC. FROM IDENT. NO: 33: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 495 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 33: (xi) SEQUENCE DESCRI PTION: SEQ ID NO: 33: ATGTGCAATG ATATGACTCC TGAACA? ATG GCTACCAATG TCAACTGTTC CTCTCCGG? G 60 CGCC? CACCC GG? GTTACGA TTACATGGAA GGTGGGGATA TTCGCGTACG TCGTCTGTTC 120 TGC GT? CCC? GTGGTACCT GCGTATCGAC AAACGCGGCA AAGTCAAGGG CACCCAAGAG 180 ATG? AAAAC? ACT? CAATAT TATGGAAATC CGT? CTGTTG CTGTTGGTAT CGTTGCAATC 240 AAAGGTGTTG AATCTGAATT CTATCTTGCA ATGAACAAGG AAGGAAAACT CTATGCAAAG 300 AAAGAATGCA ATGAAGATTG TAACTTCAAA GAACTAATTC TGGAAAACCA TTACAACACA 3 60 TATGCATCTG CTAAATGGAC CCACAACGGT GGTGAAATGT TCGTTGCTCT GAACCAGAAA 420 GGT? TCCCTG TTCAAGGTAA GAAAACCAAG AA? GA? CAGA? AACCGCTCA CTTCCTGCCG 480 ? TGGC ?? TCA CTT? A 495 (2) INFORMATION FOR. SEC. FROM IDENT. NO: 34: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 164 amino acids (B) TYPE: amino acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 34: Mee Cys Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Asn Cys 1 5 10 15 Ser Ser Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly 20 25 30 Asp He Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg 35 40 45 He Asp Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn 50 55 60 Tyr Asn He Mee Glu He Arg Thr Val Wing Val Gly He Val Wing He 65 70 75 80 Lys Gly Val Glu Ser Glu Phe Tyr1 Leu Ala Met Asn Lys Glu Gly Lys 85 90 95 Leu Tyr Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu 100 105 110 lie Leu Glu Asn Kis Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr His 115 120 125 Asn Gly Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly He Pro Val 130 135 140 Gln l Gly Lys Lys Thr Lys Lys Glu Gln Lys Thr Wing His Phe Leu Pro 145 150 155 160 Met Ala He Thr (2) INFORMATION FOR SEC. FROM IDENT. NO: 35: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 495 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 35: ATGTCTAATG ATATGACTCC GGAACAGATG GCTACCAACG TTAACTCCTC CTCCCCGGAA 60 CGTCACACGC GTTCCTACG? CTACATGGAA GGTGGTGACA TCCGCGTACG TCGTCTGTTC 120 TGCCGTACCC AGTGGTACCT GCGTATCGAC AAACGCGGCA AAGTCAAGGG CACCCAAGAG 180 ATGA? AAACA ACTACAATAT TATGGAAATC CGTACTGTTG CTGTTGGTAT CGTTGCAATC 240 AAAGGTGTTG AATCTGAATT CTATCTTGCA ATGAACAAGG AAGGAAAACT CTATGCAAAG 300 ? AAG? ATGCA ATGAAGATTG TAACTTCAAA GAACTAATTC TGGAAAACCA TTACAACACA 360 T? TGCATCTG CTAAATGGAC CC? CAACGGT GGTGAAATGT TCGTTGCTCT GAACCAG? AA 420 GGTATCCCTG TTCAAGGTAA GAAAACCAAG AAAGAACAGA AAACCGCTCA CTTCCTGCCG 480 ATGGCAATCA CTTAA '495 (2) INFORMATION FOR SEC. FROM IDENT. NO: 36: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 164 amino acids (B) TYPE: amino acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TI PO OF MOLECULE: protein (xi) DESCRI PECTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 3ß: Mee Being Asn A = p Met Thr Pro Glu Gln Met Ala Thr Asn Val? Sn Ser 1 5 10 15 Being Ser Pro Glu Arg His Thr Arg Being Tyr Asp Tyr Met Glu Gly Gly 20 25 30? Sp He Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg 35 40 45 He? Sp Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn 50 55 60 Tyr Asn He Met Glu He Arg Thr Val Wing Val Gly He Val Wing He 65 70 75 80 Lys Gly Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys 85 90 95 Leu Tyr Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu Leu 100 105 110 He Leu Glu A-rp His Tyr Asn Thr Tyr Ala Be Wing Lys Trp Thr His 115 120 125 Asn Gly Gly Glu Met Phe Val Wing Leu Asn Gln Lys Gly He Pro Val 130 135 140 Gln Gly Lys Lys Thr Lys Lys Glu Gln Lys Thr Wing His Phe Leu Pro 145 150 155 160 ;: et Ala He Thr (2) INFORMATION FOR SEC. FROM IDENT. NO: 37: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 450 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: CADN (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 37: ATGTCTTCTC CTGAACGTCA TACGCGTTCC TACGACTACA TGGAAGGTGG TGACATCCGC 60 GTACGTCGTC TGTTCTGCC3.TACCCAGTGG TACCTGCGTA TCGACAAACG CGGCAAAGTC 120 ?? GGGCACCC AAGAGATGAA AAACAACTAC AATATTATGG AAATCCGTAC TGTTGCTGTT 180 GGTATCGTTG CAATCAAAGG TGTTGA? TCT GAATTCTACC TGGCAATGAA CA? AGAAGGT 240 ? A? CTGT? CG C? AAA ?? AGA ATGC? ACGAA GACTGCAACT TCAAAGAACT GATCCTGGAA 300 ?? CCACTAC? ACACCTACGC ATCTGCTAAA TGGACCCACA ACGGTGGTGA AATGTTCGTT 360 GCTCTGAACC? GAAAGGTAT CCCGGTTCGT GGT? AAAAAA CCAAAAAAGA ACAGAAAACC 420 GCTCACTTCC TGCCGATGGC AATCACTTAA 450 (2) INFORMATION FOR SEC. FROM IDENT. NO: 38: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 149 amino acids (B) TYPE: amino acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TI PO OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 38: Met Ser Ser Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly 1 5 0. 15 Gly Asp He? Rg Val? Rg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu 20 25 30 Arg He Asp Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn 35 40 45 Asn Tyr Asn He Mee Glu He Arg Thr Val Wing Val Gly He Val Wing 50 55 60 He Lys Gly Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly 65 70 75 80 Lys Leu Tyr Ala 'Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu 85 90 95 Leu He Leu Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp Thr 100 105 110 His Asn Gl? Gly Glu Met Phe Val Ala Leu Asn Gln Lys Gly He Pro (115 120 125 Val Arg Gly Lys Lys Thr Lys Lys Glu Gln Lys Thr Wing His Phe Leu 130 135 140 Pro Met Wing He Thr 145 (2) INFORMATION FOR SECTION ID: 39: (i) CHARACTERISTICS OF THE SEQUENCE : (A) LENGTH: 426 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF SEQUENCE: SEC. NO: 39: ATGTCCTACG ACTAC? TGGA AGGTGGTGAC ATCCGCGT? C GTCGTCTGTT CTGCCGTACC 60 C? GTGGTACC TGCGTATCGA CAAACGCGGC AAAGTCAAGG GCACCCAAGA G? TGA? AAAC 120 ? ACTAC? T? TTATGG? AAT CCGTACTGTT GCTGTTGGTA TCGTTGCAAT C? AAGGTGTT 180 GAATCTGAAT TCTACCTGGC AATGAACAAA GAAGGTAAAC TGTACGCAAA AAAAGAATGC 240 AACGAAGACT GCAACTTCAA AGAACTGATC CTGGAAAACC ACTACAACAC CTACGCATCT 300 GCTAAATGGA CCCACAACGG TGGTGAAATG TTCGTTGCTC TGAACCAGAA AGGTATCCCG 360 GTTCGTGGTA AAAAAACCAA AAAAGAACAG AAAACCGCTC ACTTCCTGCC GATGGCAATC '420 ? CTT ?? 426 (2) INFORMATION FOR SEC. FROM IDENT. NO: 40: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 141 amino acids (B) TYPE: amino acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 40: Met Ser Tyr Asp Tyr Met Glu Gly Gly Asp He Arg Val Arg Arg Leu 1 5 • 10 15 Pha Cys Arg Thr Gln Trp Tyr Leu Arg He Asp Lys Arg Gly Lys Val 20 25 30 Lys Gly Thr Gln Glu Met Lys Asr. Asn Tyr Asn He Met Glu He Arg 35 40 45 Thr Val Wing Val Gly He Val Wing He Lys Gly Val Glu Ser Glu Phe 50 55 60 Tyr Leu Wing Met Asn Lys Glu Gly Lys Leu Tyr Wing Lys Lys Glu Cys 65 70 75 80 Asn Glu Asp Cys Asn Phe Lys Glu Leu He Leu Glu Asn His Tyr Asn 85 90 95 Thr Tyr Ala Ser Ala Lys Trp Thr His Asn Gly Glu Glu Mee Phe Val 100 105 110 Ala Leu Asn Gln Lys Gly He Pro Val Arg Gly Lys Lys Thr Lys Lys 115 120 125 Giu Gln Lys Thr Wing His Phe Leu Pro Met Wing He Thr 130 135 140 (2) INFORMATION FOR SEC. FROM IDENT. NO: 41: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 426 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 41: ATGTCCTACG ACTACATGGA AGGTGGTGAC ATCCGCGTAC GTCGTCTGTT CTGCCGTACC 60 C? GTGGTACC TGCGTATCGA CAA? CGCGGC AAAGTCAAGG GCACCCAAGA GATGAAAAAC 120 AACTACA? T? TTATGC-? A? T CCGTACTGTT GCTGTTGGTA TCGTTGCAAT CAAAGGTGTT '180 GAATCTGAAT TCTATCTTGC AATGAACAAG GAAGGAAAAC TCTATGCAAA GAAAGAATGC 240 AATGAAGATT GTAACTTCAA AGAACTAATT CTGGAAAACC ATTACAACAC ATATGCATCT 300 GCTAAATGGA CCCACAACGG TGGTGAAATG TTCGTTGCTC TGAACCAGAA AGGTATCCCT 360 GT7CA? G3TA AG? AAACCAA GAA? GAACAG AA? ACCGCTC ACTTCCTGCC GATGGCAATC 420 ACTTAA 426 (2) INFORMATION FOR SEC. FROM IDENT. NO: 42: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 141 amino acids (B) TYPE: amino acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 2 Mee Ser Tyr? Sp Tyr Mee Glu Gly Gly Asp He Arg Val Arg Arg Leu 1 5 10 15 Phe Cys Arg Thr Gln Trp Tyr Leu Arg He Asp Lys Arg Gly Lys Val 20 25 30 Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn He Mee Glu He Arg 35 40 45 Thr Val Wing Val Gly He Val Wing He Lys Gly Val Glu Ser Glu Phe 50 55 60 Tyr Leu Wing Met Asn Lys Glu Gly Lys Leu Tyr Wing Lys Lys Glu Cys 65 70 75 80 Asn Glu Asp Cys? Sn Phe Lys Glu Leu He Leu Glu Asn His Tyr Asn 85 90 95 Thr Tyr Ala Ser Wing Lys Trp Thr His Asn Gly Gly Glu Met Phe Val 100 105 110 AI Leu Asn Gln Lys Gly He Pro Val Gln Gly Lys Lys Thr Lys Lys 115 120 125 Glu Gln Lys Thr Wing His Phe Leu Pro Met Wing He Thr 130 135 140 (2) INFORMATION FOR SEC. FROM IDENT. NO: 43: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 43: GAGCTCACTA GTGTCGACCT GCAG 24 (2) INFORMATION FOR SEC. FROM IDENT. NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (ix) ) CHARACTERISTICS: (A.) NAME / KEY: - (B) LOCATION: complement (1..24) (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 44: CTGCAGGTCG ACACTAGTGA GCTC 24 (2) INFORMATION FOR SEC. FROM IDENT. NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 45: CAATCTACAA TTCACAGA 18 (2) INFORMATION FOR SEC. FROM IDENT. NO: 46: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 46: TTAAGTTATT GCCATAGG 18 (2) INFORMATION FOR SEC. FROM IDENT. NO: 47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 47: AACAAAGCTT CTACAATTCA CAGATAGGA 29 (2) INFORMATION FOR SEC. FROM IDENT. NO: 48: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: sEC. FROM IDENT. NO: 48: AACAAGATCT TAAGTTATTG CCATAGG 27 (2) INFORMATION FOR SEC. FROM IDENT. NO: 49: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 49: CGGTCTAGAC CACCATGCAC AAATGGATAC TGACATGG 38 (2) INFORMATION FOR SEC. FROM IDENT. NO: 50: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: cDNA (xi) ) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 50: GCCGTCGACC TATTAAGTTA TTGCCATAGG AAG (2) INFORMATION FOR SEC. FROM IDENT. NO: 51: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE :: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 51: Xaa Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Xaa 1 5 10 Xaa Ser 15 (2) INFORMATION FOR SEC. FROM IDENT. NO: 52: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) POLARITY: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 52 Ser Tyr Asp Tyr Met Glu Gly Gly Asp lie Arg Val 1 5 10 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following is claimed as property.

Claims

1. A method for purifying the keratipocyte growth factor (KGF), characterized in that it consists of the following steps: a) obtaining the solution containing the KGF; b) bonding the KGF of the solution from point (a) to a cation exchange resin; c) elution of KGF in an eluate solution of the ion exchange resin; d) passing the solution of point (c) through an appropriate molecular weight exclusion binder; and e) recovering the KGF from the appropriate molecular weight exclusion binder.

2. The method according to claim 1, characterized in that KGF is produced in prokaryotic cells.

3. The method according to claim 1, characterized in that KGF is produced in E. coli.

. The method according to claim 1, characterized in that KGF is produced in mammalian cells.

5. The method according to claim 1, characterized in that KGF is produced in Chinese hamster ovary cells.

6. The method for purifying the keratipacyte growth factor (KGF), characterized in that it consists of the following steps: a) obtaining the solution containing the KGF; b) bonding the KGF of the solution from point (a) to a cation exchange resin; c) elution of KGF in an eluate solution of the ion exchange resin; d) carrying out the hydrophobic interaction chromatography in the eluate solution of point (c); and e) recovering the KGF from the hydrophobic interaction chromatography of point (d). The method according to claim 6, characterized in that it comprises an additional oxidation step of the sulfhydryl groups in the KGF. The method according to claim 6, characterized in that KGF is produced in prokaryotic cells. The method according to claim 7, characterized in that the KGF is produced in E. coli. . The method according to claim 6, characterized in that KGF is produced in mammalian cells. . The method according to claim 10, characterized in that the KGF is produced in Chinese hamster ovary cells.