CN1169732A - Keratinocyte growth factor analogs - Google Patents

Abstract

Novel analogs of proteins of KGF are provided, comprising a charge-change by the deletion or substitution of one or more of amino acid residues 41-154 of Figure 2 (amino acids 72-185 of SEQ ID NO:2). These analogs are more stable than the corresponding parent molecule KGF.

Description

Keratinocyte growth factor analogs

Invention field

The present invention relates to recombinant DNA technology and protein engineering.Particularly, the utilization recombinant DNA method is to produce the polypeptide analog of keratinocyte growth factor (KGF), and it is a kind of mitogen of effective non-one-tenth fiber epithelial cell growth, and wherein this analogue has been compared improved stability with parental generation KGF.

Background of invention

Tissue generates and the regenerated complex process is mediated by a series of rho factors, and these factors are also referred to as the soft tissue growth factor sometimes.These molecules usually by a class cell discharged and influenced other cell type propagation (Rubin etc. (1989) institute of NAS periodical, 86:802-806).Some soft tissue growth factor by some specific type cells secreted and in the growth course of multi-cell organism, influence the effector cell propagation, differentiation and/or maturation (Finch etc. (1989), science, 245:752-755).Except they effect on the organism of growing, the maintenance of some soft tissue growth factor in continuing healthy and more sophisticated system is more important on.Cell conversion fast for example takes place in many systems in mammalian body.These systems comprise skin, gi tract, and they all include epithelial cell.The included factor of this class soft tissue growth factor is the protein families of fibroblast growth factor (FGFs).

Present eight known FGF family members have dependency on primary structure, they are: Prostatropin, bFGF (Abraham etc. (1986), EMBO J.5:2523-2528); Acid fibroblast growth factor, and aFGF (Jaye etc. (1986), science, 233:541-545); The int-2 gene product, and int-2 (Dickson and Peters (1987), nature, 326:833); Hst/kFGF (Delli-Bovi etc. (1987), cell, (1987) such as 50:729-737 and Yoshida, institute of NAS periodical, 84:7305-7309); FGF-5 (Zhan etc. (1988), molecular cytobiology, 8:3487-3495); FGF-6 (Marics etc. (1989), oncogene, 4:335-340); Keratinocyte growth factor (Finch etc. (1989) and hisactophilin (hisactophilin) (Habazzettl etc. (1992), nature, 359:855-858).

In the FGF protein families, keratinocyte growth factor (" KGF ") is the unique effector that originates from the non-one-tenth fiber of mescenchymal tissue epithelial cell (particularly keratinocyte) propagation.Term " natural KGF " is meant a kind of natural mankind (hKGF) or recombinant polypeptide (rKGF) (it has or do not have signal sequence), and its aminoacid sequence is the sequence shown in the SEQ ID NO:2 or its allelic variation sequence.(unless stated otherwise, number in the mature form (for example removing signal sequence) of the natural molecule that 32 to 194 amino acids show among its amino acid of molecule described herein numbering and the SEQ ID NO:2 corresponding.)

Natural KGF can separate by natural human resource and obtains (hKGF) or by recombinant DNA technology production (rKGF) (Finch etc. (1989), Supra; Rubin etc. (1989), Supra; Ron etc. (1993), journal of biological chemistry 268 (4): 2984-2988; With (1991) such as Yan, cell in vitro developmental biology, 27A:437-438).

Well-known natural KGF at water-soluble state unstable relatively and handling and storage process in take place chemistry, mechanical degradation cause bioactive forfeiture (Chen etc. (1994), study of pharmacy, 11:1582-1589).

Natural KGF when temperature raises, be easy to assemble and under acidic conditions inactivation (Rubin etc. (1989), institute of NAS periodical, 86:802-806).The gathering of natural KGF also causes protein inactivation in the aqueous solution.This point is very inconvenient, because inactivation makes water-soluble preparation stored long period of natural KGF protein and protein use the long period to realize.And this point especially becomes problem when producing pharmaceutical preparation, and (Cleland etc. (1993), Crit. curative drug carrier system identify property comment, 10:307-377 because known accumulative protein has immunogenicity; Robbins etc. (1987), diabetes, 36:838-845; Rinckard etc. (1967), the clinical experiment immunology, 2:331-340).

Recombinant DNA technology has been used for modifying various FGF family members' sequence.For example, by deleting or replacing some positively charged residues and modify bFGF or aFGF, these residues are very important for the amino acid whose heparin of combined belt neutral charge or negative charge.It is reported that these decorating molecules descend heparin binding activity.Correspondingly, known in patient's body by heparin and or the decorating molecule quantity integrated of its analogue can reduce, therefore along with more FGF reaches its target acceptor and improves its effect (EP 0298723).

In order to improve or change one or more characteristics of natural KGF, can adopt protein engineering method.Ron etc. are at journal of biological chemistry 268 (4): reported the modified KGF polypeptide in N- end 3,8,27,38 or 49 amino acids disappearance among the 2984-2988.The polypeptide of those disappearance N- ends 3,8 or 27 amino acids residues still possesses heparin binding activity, and all the other then lose this activity.And it is all active to it is reported that the polypeptide of 3,8 residues of disappearance keeps, but the polypeptide that lacks 27 residues then makes the active decline 10-20 of mitogen doubly, and the polypeptide that lacks 38 or 49 amino acids is then lost whole mitogen activity.But its stability of KGF polypeptide of modifying is not seen discussion or report.

Above disclosed PCT application number 90/08771, supra also reports a kind of chimeric protein, it is combined by the C-terminal part (about 140 amino acid) of terminal preceding 40 amino acid of sophisticated natural KGF N-and aFGF.This block polymer it is reported with the same keratinocyte that is positioned to of KGF, but insensitive to heparin, and this is aFGF but not the characteristics of KGF.And this block polymer stability is not seen discussion and report.

Natural relatively KGF, its stability has significantly improved modification KGF molecule not see that written report is arranged.And do not had any bibliographical information enough example or evidence are to provide the reasonable prospect that can successfully produce the KGF molecule that possesses desired character.

At present still can not be only according to the feature of its primary structure predicted protein matter.For example, aFGF mitogen activity strengthens greatly when heparin is arranged, and the bFGF activity only has a small amount of increase, [(Burgess and Maciag (1989), biological chemistry yearbook, 58:575-606 although heparin can be combined closely with bFGF; Schreiber etc. (1985), institute of NAS periodical, 82:6138-6142; And Gospodarowizc and Cheng (1986), stechiology, 128:475-485); With PCT 90/00418)].On the contrary, its thymidine deoxynucleoside of the BALB/MK cell of growing in containing KGF and heparin substratum mixes and is suppressed.

Usually bioactive effect is decided by series of factors to protein to change amino acid, comprises whether protein three-dimensional structure and this modification locate the prlmary structure of protein sequence at heparin binding domain or receptor binding domains etc.In view of the three-dimensional structure of natural KGF with and heparin binding domain or receptor binding domains etc. locate the primary structure sequence and there is no report, even at these protein of classifying and distinguishing usually, the knowledge in this field also can't carry out summing up based on the amino acid modified effect to natural KGF.

Purpose of the present invention promptly provides the nucleic acid molecule of the polypeptide analog of KGF and this analogue of encoding, and it compares the stability (for example, being in typical pH temperature and/or other storage requirement) that performance improves with natural KGF.

Summary of the invention

The invention provides the bioactive KGF polypeptide analog of new tool.In the present invention, term " KGF " comprises natural KGF and some protein, the peptide sequence that it is characterized in that its peptide sequence and natural KGF is basic identical, and has kept the part or all of biologic activity of natural KGF, especially is non-one-tenth fiber epithelial cell proliferation activity." peptide sequence that it is characterized in that peptide sequence and natural KGF is basic identical " refer to remain with SEQ ID NO:2 in Arg ⁴¹, Gln ⁴³, Lys ⁵⁵, Lys ⁹⁵, Asn ¹³⁷, Gln ¹³⁸, Lys ¹³⁹, Arg ¹⁴⁴, Lys ¹⁴⁷, Gln ¹⁵², Lys ¹⁵³And Thr ¹⁵⁴Corresponding residue, and by one can be coded with the dna sequence dna of 201 to 684 nucleotide sequence hybridizations among the SEQ ID NO:1, preferably be controlled under the strict hybridization conditions.

For determining corresponding amino acid position between two aminoacid sequences, these two sequence contrasts should be arranged and make it to reach maximum residue coupling, comprise the displacement of N-terminal and/or C-terminal, in thing to be selected, introduce the residue that suitable gap and/or deletion are inserted.Database retrieval, sequential analysis and operation can use well-known routine in order to determine that sequence homology/identity scans arithmetic routine and (for example: Pearson and Lipman (1988), institute of NAS periodical, 85:2444-2448 carries out; Altschul etc. (nineteen ninety), molecular biology magazine, 215:403-410; Lipman and Pearson (1985), science, (1984) such as 222:1435 or Devereux, nucleic acids research, 12:387-395).

Stringent condition during hybridization refers to salt, temperature, organic solvent and other stringent condition that typical control parameter etc. combines in hybridization.The stringent condition of demonstration is 4 * SSC62-67 ℃ of hybridization, and 0.1 * SSC cleans in 62-67 ℃ then, and the time is about 1 hour.Perhaps, the stringent condition of another demonstration is at the 45-55% methane amide, 4 * SSC and 40-45 ℃ hybridization.(see T.Maniatis etc., Molecular Cloning (A LaboratoryManual); Cold Spring Harbor Laboratory (nineteen eighty-two), the 387-389 page or leaf).

Like this, this protein comprises amino acid whose allelic variation among the natural KGF, or disappearance, replacement and insertion, also comprises the fragment of natural KGF molecule, chimeric and hybrid molecule.The included proteinic example of KGF is corresponding to Cys among the sequence SEQ ID NO:2 ¹And Cys ¹⁵The position is replaced or is lacked, and gained protein is compared its stability and improved (at the U.S.S.N.08/487 that enjoys jointly, be illustrated in 825, submit to July 7 nineteen ninety-five) greatly with parental generation protein.Concrete disclosed molecule comprises: C (1,15) S, and the amino acid of this KGF on 1 and 15 has been replaced halfcystine by Serine; Δ N15-Δ N24, this KGF deletes any one in preceding 15 to 24 amino acid of natural KGF N-terminal; Δ N3/C (15) S, this KGF delete preceding 3 amino acid of natural KGF N-terminal and replace halfcystine by Serine on 15; Δ N3/C (15)-, this KGF deletes preceding 3 amino acid of natural KGF N-terminal and deleted halfcystine on 15; Δ N8/C (15) S, this KGF have deleted terminal preceding 8 amino acid of natural KGFN and replaced halfcystine by Serine on 15; Δ N8/C (15)-, this KGF has deleted preceding 8 amino acid of natural KGF N-terminal and deleted halfcystine on 15; C (1,15,40) S, this KGF is 1,15, and 40 upper amino acids are replaced halfcystine by Serine; C (1,15,102) S, this KGF is 1,15, and 102 upper amino acids are replaced halfcystine by Serine; And C (1,15,102,106) S, this KGF is 1,15, and 102,106 upper amino acids are replaced halfcystine by Serine.

Another example of KGF comprises these protein, and its () can become the higher amino acid of ring trend to replace Asn by at least one ¹¹⁵-His ¹¹⁶-Tyr ¹¹⁷-Asn ¹¹⁸-Thr ¹¹⁹At least one amino acid within this Cheng Huanqu the and obtain (U.S.S.N.08/323 that is owning together, be illustrated in 473, submit on October 13rd, 1994), comprise H (116) G especially, this KGF replaces Histidine at 116 upper amino acids of natural KGF by glycine.

Another example also comprises these protein, and they have one or more amino acid in the 123-133 district of natural KGF (amino acid/11 54-164 among the SEQ ID NO:2) replaces, disappearance or interpolation; These protein have agonist or antagonistic activity.

Curiously, when a KGF molecule (promptly, the parental generation molecule) deletion or (that is: replace electronegative residue with neutral residue or positively charged residue when replacing neutral or electronegative peptide with positively charged residue, replace neutral residue with positively charged residue), gained KGF analogue has better stability than parental generation KGF molecule.Preferably, except stability increased, the present invention also made these analogues compare with natural KGF and still shows all biological active (that is, keeping essentially identical receptors bind or avidity at least).

Another aspect of the present invention has been described the isolation and purification of the Nucleotide of the active KGF polypeptide analog of coding various biological.In one embodiment, the cloned dna molecule of such Nucleotide composition enters the plasmid or the virus vector of biological function.In another embodiment, utilize the construct of Nucleotide stably to transform protokaryon or eukaryotic host cell.In the another one embodiment, the present invention also is included in suitable culture condition and cultivates with a kind of nucleic acid molecule stable conversion protokaryon (preferred intestinal bacteria) or eukaryotic host cell, makes it expressing K GF analogue.After the expression, the gained recombinant polypeptide can separate and purify.

The KGF analogue that includes therapeutically effective amount and the pharmaceutical preparation of pharmaceutically acceptable carrier of relating in one aspect in addition of the present invention.This preparation can be used for treating the patient of epithelial diseases and damage.

In this case, also relate to the similar method of KGF of using effective in cure amount to the patient on the other hand with the stimulation epithelial cell growth.In one embodiment, the epithelial propagation of non-one-tenth fiber is activated.These epithelial cells comprise: annex cell, pancreatic cell, liver cell and respiratory tract and gastrointestinal mucosal epithelium (cell).

The accompanying drawing summary

Fig. 1 illustrates the Nucleotide (SEQ ID NO:1) of natural KGF and amino acid (SEQID NO:2) sequence, and (it is expressed that the nucleotides sequence of the natural KGF of encoding mature form is classified among the SEQ IDNO:1 201 to 684 bit bases as; The aminoacid sequence of the natural KGF of mature form is that 32 to 194 amino acids residues are expressed among the SEQ ID NO:2).

Fig. 2 A, 2B, 2C illustrate plasmid pCFM 1156 respectively, the collection of illustrative plates of pCFM 1656 and pCFM3102.

Fig. 3 illustrates nucleotide sequence (SEQ ID NO:3) and the aminoacid sequence (SEQ ID NO:4) of construct RSH-KGF.

Fig. 4 illustrates nucleotide sequence included among the plasmid KGF (SEQ ID NO:5) and amino acid (SEQ ID NO:6) sequence.

Fig. 5 illustrates the oligonucleotide fragment (OLIGO#6 to #11 of chemosynthesis; Should be SEQID NO:12-17 mutually), they are used to replace the KpnI of plasmid KGF and the dna sequence dna between EcoRI site (46 to 85 amino acids among the SEQ ID NO:6) to obtain plasmid KGF (dsd).

Fig. 6 illustrates the oligonucleotide fragment (OLIGO#12 to #24 of chemosynthesis; Should be SEQID NO:18-30 mutually), they are in order to make up KGF (the suitableeest codon).

Fig. 7 illustrates nucleotide sequence (SEQ ID NO:31) and the aminoacid sequence (SEQ ID NO:32) of R (144) Q, and this KGF analogue is replaced arginine at natural KGF 144 amino acids places by glutamine.

Fig. 8 illustrates C (1,15) nucleotide sequence of S/R (144) E (SEQ ID NO:33) and aminoacid sequence (SEQ ID NO:34), this KGF analogue is in natural KGF1 position and 15 amino acids places replace halfcystine by Serine and 144 amino acids places replace arginine by L-glutamic acid.

Fig. 9 illustrates C (1,15) nucleotide sequence of S/R (144) Q (SEQ ID NO:35) and aminoacid sequence (SEQ ID NO:36), this KGF analogue is at 1 of natural KGF and 15 amino acids places replace halfcystine by Serine and 144 amino acids places replace arginine by glutamine.

Figure 10 illustrates nucleotide sequence (SEQ ID NO:37) and the aminoacid sequence (SEQ ID NO:38) of Δ N23/R (144) Q, and this KGF analogue lacks preceding 23 amino acid and 144 amino acids places replace arginine by glutamine at the N-terminal of natural KGF.

Figure 11 illustrates soluble protein quantity, and it is a function 37 ℃ of incubation times by size exclusion high performance liquid chromatography (HPLC).

Figure 12 illustrates natural KGF, and the estimation melting temp of C (1,15) S/R (144) Q and C (1,15) S/R (144) E is the function of pH.

Figure 13 illustrates the typical mitogen activity curve of R (144) Q, and the incorporation of 3H-Thymine deoxyriboside determines when being synthesized by mensuration DNA, and it is compared with the typical curve of natural KGF.

Figure 14 illustrates the typical mitogen activity curve of Δ N23/R (144) Q, and the incorporation of 3H-Thymine deoxyriboside determines when being synthesized by mensuration DNA, and it is compared with the typical curve of natural KGF.

Figure 15 illustrates the typical mitogen activity curve of C (1,15) S/R (144) Q, and 3H-Thymine deoxyriboside incorporation determines when being synthesized by mensuration DNA, and it is compared with the typical curve of natural KGF.

Figure 16 illustrates the typical mitogen activity curve of C (1,15) S/R (144) E, and 3H-Thymine deoxyriboside incorporation determines when being synthesized by mensuration DNA, and it is compared with the typical curve of natural KGF.

Figure 17 illustrates nucleotide sequence (SEQ ID NO:41) and the aminoacid sequence (SEQ ID NO:42) of Δ N23/N (137) E, this KGF analogue lacks preceding 23 amino acid at the N-terminal of natural KGF, and replaces l-asparagine at 137 amino acids places by L-glutamic acid.

Figure 18 illustrates nucleotide sequence (SEQ ID NO:43) and the aminoacid sequence (SEQ ID NO:44) of Δ N23/K (139) E, this KGF analogue lacks preceding 23 amino acid at the N-terminal of natural KGF, and replaces Methionin at 139 amino acids places by L-glutamic acid.

Figure 19 illustrates nucleotide sequence (SEQ ID NO:45) and the aminoacid sequence (SEQ ID NO:46) of Δ N23/K (139) Q, this KGF analogue lacks preceding 23 amino acid at the N-terminal of natural KGF, and replaces Methionin at 139 amino acids places by glutamine.

Figure 20 illustrates nucleotide sequence (SEQ ID NO:47) and the aminoacid sequence (SEQ ID NO:48) of Δ N23/R (144) A, this KGF analogue lacks preceding 23 amino acid at the N-terminal of natural KGF, and replaces arginine at 144 amino acids places by L-Ala.

Figure 21 illustrates nucleotide sequence (SEQ ID NO:49) and the aminoacid sequence (SEQ ID NO:50) of Δ N23/R (144) L, this KGF analogue lacks preceding 23 amino acid at the N-terminal of natural KGF, and replaces arginine at 144 amino acids places by leucine.

Figure 22 illustrates nucleotide sequence (SEQ ID NO:51) and the aminoacid sequence (SEQ ID NO:52) of Δ N23/K (147) E, this KGF analogue lacks preceding 23 amino acid at the N-terminal of natural KGF, and replaces Methionin at 147 amino acids places by L-glutamic acid.

Figure 23 illustrates nucleotide sequence (SEQ ID NO:53) and the aminoacid sequence (SEQ ID NO:54) of Δ N23/K (147) Q, this KGF analogue lacks preceding 23 amino acid at the N-terminal of natural KGF, and replaces Methionin at 147 amino acids places by glutamine.

Figure 24 illustrates nucleotide sequence (SEQ ID NO:55) and the aminoacid sequence (SEQ ID NO:56) of Δ N23/K (153) E, this KGF analogue lacks preceding 23 amino acid at the N-terminal of natural KGF, and replaces Methionin at 153 amino acids places by L-glutamic acid.

Figure 25 illustrates nucleotide sequence (SEQ ID NO:57) and the aminoacid sequence (SEQ ID NO:58) of Δ N23/K (153) Q, this KGF analogue lacks preceding 23 amino acid at the N-terminal of natural KGF, and replaces Methionin at 153 amino acids places by glutamine.

Figure 26 illustrates nucleotide sequence (SEQ ID NO:59) and the aminoacid sequence (SEQ ID NO:60) of Δ N23/Q (152) E/K (153) E, this KGF analogue lacks preceding 23 amino acid at the N-terminal of natural KGF, and at 152 amino acids places and 153 amino acids places replace glutamine and Methionin by L-glutamic acid respectively.

Detailed Description Of The Invention

According to the present invention, provide new KGF analog. Lack or replace production KGF analog by one or more specific, positively charged residue place in KGF.

The KGF analog has the stability of improvement under at least a condition in a series of purifying and/or storage requirement except other characteristics. For example, the KGF analog produces with protein a large amount of, that folded form is correct usually. And, in case after the material purifying, compare under the conditions such as (equal) pH and temperature more stable with its parent's molecule. Embodiment part ([R (144) Q and R (144) E) that will describe below], replace arginine at 144 amino acids places by glutamine or glutamic acid respectively) show in the analog, compare with natural KGF, their (1) were increased to 37.2 days in the half-life of 37 ℃ of storages by 35 days; (2) its hot thaw temperature rising 7.5-9.5% in the process of pyrolysis folding; (3) rising of Tm in certain limit pH value.

Although do not want to be fettered by theoretical, R (144) Q and the stability-enhanced possible cause of R (144) E be from the minimizing of cluster alkaline residue in the total charge density, and these residues are intrinsic unstable by electrical charge rejection in without the situation of heparin. Result given below shows that 144 arginine residues may be corresponding with a residue among the bFGF, according to this residue of X-light crystallography research report combination (Ago etc. (1991) close or mediation heparin in the cluster alkaline residue, journal of biological chemistry, 110:360-363; With (1993) such as Eriksson, protein science, 2:1274-1284).

Natural KGF has 46 charged residues, wherein 27 positively charged. For observation post gets the result of KGF analog, natural KGF primary structure and bFGF primary structure compared shows that these 27 charged residues also form in the bFGF primary structure identical bunch. According to the position of these residues at protein three-dimensional structure, realize improving stability thereby replace the electrostatic interaction that one or more residue in this cluster residue may change between the contiguous residue with electronegative or neutral amino acid.

Except preferred R (144) Q of special proposition herein, the present invention has also considered other analog. Used " KGF analog " or " polypeptide analog of KGF " refer to the polypeptide that has one or more residue generation electric charge to change between 41-154 amino acids (72-185 amino acids among the SEQ ID NO:2) in the present invention, are lacked or select neutrality or negative electrical charge residue to replace to affect the protein that positive charge reduces particularly including 123-133 amino acids residue (154-164 amino acids among the SEQ ID NO:2). Preferred residue is Arg during modification⁴¹，Gln ⁴³，Lys ⁵⁵，Lys ⁹⁵，Lys ¹²⁸，Asn ^l37，Gln ^l38，Lys ¹³⁹，Arg ¹⁴⁴，Lys ¹⁴⁷， Gln ¹⁵²，Lys ¹⁵³，Thr ^l54, more preferably be Gln¹³⁸，Lys ¹³⁹，Arg ^l44，Lys ¹⁴⁷，Gln ¹⁵²， Lys ¹⁵³, most preferably be Arg¹⁴⁴ Comprise glutamic acid, aspartic acid, glutamine, asparagine, glycine in order to the preferred amino acid of replacing, alanine, valine, leucine, isoleucine, serine and threonine, its Glutamic Acid, glutamine, aspartic acid, asparagine and alanine are particularly preferred.

Arbitrary modification should be taken into account that all the electrical charge rejection of the three-dimensional structure that makes this molecule reduces; Most preferably analog is compared with parent's molecule and will stability be improved. Clearly, disappearance can not too much can not make residue be too near to and makes between two negative electrical charge residues and produce electrical charge rejection with replacing.

By biological method production when this KGF is similar, such as the product of cellular expression but not during the derivative such as the hydrolysis of solid phase synthetic product or natural generation product or enzymolysis, it is different that the nucleotides of these polypeptide of encoding and the nucleotide sequence of natural KGF have been compared one or more base. These nucleotides can be expressed, and resulting polypeptide can be by one of a series of recombinant technique methods of knowing for those skilled in that art purifying.

Encode the dna sequence dna of all or part of KGF analog except some other structure, also may be included in " preferably " codon (for example, Bacillus coli expression codon) of integrating when expressing among the selected host; The cleavage site of restriction enzyme; But and be convenient to make up expression vector additional initial, stop and middle nucleotide sequence (for example at the initial methionine residues of expression in escherichia coli. )

The present invention also provides to express recombinant molecule or the carrier of polypeptide. These carriers comprise DNA or RNA, and its character can be annular, wire, strand or two strands, can be natural generation or be assembled by a series of natural generations or synthetic part.

This class expression vector is known many examples. The various compositions of carrier, replicon for example, Select gene, enhancer, promoters etc. can by natural resources or known steps is synthetic obtain. In each example, expression carrier used thereof comprises an expression regulation element relevant with the encoded K GF analog nucleotide sequence function of inserting at least among the present invention. This controlling element can be controlled nucleotides developed by molecule polypeptide among the present invention. Useful controlling element comprises, the lac system, the trp system, the promoter of bacteriophage lambda and operon, glycolysis Yeast promoter, leavening acid acid phosphatase promoter, yeast α conjugative element, adenovirus, Epstein-Barr virus, the promoter of polyomavirus and simian virus etc., and the promoter of miscellaneous retroviruses. Certainly, other multiple carrier and control element that is suitable for protokaryon or eukaryotic cell expression known in the art all can be used in this working of an invention.

The embodiment of suitable procaryotic clone carrier comprises from colibacillary plasmid (pBR322 for example, colE1, pUC and F-factor), be preferably pCFM 1156 (ATCC 69702), pCFM1656 (ATCC 69576) and pCFM 3102 plasmids such as (embodiment below partly are illustrated).Other suitable expression also can be in order to realize this purpose as the well-known type of expressing in mammals, insect, yeast, fungi and bacterium.Can be with these carrier transfection appropriate host cell so that the expression of KGF analogue polypeptide.

Useful host microorganism can be protokaryon or eukaryote among the present invention.Suitable prokaryotic hosts comprises various intestinal bacteria (for example FM5, HB101, DH5 α, DH10 and MC1061), pseudomonas, genus bacillus, and streptomycete bacterial strain, wherein preferred intestinal bacteria.Suitable eukaryotic host cell comprises yeast and other fungi, insect cell, vegetable cell, zooblast be COS (COS-1 and COS-7) cell and the strain of CV-1 MC for example, from the Swiss cell strain, the 3T3cells of Balb-c or NIH, HeLa and L-929 mouse cell and CHO, BHK or Hak hamster cell.According to selected host, may or glycosylation not take place by mammals or other eukaryotic carbohydrate glycosylation by the recombinant polypeptide that it produced.

Preferred production methods changes according to different factors and consideration; Is conspicuous to the one skilled in the art to the suitableeest production stage of a certain specified conditions after by a few experiments.The utilization means known in the art can make the gained expression product be purified to and be bordering on homogeneous.Typical case comprises with high pressure or additive method fracturing cell walls by the purification step of prokaryotic cell prokaryocyte production, and is centrifugal or remove by filter cell debris, then allows supernatant or filtrate cross ion exchange chromatography, passes through hydrophobic interaction chromatography at last.If expression product is insoluble form, the step of purification comprises that then dissolving contains the inclusion body of analogue and subsequently ion-exchange chromatography chromatography, protein refolding and hydrophobic interaction chromatography.The enforcement example of purification technique is being enjoyed jointly, and the U.S.S.N.08/323 that submits on October 13rd, 1994 is illustrated in 339.Generally, U.S.S.N.08/323,339 have illustrated the method for the purifying keratinocyte growth factor that comprises the steps: the solution that (a) obtains to contain KGF; (b) KGF in the gained solution in (a) is bonded on the Zeo-karb; (c) with elutriant wash-out KGF from the resin cation (R.C.); (d) with gained solution in (c) by suitable molecular weight exclusion medium or carry out the hydrophobic interaction chromatography chromatography; (e) reclaim KGF by molecular weight exclusion medium or hydrophobic interaction chromatography chromatography.

Certainly, these analogues can be assessed their physical property through rapid screening.Although it is unimportant to be used for detecting the specific experiment of analogue, has still provided various well-known stability among the embodiment and measured.In addition, level of biological activity (for example receptors bind and/or affinity, mitogen activity, cell proliferation and/or activity in vivo) also can detect by using multiple mensuration, propose some of them (method) in an embodiment.Known have several different methods can be used for rapid screening KGF analogue to determine whether they have acceptable biologic activity.In the experiment of this class specific experiment can detect the KGF analogue with ¹²⁵The competition of I-KGF marker is in conjunction with activity (Bottaro etc. (nineteen ninety), journal of biological chemistry, the 256:12767-12770 of KGF acceptor (KGFR); Ron etc. (1993), journal of biological chemistry, 268.:2984-2988).Detect interactional another method of KGFR/KGF analogue relate to use so-called biologic specificity transactional analysis (BIA) technology in real time (Felder etc. (1993), molecule and cytobiology, 13:1449-1455).And mitogen experiment can be used to measure the KGF analogue stimulate DNA synthetic ability (Rubin etc. (1989), above).At last, cell proliferation experiment can be used to detect the ability that the KGF analogue stimulates cellular proliferation (Falco etc. (1988), oncogene, 2:573-578).Use above-mentioned any one experimental system, all the biologic activity of energy rapid screening KGF analogue.

The KGF analogue also can further be modified and contain the unexistent additional chemical ingredients of normal polypeptide.These deutero-compositions may improve its solvability, adsorptivity and biological half time etc.The side effect of not expecting of protein etc. may be eliminated or alleviate to these compositions.(chemistry) composition that can mediate these effects is at REMINGTON ' S PHARMACEUTICAL SCIENCES, 18 editions, Mack publishing company, Easton, PA (nineteen ninety).Use can carry out introducing covalent modification (T.E.Creighton (nineteen eighty-three), protein: structure and characterization of molecules, W.H.Freeman ﹠amp with the organic derivatizing agent and the target amino acid residue on the peptide of side chain of selecting and terminal residue reaction in molecule; Co., San Francisco, 79-86 page or leaf).Polyoxyethylene glycol (PEG) is to be used for one of chemical ingredients for preparing the human cytokines product.In some protein, additional polyoxyethylene glycol can prevent proteolysis, Sada etc. (1991) fermenting organism engineering magazine, and 71:137-139, and also it is effectively available to add the method for certain polyoxyethylene glycol composition.See U.S. Patent number, 4,179,337, Davis etc. " immunogenic polypeptide ", distribution on December 18th, 1979; And U.S. Patent number 4,002,531, Royer " polyethyleneglycol modified enzyme and consequent product ", distribution on January 11st, 1977.See the summaries in Enzymes asdrugs (Holcerberg and Roberts, 367-383 page or leaf (1981)) such as Abuchowski.For polyoxyethylene glycol, can use several different methods to add it to protein.Put it briefly, polyoxyethylene glycol can be connected on the protein by the reactive group on the protein.For example the amino on Methionin and the N-terminal residue is that this type of adds group very easily.For example, Royor (U.S. Patent number 4,002,531) illustrates that reductive alkylation can be used to add polyoxyethylene glycol to enzyme.EP 0539167, published on April 28th, 1993, " Peg imidoether (imidate) and the protein derivatives thereof " of Wright illustrate that the peptide and the organic compound that have the free amino group group can be modified by the imidic acid derivative that gathers ethanol or other water-soluble organic polymer.U.S. Patent number 4,904,584, Shaw submitted to February 27 nineteen ninety, related to by the free amino group group adding the quantity that peg molecule is modified Methionin to the protein.

In another embodiment, the present invention is directed to the pharmaceutical preparation of the single dose unit of taking medicine, it can treat the disease (for example people) of warm-blooded animal safely by gi tract external administration and oral administration.This pharmaceutical preparation is the treatment preparation or the diagnostic preparation of dry powder form or other dehydrated forms, and it can be rebuild by adding physiologically acceptable solvent.This solvent can be a sterilized water, physiological saline, glucose solution or other carbohydrate liquid (N.F,USP MANNITOL for example, polyvalent alcohol such as Xylitol and glycerine) etc. can dissolve the arbitrary medium of dried composition, it can be complementary with selected route of administration and not have the negativity interference effect for the reconstruction of stability agent of effective constituent and use.In a specific embodiment, the present invention is directed to the test kit that produces single dose administration unit.This reagent comprises two containers, is protein dry powder in first container, is the liquid preparation that contains the reconstruction of stability agent in second container.As for protein concn in the solution, fill the volume and the volume of a container (these relevant parameters can suitably be adjusted according to the required concentration of effective constituent final dose unit) of solution in each container, these may in very large range change and be well known to those skilled in the art.

Can be according to KGF analogue of the present invention as the treatment preparation, diagnostic preparation and research preparation.These KGF analogues can be used for external or the in-vivo diagnostic experiment separates the cell of expressing KGFR with quantity and/or the mensuration of KGF in quantitative measurment tissue or the organ samples.(Bottaro etc. (nineteen ninety), journal of biological chemistry, 256:12767-12770; Ron etc. (1993), journal of biological chemistry, 268:2984-2988).In the experiment of tissue or organ ¹²⁵I-KGF analogue and KGFR bonded radioactivity are than standard ¹²⁵The typical curve of I-KGF analogue radioactivity is low, and its reason is that unlabelled natural KGF also combines with KGFR.Equally, ¹²⁵The I-KGF analogue also can be used for detecting the situation that exists of KGFR in the various dissimilar cells.

The present invention considers also and utilizes the KGF analogue to produce the antibody at this peptide that this antibody also combines with natural KGF.In the present embodiment, antibody character can be divided into mono-clonal and polyclonal antibody and be produced by the KGF analogue.Gained antibody preferentially combines with natural KGF, when especially this protein is in native conformation (biologic activity).These antibody can be used for detecting or purifying KGF.

In addition, the present invention also considers to utilize the KGF analogue to find to have the high-affinity or the low-affinity KGF binding molecule of treatment using value, for example uses its conduct KGF medication or KGF activity inhibitor effectively.The thermostability of KGF analogue under physiological condition (for example 37 ℃) identify that this class binding molecule is very important because their temperature dependencies very strong to the affinity tool of KGF then can't be predicted on affinity in the time of 4 ℃.

When using in vivo, need additive during KGF analogue prescription.These additives comprise: buffer reagent, carrier, stablizer, vehicle, sanitas, (for example viscosity modifier and weighting agents) such as tension regulator and antioxidants.The selection of special additive depends on the administering mode of storage form (for example liquid or dry powder) and KGF analogue.This area known proper formulation can be from REMINGTON ' S PHARMACEUTICAL SCIENCES (latest edition), Mack publishing company, Easton, PA.

The KGF analogue can be applied in the tissue by dose therapeutically effective specifically, it is characterized in that this tissue has the non-one-tenth fiber epithelial cell of damage or the enough amounts of clinical apodia.Because KGF combines with heparin, thus possible heparin, heparin sulfate, heparin class glycosaminglycans and heparin class glycosaminoglycan compounds etc. exists the molecule of born of the same parents' external environment also can combine in vivo with KGF.The KGF analogue that reduces with heparin binding activity will have enhanced effectiveness like this because there is more KGF will reach its target acceptor not by the heparin of born of the same parents' external environment or heparitin compound institute chelating.These analogues are more effective in treatment, because it is lower to treat required specific KGF dosage at every turn.

The KGF analogue can be applied in the tissue by dose therapeutically effective, and this tissue is characterized in that having the damage or the non-one-tenth fiber epithelial cell of the enough amounts of apodia clinically.The KGF analogue can successfully use the zone that comprises but do not limit to have: stimulation, hyperplasia and the differentiation of accessory structures such as the hair follicle of burn and shallow degree, degree of depth wounded patient, sweat gland, sebiferous gland; Acceleration is steeped the epithelium regeneration that (Dullosa) causes wound by epidermolysis, and this epidermolysis bubble is epidermis and its lower floor's corium adhesion defects, can cause having opening, the painful blister of serious sickness rate; Prevent baldness and the treatment male sex's bald (disease) and the alopecia of carrying out property of sex of chemotherapy induction; The treatment gastric and duodenal ulcer; Treatment such as Crohn ' s disease (mainly infecting small intestine) and ulcerative colitis enteritis such as (mainly infecting large intestine); Prevent from or alleviate to radiate and chemotherapeutical internal organ toxicity inducing cell protection and/or cell regeneration by treating (for example before the treatment and/or treatment back); Stimulate whole gi tract to produce mucus; Induce the hyperplasia and the differentiation of II type pneumonocyte, but assisting therapy or prevent premature infant's transparent film disease (for example depleted syndromes of infant breathes and pulmonary branches tracheae heteroplasia); Stimulate pulmonary insufficiency acute or that chronic pulmonary is damaged or inhalation injury (comprising the hyperoxia level) causes, pulmonary emphysema are used the chemotherapy of damage lung, the generation and the differentiation of bronchiole/respiratory epithelium cell under ventilator wound or other injury of lung situations; Improve liver function with treatment or prevent damage that liver cirrhosis, fulminant hepatic failure, acute viral hepatitis cause and/or the infringement of toxic liver; Induce corneal cell regeneration, for example treat corneal abrasion; Induce epithelial cell regeneration with carrying out property of treatment gum disease; Induce tympanum epithelial cell regeneration with the damage of treatment eardrum and treatment with prevent that diabetes from showing effect or auxiliary agent during as islet cell transplantation.

Need the epithelial patient of the non-one-tenth fiber of hyperplasia will take the KGF analogue of effective dose." effective dose " refers to and can excited the required KGF analogue dosage of intended effect by the treatment patient, and determined by the attending doctor usually.The factor that influences KGF analogue application dosage generally includes patient's age and general situation, the disease of treatment etc.Typical dosage is 0.001mg/ kg body weight～500mg/ kg body weight.

The KGF analogue can gi tract outer (for example by approach such as intravenous drip, tracheae suction, intramuscular injection, subcutaneous injection or abdominal injections), oral or surperficial use etc. be applied to warm-blooded animal (for example people) safely.The KGF analogue can use one or many to use, and mainly is according to the state of an illness and status of patient.In some case, the KGF analogue can be used for the auxiliary of other treatment and uses jointly with the other drug preparation.

During the following example is included in the present invention is more fully illustrated.Be appreciated that the operation steps that proposes below to modify and do not deviate from aim of the present invention.

Embodiment

The standard method of many operation stepss of Miao Shuing or suitable replacement operation step in the following embodiments, can provide by the molecular biology operational manual of extensive approval, they comprise molecular cloning, second edition, Sambrook etc., press of cold spring harbor laboratory (1987) and modern molecular biology method, Ausabel etc., Greene Publishing Associates/WileyInterscience, New York (nineteen ninety).

Embodiment 1: the DNA of preparation encoded K GF and KGF analogue

Use from zooblast RNA and chemosynthesis (the suitableeest codon of intestinal bacteria) oligonucleotide (OLIGOS) by polymerase chain reaction (PCR) the method KGF full length gene fragment (the natural KGF polypeptide of sequence of encoding) of cloning people.Two methods are described below:

Use is carried out pcr amplification by extracting RNA in the cell of known this polypeptide of generation.At first, human fibroblasts's strain AG 1523A (is obtained by Human Genetic Mutant Cell CultureRepository Institute For Medical Research, Camden, NewJersey) cell is by the guanidine thiocyanate fragmentation, extracting subsequently (according to (1987) biological chemistry yearbooks such as Chomyzinski, the method among the 172:156).Use obtains the cDNA of KGF to the stdn reverse transcription operation steps of whole RNA.Use KGF cDNA as template and encoded K GF gene next-door neighbour 5 ' and the primer 1 (OLIGO#1) of the dna sequence dna of 3 ' end and primer 2 (OLIGO#2) carry out pcr amplification (PCR#1) [9600 type thermo cyclers (and Perkin-Elmer Cetus, Norwalk, CT); 28 circulations; Each circulation comprises 94 ℃ of sex change in following 1 minute, 60 ℃ of annealing in following 2 minutes, 72 ℃ of extensions in following 3 minutes].Get 1 little equal portions in the product of PCR#1 and carry out the pcr amplification second time (PCR#2) as template, cycling condition is except that 50 ℃ of annealing temperatures, and all the other are all same as above.Be the KGF gene of cloning by expression, use nested primers so that at KGF gene two ends generation restriction enzyme site.Use the KGF DNA product among OLIGO#3 and the OLIGO#4 modified PCR #2, make it have the restriction site [PCR#3 of MluI and BamHI at 5 ' end, 3 ' end respectively; 30 circulations; Each circulation comprises 94 ℃ of sex change in following 1 minute, 60 ℃ of annealing in following 2 minutes and 72 ℃ of extensions in following 3 minutes].This DNA is subsequently with MluI and BamHI cutting, phenol extracting, ethanol sedimentation.Again be connected (using the T4 ligase enzyme) after suspending with pCFM 1156 plasmids (Fig. 2 A) that contain " RSH " signal sequence and make up RSH-KGF (Fig. 3).

Connecting product conversion (according to Hanahan (nineteen eighty-three), molecular biology magazine, the method in the 166:557 page or leaf) enters intestinal bacteria FM5 bacterial strain (ATCC:53911) and be laid on LB+ kantlex flat board under 28 ℃.Select some transformant to make it in the liquid nutrient medium that contains 20 μ g/ml kantlex, to grow.By separating RSH-KGF plasmid and order-checking in each culture cell.Because there is a NdeI site KGF gene inside, enter between the NdeI and BamHI site of the expressed plasmid of gained so natural KGF gene directly can not be cloned.This can be connected by three sections (three-way) finishes.BsmI and SstI cutting plasmid RSH-KGF so that single Restriction Enzyme point of contact only to be arranged obtain 3kbp dna fragmentation (containing KGF gene 3 ' end) by the separation of 1% agarose gel electrophoresis.Replace OLIGO#3 to carry out PCR (PCR#4) as mentioned above once more with OLIGO#5.The PCR product separates the dna fragmentation that obtain 331bp with BsmI cutting back by 4% agarose gel electrophoresis with NdeI.The 3rd section that is used for connecting is that plasmid pCFM 1156 cuts the 1.8kbp dna fragmentation that the back is separated to obtain by 1% agarose gel electrophoresis by NdeI with SstI.Connect back (use the T4 ligase enzyme), as above transform, kantlex selection and dna sequencing, a clone who selects contains structure shown in Fig. 4, and this plasmid is called KGF.Because the ribosome bind site of an inside can produce the brachymemma product, the KGF dna sequence dna between single KpnI and EcoRI replaces with the oligonucleotide of chemosynthesis that (OLIGO#6～OLIGO#11) is with the use (Fig. 5) in minimizing internal start site.

Oligonucleotide #1 (SEQ ID NO:7): 5 ＇-CAATGACCTAGGAGTAACAATCAAC-3 ＇

Oligonucleotide #2 (SEQ ID NO:8): 5 ＇-AAAACAAACATAAATGCACAAGTCCA-3 ＇

Oligonucleotide #3 (SEQ ID NO:9): 5 ＇-ACAACGCGTGCAATGACATGACTCCA-3 ＇

Oligonucleotide #4 (SEQ ID NO:10):

5＇-ACAGGATCCTATTAAGTTATTGCCATAGGAA-3＇

Oligonucleotide #5 (SEQ ID NO:11):

5＇-ACACATATGTGCAATGACATGACTCCA-3＇

Oligonucleotide #6 (SEQ ID NO:12):

5＇-CTGCGTATCGACAAACGCGGCAAAGTCAAGGGCACCC-3＇

Oligonucleotide #7 (SEQ ID NO:13):

5＇-AAGAGATGAAAAACAACTACAATATTATGGAAATCCGTACTGTT-3＇

Oligonucleotide #8 (SEQ ID NO:14):

5＇-GCTGTTGGTATCGTTGCAATCAAAGGTGTTGAATCTG-3＇

Oligonucleotide #9 (SEQ ID NO:15):

5＇-TCTTGGGTGCCCTTGACTTTGCCGCGTTTGTCGATACGCAGGTAC-3＇

Oligonucleotide #10 (SEQ ID NO:16):

5＇-ACAGCAACAGTACGGATTTCCATAATATTGTAGTTGTTTTTCATC-3＇

Oligonucleotide #11 (SEQ ID NO:17):

5＇-AATTCAGATTCAACACCTTTGATTGCAACGATACCA-3＇

These oligonucleotide are with the sex change of T4 polynucleotide kinase phosphorylation post-heating.Temperature is slowly reduced to room temperature makes single stranded oligonucleotide form double-stranded fragment.Use T4 ligase enzyme covalently bound oligonucleotide internal viscosity end and double chain oligonucleotide fragment KGF plasmid to KpnI and EcoRI cutting.Novel plasmid is called KGF (dsd).

Use chemosynthesis OLIGO#12～24 to make up the KGF gene of suitable codon of complete intestinal bacteria by pcr amplification.

Oligonucleotide #12 (SEQ ID NO:18): 5 ＇-AGTTTTGATCTAGAAGGAGG-3 ＇

Oligonucleotide #13 (SEQ ID NO:19): 5 ＇-TCAAAACTGGATCCTATTAA-3 ＇

Oligonucleotide #14 (SEQ ID NO:20):

5＇-AGTTTTGATCTAGAAGGAGGAATAACATATGTGCAACGACATG-

ACTCCGGAACAGATGGCTACCAACGTTAACTGCTCCAGCCCGGAACGT-3＇

Oligonucleotide #15 (SEQ ID NO:21):

5＇-CACACCCGTAGCTACGACTACATGGAAGGTGGTGACATCCGT-

GTTCGTCGTCTGTTCTGCCGTACCCAGTGGTACCTGCGTATCGACAAA-3＇

Oligonucleotide #16 (SEQ ID NO:22):

5＇-CGTGGTAAAGTTAAAGGTACCCAGGAAATGAAAAACAACTACAACATC-

ATGGAAATCCGTACTGTTGCTGTTGGTATCGTTGCAATCAAA-3＇

Oligonucleotide #17 (SEQ ID NO:23):

5＇-GGTGTTGAATCTGAATTCTACCTGGCAATGAACAAAGAAGGTAAACT-

GTACGCAAAAAAAGAATGCAACGAAGACTGCAACTTCAAAGAA-3＇

Oligonucleotide #18 (SEQ ID NO:24):

5＇-CTGATCCTGGAAAACCACTACAACACCTACGCATCTGCTAAATGGAC-

CCACAACGGTGGTGAAATGTTCGTTGCTCTGAACCAGAAAAGGT-3＇

Oligonucleotide #19 (SEQ ID NO:25):

5＇-ATCCCGGTTCGTGGTAAAAAAACCAAAAAAGAACAGAAAACCGCTC-

ACTTCCTGCCGATGGCAATCACTTAATAGGATCCAGTTTTGA-3＇

Oligonucleotide #20 (SEQ ID NO:26): 5 ＇-TACGGGTGTGACGTTCCGGGG-3 ＇

Oligonucleotide #21 (SEQ ID NO:27): 5 ＇-CTTTACCACGTTTGTCGATA-3 ＇

Oligonucleotide #22 (SEQ ID NO:28): 5 ＇-ATTCAACACCTTTGATTGCA-3 ＇

Oligonucleotide #23 (SEQ ID NO:29): 5 ＇-CCAGGATCAGTTCTTTGAAG-3 ＇

Oligonucleotide #24 (SEQ ID NO:30): 5 ＇-GAACCGGGATACCTTTCTGG-3 ＇

OLIGO#12～OLIGO#24 designs like this, and the dna sequence dna of the natural KGF total length of promptly encoding just can obtain the double chain DNA sequence (Fig. 6) of expectation respectively by " Waston " chain or the representative of " Crick " chain through pcr amplification.[PCR#5, Model 9600 thermo cyclers, Perkin-Elmer Cetus]; 21 circulations, each circulation are included in 94 ℃ of sex change in following 31 seconds, 50 ℃ of annealing in following 31 seconds and 73 ℃ of extensions in following 31 seconds; Extension step last after 21 circulations finish was still carried out 7 minutes].Behind the pcr amplification, dna fragmentation obtains the 521bp fragment with XbaI and BamHI cutting and is connected with the expression plasmid pCFM 1156 that cuts with same enzyme.PCR#5 utilizes the KGF template of outside primer (100pmoles/100 μ l rxn) oligonucleotide #12 (OLIGO#12) and oligonucleotide #13 (OLIGO#13) and 1 μ l/100 μ l rxn, use OLIGO#20～OLIGO#24 as auxiliary oligonucleotide band (Jayaraman etc. simultaneously, (1992), Biotechniques 12:392) connects OLIGO#14～OLIGO#19 (OLIGO#15～OLIGO#18 is by T4 polynucleotide kinase phosphorylation).Final plasmid is called KGF (the suitableeest codon).

Its dna sequence dna part of all KGF analogues as described herein is from KGF (dsd) or KGF (the suitableeest codon), and perhaps both is compound.These sequences are further modified by inserting in suitable restriction enzyme site to the class dna sequence dna, the amino acid of the KGF analogue that such dna sequence encoding is specific and be to utilize a kind ofly to create to multiple above-mentioned synthetic DNA fragment technology.Any analogue all can intactly be obtained by above-mentioned technology.Although the suitableeest e. coli codon is used in suitable place as the part of general oligonucleotide design, the existence of it part or all in any detected gene can not improve proteinic output in the bacterial cell by these cultivations significantly.Fig. 7-10 and Figure 17-26 proposed specific KGF analogue nucleotide sequence and aminoacid sequence structure one suitable embodiment: R (144) Q (Fig. 7); C (1,15) S/R (144) E (Fig. 8); C (1,15) S/R (144) Q (Fig. 9); Δ N23/R (144) Q (Figure 10); Δ N23/N (137) E (Figure 17); Δ N23/K (139) E (Figure 18); Δ N23/K (139) Q (Figure 19); Δ N23/R (144) A (Figure 20); Δ N23/R (144) L (Figure 21); Δ N23/K (147) E (Figure 22); Δ N23/K (147) Q (Figure 23); Δ N23/K (153) E (Figure 24); Δ N23/K (153) Q (Figure 25); Δ N23/Q (152) E/K (153) E (Figure 26).All are described its dna sequence dna of KGF analogue plasmid herein and all verify.

Embodiment 2: the production in the intestinal bacteria

Use three kinds of different expression plasmids during clone KGF analogue gene.They are pCFM1156 (ATCC#69702), pCFM1656 (ATCC#69576) and pCFM3102 (being respectively Fig. 2 A, 2B and 2C).Use the overlapping oligonucleotide sudden change of PCR method that plasmid pCFM1656 is carried out the conversion of a series of fixed point base and can obtain plasmid p3102.By next-door neighbour 5 ' BglII site (pCFM1656 plasmid base #180) to plasmid replication promotor PcopB, towards the plasmid replication gene, the base pair conversion is as follows: base pair changes base pair among the pCFM 3102 among the pCFM 1656 base pCFM 1656

#??204??????????????T/A??????????????????C/G

#??428??????????????A/T??????????????????G/C

#??509??????????????G/C??????????????????A/T

# 617--insert two G/C base pairs

#??677??????????????G/C??????????????????T/A

#??978??????????????T/A??????????????????C/G

#??992??????????????G/C??????????????????A/T

#?1002??????????????A/T??????????????????C/G

#?1005??????????????C/G??????????????????T/A

#?1026??????????????A/T??????????????????T/A

#?1045??????????????C/G??????????????????T/A

#?1176??????????????G/C??????????????????T/A

#?1464??????????????G/C??????????????????T/A

# 2026 G/C base pair disappearance

#?2186??????????????C/G??????????????????T/A

#?2479??????????????A/T??????????????????T/A#?2498-2501?????????????AGTG?????????????????GTCA

TCAC CAGT# 2641-2647 TCCGAGC base pair disappearance

AGGCTCG# 3441 G/C A/T# 3452 G/C A/T# 3649 A/T T/A# 4556--insert base pair

(SEQ?ID?NO：39)5′-GAGCTCACTAGTGTCGACCTGCAG-3′

(SEQ?ID?NO：40)3′-CTCGAGTGATCACAGCTGGACGTC-5′

As above finding, pCFM 1156, and pCFM 1656 and pCFM 3102 are closely similar and contain a plurality of identical restriction sites.The selection of plasmid is for convenient, and the composition of carrier DNA can the exchange easily with novel plasmid.The host cell that is used for cloning is an intestinal bacteria FM5 bacterial strain (ATCC:53911), and its method for transformation illustrates with reference to (Hanahan (nineteen eighty-three), above method) or with reference to the manufacturer and uses Gene Pulser ^TM(Hercules CA) carries out electroelution to the transfection device for Bio-RadLaboratories, Inc..

At first, by taking out 0.1ml in the frozen suitable bacterial strain storage tube of glycerine to 2 liters of flasks that contain 500ml Luria meat soup, the inoculation culture that beginning is in a small amount fresh, the intestinal bacteria recombinant clone of this expectation have any of the constructed plasmid of pCFM carrier of three expectations.This culture is 30 ℃ of down vibrations 16 hours, be transferred to subsequently fill the 8L sterilization in batches substratum (Tsai etc. (1987), the industrial microorganism magazine is in 15L fermentor tank 2:181-187).

With food ingredient in the Feed#1 substratum give cultivate in batches batching (Tsai etc. (1987), supra).When OD 600 reaches 35, with culture temperature carry fast to 37 ℃ 2 hours with induce the expectation KGF expression, rise to 42 ℃ then so that the sex change of CI repressor.Stop the interpolation of Feed1 and change Feed 2 into, initial feed rate is 300ml/hr.Feed 2 comprises that the 175g/l pancreatin separates peptone, 87.5g/l yeast extract and 260g/l glucose.At 42 ℃ after 1 hour, culture temperature is reduced to 36 ℃, and after this this culture temperature was kept 6 hours.

Stop fermentation, will put into the cell centrifugation pipe of 1L by the centrifugal plastics bag of collecting.At centrifugal 60 minutes sedimentation cells of 400 rpm, go supernatant with the cell paste put-90 ℃ freezing.

At expression in escherichia coli after the various KGF analogues, use following operation steps purifying natural KGF, R (144) Q, C (1,15) S/R (144) E, C (1,15) S/R (144) Q and Δ N23/R (144) Q albumen.By high-density cells fermentation gained cell paste 4 ℃ with 0.2MNaCl, 20mM NaPO ₄, make 10-20% aaerosol solution (mass volume ratio) by suitable high speed shear mixing tank in pH 7.5 solution.The suspension cell pressure-even pulp crusher (APV Gaulin, Inc., Everett, MA) in by three times with cracking.Use suitable heat exchanger that effusive homogenate is cooled to 4-8 ℃.Use JS 4.2 rotary heads that lysate is put J-6B ^TM(Bera CA) at 4 ℃ 4, removed cell residue in the centrifugal 30-60 of 200rpm minute to whizzer for BeckmanInstruments, Inc..Supernatant is poured into carefully prepares 450ml (5em * 23cm) and with 0.2MNaCl, 20mM NaPO in advance ₄, pH 7.5 solution are at 4 ℃ of equilibrated S-Sepharoes FastFlow ^TMResin column (Pharmacia, Piscataway, NJ).At 4 ℃ of 0.4M NaCl, 20mM NaPO with 5 times of column volumes (2250ml) ₄, pH 7.5 solution are washed post, with 5L 0.5MNaCl, and 20mM NaPO ₄, pH7.5 is at 4 ℃ of desired protein of wash-out.Fraction collection 50ml is at the A of monitoring stream fluid ₂₈₀Value.By A ₂₈₀The fraction collection sample that evaluation contains the wash-out material carries out 14% sodium laurylsulfonate-polyacrylamide gel (SDS-PAGE) electrophoresis to confirm existing of expectation polypeptide.

Merge the fraction collection liquid that contains proteins of interest, add isopyknic distilled water subsequently.The sample of dilution is poured into and prepares 450ml (5cm * 23cm) and with 0.4M NaCl, 20mM NaPO in advance ₄, pH 6.8 solution are at 4 ℃ of equilibrated S-Sepharose Fast Flow resin columns.At 4 ℃ of 0.4M NaCl, 20mM NaPO with 5 times of volumes (2250ml) ₄, pH6.8 solution is washed post, with 20 times of column volume 0.4M NaCl, 20mM NaPO ₄, pH6.8 to 0.6M NaCl, 20mM NaPO ₄, the linear gradient elution protein of pH6.8.Equally, at A ₂₈₀Fraction collection 50ml liquid when the place continues to monitor effluent liquid.Those contain proteinic fraction collection liquid merging back, and (Mayberry MA) makes concentrated volume to 30-40ml by YM-10 film (isolating molecular weight is 10,000) for Amicon, Inc. in the 350cc stirred vessel.

Sample to 1 on the concentrated solution, and 300ml (4.4cm * 85cm) to contain 1 * PBS (Dulbecco ' s phosphate buffered saline(PBS), " D-PBS ", no Ca ⁺⁺And Mg ⁺⁺) or 0.15MNaCl, 20mM NaPO ₄, the Superdex-75 of the post damping fluid pre-equilibration of pH7.0 ^TMResin (Pharmacia) post.Treat that sample enters in the post fully, use the post damping fluid by elute protein in the gel filter medium.After this, branch reclaims 10ml and merges those collection liquid that contain analogue (being determined by the 14%SDS-PAGE method).Aforesaid operations such as non-ly refer in particular in addition all carries out under 4-8 ℃.

Analyze

Analysis is derived from colibacillary natural KGF; R (144) Q, C (1,15) S/R (144) E; C (1,15) S/R (144) Q and Δ N23/R (144) Q.

Conformational stability

These protein are at package stability, the folding transformation temperature (T of pyrolysis _m) and wide pH value scope in stability etc. compare.

Natural KGF, R (144) Q, C (1,15) S/R (144) E, C (1,15) S/R (144) Q and Δ N23/R (144) Q also detect in the anti-accumulative effect of elevated temperature.Contain the proteinic sample of 0.5mg/ml with the D-PBS preparation.Every 0.5ml sample adds to respectively in the 3cc type-1 vial.Vial adds behind the rubber plug and seals up 13mm (flip-off) aluminium matter bag mouth.Bottle is put in 37 ℃ of incubators.Take out the soluble proteins that the bottle analysis is lost after placing preset time.By 0.22 μ M Spin-X filter (Costar, Cambridge, MA) the centrifugal visible precipitation of removing in each 250 μ l sample.Subsequently soluble proteins in the filtered soln is carried out the size exclusion high performance liquid chromatography.Quadrature and the incubation time during to 37 ℃ is function construction with its result for gained peak area in the high performance liquid chromatography.

Kinetic curve can estimate this transformation period of having lost the monomeric protein of solubility in view of the above.Table 1 illustrates these protein still keep solvable state KGF when storing for 37 ℃ transformation period.

Table 1

The forfeiture proteic transformation period of soluble and monomeric

Protein	Transformation period (my god)
		Natural KGF R (144) Q C (1,15) S/R (144) Q Δ N23/R (144) Q C (1,15) S/R (144) E	????0.6 ????4.1 ????13.3 ????22.3 ????38.0

As top table 1 and Figure 11 finding, natural KGF assembles the fastest, and the transformation period is 0.6 day.R (144) Q improves transformation period to 4.1 day.C (1,15) S/R (144) Q, Δ N23/R (144) Q and C (1,15) S/R (144) E then show sizable raising, the transformation period was respectively 13.3,22.3,38 days.

Pyrolysis is folding

Pyrolysis is folding, and (Easton is MD) in its garden dichroism (CD) of 230nm place monitoring for Jasco, Inc. by the J-720TM spectropolarimeter of being furnished with PTC-343 Peltier-type temperature controlling system.When carrying out the CD analysis, the single sample that contains the 0.1mg/ml polypeptide that is used for analyzing is by D-PBS (Life Technologies, Inc., Grand Island, NY) preparation.The about 2.5ml of each sample optical path length of packing into is the right angle Suprail of 10mm ^TMQuartzy (HeraeusQuarzschmelze, GmbH, Hanau, Germany) the fluorescence cup (Hellma Cells, Inc., Jamaica, NY).Then this glass put in the Peltier type temperature controlling system of spectropolarimeter.The pyrolysis folding rate is 50 ℃/hour.Change in 230nm place monitoring ovality and to show and separate folded situation.Be tested and appraised the T that under a certain temperature, has 50% protein unfolding to estimate each sample in the solution _mValue (Biophysical Chemistry, Cantor and Schimmel (eds), W.H.Freeman and Co. San Francisco (1980)).Three kinds of proteinic estimation T _mValue is listed in table 2.

Table 2

The melting temp of estimation

Protein	Melting temp (℃)
		Natural KGF R (144) Q C (1,15) S/R (144) Q Δ N23/R (144) Q C (1,15) S/R (144) E	????54.0 ????61.5 ????62.5 ????63.0 ????63.5

Shown in the result, R (144) Q compares its T with natural KGF _mIt is high more than 7 ℃ that value is wanted.If R (144) Q is replaced to then T of C (1,15) S/R (144) Q or Δ N23 _mValue raises 1 ℃ and higher more than 8 ℃ than natural KGF at least.And C (1,15) S/R (144) E is then high more than 9 ℃ than natural KGF equilibrium temperature.Therefore, 144 amino acids are become neutrality by positively charged residue or the negative charge residue can strengthen polypeptide stability greatly.

pH

Add concentrated hydrochloric acid or concentrated sodium hydroxide and transfer D-PBS, relatively the acid acceptance of C (1,15) S/R (144) Q and C (1,15) S/R (144) E and natural KGF to different pH scopes.The D-PBS of the different pH values of about 2.35ml mixes in quartz curette with 100 μ l 2.45mg/ml KGF protein.It is folding and in monitoring garden, 230nm place dichroism that these samples carry out pyrolysis with 50 ℃/speed at one hour rating.Figure 12 shows the T of natural KGF, C (1,15) S/R (144) Q and C (1,15) S/R (144) E _mFunction to pH.In the pH scope of being tested, the T of C (1,15) S/R (144) Q and C (1,15) S/R (144) E _mValue all is higher than KGF.

The extracorporeal biology activity

By the Balb/MK cellular uptake [ ³H]-half maximum concentration of Thymine deoxyriboside and the function of protein concn detected R (144) Q, Δ N23/R (144) Q, C (1,15) S/R (144) Q and C (1,15) the external mitogen activity of S/R (144) E (with reference to (1989) such as Rubin, method above).

Usually each KGF analogue its concentration relevant with known natural KGF can be used the external biological assay determination.Each KGF analogue dilution back is used Balb/MK mitogen to test and is detected its biologic activity.At first with the dilution of bioanalysis substratum, this substratum comprises 50% Eagle ' s MEM customized to each sample, 50% Eagle ' s MEM customized, 5 μ G/ml Transferrins,iron complexess, 5ng/ml Sodium Selenite, 0.0005% HSA and 0.005% polysorbas20.The KGF sample is added inoculation subsequently to be had in Falcon primeria 96 orifice plates of Balb/MK cell.When mensuration DNA is synthetic [ ³H]-Thymine deoxyriboside mixes and the natural KGF typical curve of reference is changed the natural KGF concentration that is counted as adding.The result is shown in the 13-16.As Figure 13-16 finding, every kind of KGF analogue all has the mitogen activity.

Although above description of the present invention is generality and optimization embodiment, is appreciated that according to foregoing description those skilled in the art and still can carries out other variations and improve.

Claims (16)

1. the polypeptide analog of a natural KGF includes the change in electrical charge that one or more amino-acid residue causes by lacking or replacing in the 41-154 amino acids residue shown in Fig. 2 (72-185 amino acids among the SEQ ID NO:2).

2. according to the polypeptide analog of claim 1, wherein the amino acid of disappearance or replacement is selected from Arg ⁴¹, Gln ⁴³, Lys ⁵⁵, Lys ⁹⁵, Lys ¹²⁸, Asn ¹³⁷, Gln ¹³⁸, Lys ¹³⁹, Arg ¹⁴⁴, Lys ¹⁴⁷, Gln ¹⁵², Lys ¹⁵³And Thr ¹⁵⁴

3. according to the polypeptide analog of claim 1, be selected from R (144) Q, C (1,15) S/R (144) Q, C (1,15) S/R (144) E and Δ N23/R (144) Q.

4. a pharmaceutical preparation comprises a kind of KGF polypeptide analog and the pharmaceutically acceptable carrier according to claim 1 for the treatment of significant quantity.

5. a pharmaceutical preparation comprises cryodesiccated a kind of KGF polypeptide analog according to claim 1 for the treatment of significant quantity.

6. the pharmaceutical preparation in the claim 4 also comprises pharmaceutically acceptable carrier.

7. nucleic acid molecule that is selected from DNA and RNA, the polypeptide analog of a kind of natural KGF of this nucleotide coding wherein, it includes among Fig. 2 shown in (72-185 amino acids among the SEQ ID NO:2) one or more amino acid in the 41-154 amino acids residue by disappearance or replace and the change in electrical charge that causes.

8. according to a kind of nucleic acid molecule of claim 7, wherein the amino acid of disappearance or replacement is selected from Arg ⁴¹, Gln ⁴³, Lys ⁵⁵, Lys ⁹⁵, Lys ¹²⁸, Asn ¹³⁷, Gln ¹³⁸, Lys ¹³⁹, Arg ¹⁴⁴, Lys ¹⁴⁷Gln ¹⁵², Lys ¹⁵³And Thr ¹⁵⁴

9. according to a kind of nucleic acid molecule of claim 7, wherein polypeptide analog is selected from R (144) Q, C (1,15) S/R (144) Q, C (1,15) S/R (144) E and Δ N23/R (144) Q.

10. the plasmid of a tool biological function or virus vector comprise a kind of nucleic acid molecule according to claim 7.

11. protokaryon or eukaryotic host cell with a middle tool biological function carrier transfection stably or a conversion according to Claim 8.

12. the prokaryotic host cell according to claim 11, it is intestinal bacteria.

13. the eukaryotic host cell according to claim 11, it is a mammalian cell.

14. the eukaryotic host cell according to claim 11, it is a Chinese hamster ovary cell.

15. method of producing the KGF polypeptide analog, it comprises according to the protokaryon of a kind of nucleic acid molecule stable conversion in the claim 7 or eukaryotic host cell grows under suitable nutritional condition, this mode allows the expression of coded polypeptide analogue, and separates consequent polypeptide analog.

16. a method that stimulates non-one-tenth fiber epithelial cell to produce, it comprises makes these cells contact with the KGF polypeptide analog according to claim 1 of effective dose.

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