JP2000236880A - Method for producing protein variant, method for producing protein variant library, and method for producing library of cDNA encoding protein variant - Google Patents

Abstract

(57) [Object] To construct a library of mutant proteins by inserting a peptide into an arbitrary site on the primary structure of a protein molecule, and to select a desired mutant protein from the library. Aim. The present invention encodes a variant having an amino acid or peptide introduced at a random site on the primary structure of a protein.
Create a library of cDNAs and include the cD
A method for producing a library of protein variants, comprising expressing a protein variant encoded by NA. A library of cDNAs encoding mutants in which amino acids or peptides are introduced at random sites in the primary structure of the protein is prepared, and the protein mutants encoded by the cDNAs in the library are expressed. A method for producing a protein variant, comprising selecting a cell expressing the protein variant, and causing the cell to produce the protein variant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing a mutant protein in which an arbitrary amino acid or peptide is introduced into a random site on the primary structure of a protein, a method for preparing a library of the mutant protein, and a method for producing the mutant protein. The present invention relates to a method for preparing a library of cDNAs encoding proteins.

[0002]

2. Description of the Related Art Conventionally, genetic engineering techniques have been used in addition to chemical techniques to modify the primary structure of proteins that are linear polymers of amino acids. Modification of the protein structure using genetic engineering techniques involves substitution of bases on the DNA structure,
It has been common practice to cause substitutions, deletions and introductions of amino acids by causing deletions and introductions and translating them. However, the introduction of such mutations
Only specific mutations could be introduced at the intended specific site and could not be introduced at unintended random sites.

[0003] Further, in the prior art, in order to prepare various mutant proteins, cDNAs encoding the respective proteins had to be separately prepared and isolated. In recent years, the elucidation of functional domains has been advanced by studying protein structure-activity relationships, and techniques for introducing heterologous peptides having various functions on the primary structure of any protein have been developed. In order to determine the position of introduction, it was necessary to use trial and error.

Further, in recent years, as the importance of the sugar chain of a glycoprotein to the function of the protein portion has been understood, a peptide having a sequence to which a sugar chain can be added on the primary structure of a known protein has been introduced. Techniques for causing chain addition have been developed, but optimization of the introduction position of the amino acid to which the sugar chain has been added can only be achieved by trial and error.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to obtain a protein having no structure-activity correlation information leading to a function modification or a protein having a function modified by performing a structural modification of a protein whose function modification cannot be predicted. Another object of the present invention is to provide a technique for producing a protein mutant library in which an arbitrary amino acid or peptide is introduced into a random site on the primary structure. Another object of the present invention is to provide a technique for optimizing an amino acid or peptide introduction site for obtaining an intended function-modified protein by selecting a protein having a desired function from the mutant library.

[0006] Another object of the present invention is to obtain a protein having no structure-activity relationship information leading to a function modification or a protein having a function modified by introducing a sugar chain modification to a protein whose function modification cannot be predicted. It is an object of the present invention to provide a technique for producing a sugar chain-modified protein mutant library in which a sugar chain-modified amino acid or peptide is introduced into a random site on the primary structure. Furthermore, by selecting a glycoprotein that has been changed to a desired function from the sugar chain-modified mutant library, optimization of a sugar chain-modified amino acid or peptide introduction site for obtaining an intended function-modified glycoprotein is aimed at. To provide technology.

[0007]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors cut a circular plasmid containing a cDNA encoding a protein at one random position and cut this site. Encodes an amino acid or peptide
By introducing DNA, a technique for introducing an amino acid or peptide at an arbitrary position on the primary structure of a translated protein was completed. This has made it possible to construct a mutant library in which amino acids or peptides have been introduced at various sites on the primary structure of the protein.

The present inventors applied the technique to the secreted simple protein secFGF-1 and introduced various sugar chain-added sequence amino acids to produce secFGF-1 as a sugar chain-modified protein. Successful. In this specification,
“FGF” refers to fibroblast growth factor (fibroblast growth factor).
factor). Furthermore, the present inventors have also completed a technique for selecting a function-modified secFGF-1 from the library, and as a result, have completed a technique for completing a function modification by introducing a mutation into a protein in a single step. The present invention has been completed based on such findings.

That is, the present invention encodes a protein
randomizing the primary structure of a protein, comprising incorporating the cDNA into a circular plasmid, cutting the circular plasmid at a random position, and introducing a DNA encoding an amino acid or peptide into the cut site. Coding for a variant in which an amino acid or peptide has been introduced at a unique site
A method for producing a library of NA is provided. As used herein, "cD encoding a variant having an amino acid or peptide introduced at a random site on the primary structure of a protein.
The `` NA library '' refers to a population of cDNAs encoding various mutants in which amino acids or peptides are introduced at random sites in the primary structure of the protein, and these cDNAs are
Usually, it is in a state of being integrated into a vector such as a plasmid or a virus.

The method for preparing a cDNA library of the present invention comprises the following steps: (a) a step of incorporating a cDNA encoding a protein into a circular plasmid; and (b) a step of cutting the circular plasmid at one random position. (C) introducing a DNA encoding an amino acid or peptide into the cleavage site of the plasmid, wherein the DNA encoding the amino acid or peptide is introduced;
Adding additional nucleotides to the 5 'end and / or 3' end of to obtain a clone whose amino acid or peptide frame matches the frame of the protein into which it is introduced, ( d) a step of introducing a plasmid into which the DNA encoding the amino acid or peptide has been introduced into a host cell to obtain a transformant; (e) a step of recovering the plasmid from the transformant; and (f) a step of recovering the plasmid. DNA encoding amino acids or peptides and cDNA encoding proteins
And (g) cutting out the DNA in the region containing
The method may include the step of incorporating NA into an expression vector, the step of (h) introducing the expression vector into a host cell, and the step of (i) expressing a protein variant in the host cell. In the step (c), a DNA encoding an amino acid or a peptide is introduced into a cleavage site of the plasmid in a state linked to the selection marker gene, and in the step (d), the DNA is It is preferable to select a transformant and remove the selection marker gene in the step (f).

[0011] The present invention also provides a cDNA library encoding a mutant in which an amino acid or peptide is introduced at a random site on the primary structure of a protein, and the cDNA in the library encodes the cDNA. The present invention provides a method for preparing a library of protein variants, which comprises expressing a protein variant. As used herein, the term “library of protein variants” refers to a population of various protein variants.

Further, the present invention provides a cDNA library encoding a mutant in which an amino acid or a peptide is introduced at a random site on the primary structure of a protein, and the cDNA in the library encodes the cDNA. A method for producing a protein variant, comprising expressing a protein variant, selecting cells expressing the protein variant of interest, and causing the cells to produce the protein variant. The method for producing a protein mutant of the present invention comprises the following steps: (a) preparing a cell line showing a specific response to the protein; and (b) amino acid or peptide at a random site on the primary structure of the protein. CDNA encoding the introduced mutant
A step of preparing a cDNA library by introducing an expression vector incorporating the above into the cell line, (c) a step of detecting the response and selecting a cell showing a response, and (d) a step of: The method may include a step of culturing and recovering the target protein mutant from the culture solution and / or the cultured cells.

The protein to be mutated may be any protein. As an example, a factor belonging to the FGF family and a factor closely related thereto, a factor belonging to a heparin-binding growth factor family and a factor closely related thereto can be mentioned. More specifically, the following (a) to (f) )
Protein. (A) a protein consisting of the amino acid sequence of SEQ ID NO: 2 (FGF-1a); (B) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, added or modified in the amino acid sequence of SEQ ID NO: 2, and having FGF-1 activity;

(C) a protein consisting of the amino acid sequence of SEQ ID NO: 4 (FGF-6 / 1a); (D) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, added or modified in the amino acid sequence of SEQ ID NO: 4, and having FGF-6 / 1a activity. (E) a protein consisting of the amino acid sequence of SEQ ID NO: 6 (secFGF-1); (F) a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted, added or modified in the amino acid sequence of SEQ ID NO: 6, and having secFGF-1 activity.

The amino acid or peptide to be introduced may be any amino acid or any peptide composed of two or more amino acids, but a functional peptide is particularly preferable because it can be expected to alter the protein function. For example, a sugar chain-added mutant can be prepared by introducing an amino acid or peptide capable of undergoing sugar chain addition. Peptides that can undergo sugar chain addition include Asn-X-Ser, Asn-X-Thr, and YT
hr-Pro-Z-Pro, Ser-Gly (X, Y, Z are any amino acids)
And a wide variety of peptides including these.

When the sugar chain is a sulfated polysaccharide or glycosaminoglycan, the peptide to which the sugar chain can be added is preferably a proteoglycan core protein or a part thereof. When the sugar chain is an N-type sugar chain (asparagine-linked sugar chain), the peptide to which the sugar chain can be added is preferably a peptide containing an N-type sugar chain-added amino acid sequence. When the sugar chain is an O-type sugar chain (serine / threonine-linked sugar chain), the peptide to which the sugar chain can be added is preferably a peptide containing an O-type sugar chain-added amino acid sequence.

Further, the present invention provides a novel protein mutant that can be produced by the above-described method for producing a protein mutant. Examples of the protein mutant include a mutant of a factor belonging to the FGF family or a closely related factor, a mutant of a factor belonging to the heparin-binding growth factor family or a closely related factor, and the like. For example, mutants of the above-mentioned proteins (a) to (f), specifically, SEQ ID NO: 2
FGF-1 variants comprising the amino acid sequence of 5, 27, 29, 31, 33 or 35 can be mentioned. Hereinafter, the present invention will be described in detail.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a protein into which an amino acid or peptide is introduced may be any protein. The amino acid or peptide to be introduced may be any amino acid or peptide. Hereinafter, as a specific example, heparin-binding growth factor FGF
An example of introducing a peptide containing a sugar chain binding sequence into -1 is shown, but the present invention is not limited to this. Examples of the protein into which the peptide is introduced include the following (a) to
The protein may be any one of (f).

(A) A protein comprising the amino acid sequence of SEQ ID NO: 2 (FGF-1a). (B) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, added or modified in the amino acid sequence of SEQ ID NO: 2, and having FGF-1 activity; (C) a protein consisting of the amino acid sequence of SEQ ID NO: 4 (FGF-6 / 1a); (D) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, added or modified in the amino acid sequence of SEQ ID NO: 4, and having FGF-6 / 1a activity. (E) a protein consisting of the amino acid sequence of SEQ ID NO: 6 (secFGF-1); (F) a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted, added or modified in the amino acid sequence of SEQ ID NO: 6, and having secFGF-1 activity.

The proteins having the amino acid sequences of SEQ ID NOs: 2, 4, and 6 are, for example, those of SEQ ID NOs: 1, 3, and 5,
Respectively. Some of these proteins include a peptide sequence capable of undergoing sugar chain addition and a signal peptide sequence in addition to the peptide sequence of a factor belonging to the FGF family. The heparin-binding protein and FGF referred to in the present specification are, in addition to a protein primarily defined by the cDNA described in the sequence listing, a secretory signal peptide present at the amino terminus when secreted from a cell. Includes truncated forms of the peptide sequence.

Hereinafter, a specific example in which a peptide containing a sugar chain binding sequence is introduced will be described, but the present invention is not limited thereto. Sugar chains covalently bonded to proteins include sulfated polysaccharides such as heparan sulfate and chondroitin sulfate,
Glycosaminoglycans, N-type sugar chains, and O-type sugar chains can be exemplified, but are not limited thereto. The sulfated polysaccharide referred to in the present specification extends from xylose bonded to a serine residue present in the primary structure of a protein, or extends to a non-reducing terminal side of an N-type sugar chain or an O-type sugar chain described below. It is a general term for a variety of extended or free sugar chain structures, many of which have a repeating structure of disaccharide units of amino sugar and uronic acid (or galactose), and have several hydroxyl or amino groups. Is substituted with a sulfate group.

Glycosaminoglycans have the same structure, but also include those having no substitution with a sulfate group. In the present specification, these are collectively referred to as sulfated polysaccharides and the like. The specific structure is described in “Fate of sugar chain in cell” (edited by Nagai, Hakomori and Kohata, Kodansha Scientific), and a typical sugar chain sequence is shown in FIG. The term "N-type sugar chain" as used herein is a general term for various sugar chain structures that extend from N-acetylglucosamine bound to an asparagine residue in the primary structure of a protein. The specific structure is described in “Fate of Sugar Chain in Cell” (edited by Nagai, Hakomori and Kohata, Kodansha Scientific). A typical sugar chain sequence is shown in FIG.

As used herein, the term "O-type sugar chain" is a general term for a variety of sugar chain structures extending from N-acetylgalactosamine bound to serine or threonine residues in the primary structure of a protein. It is. The specific structure is
It is described in “Fate of sugar chains in cells” (edited by Nagai, Hakomori and Kohata, Kodansha Scientific), and a typical sugar chain sequence is shown in FIG. Such as these sulfated polysaccharides,
The N-type sugar chain and the O-type sugar chain may have an addition, a deletion, a substitution or a modification in a part of the sugar chain sequence as long as they exert their functions.

As used herein, “function modification” means that the activity of a target protein is changed. Examples of the function modification include not only simple modification of physiological activities (proliferation promoting activity, differentiation promoting activity, etc.), but also a change in necessity of a cofactor required for activity expression, resistance to heat, acid or alkali. Changes in physical stability such as changes, changes in biological stability indicated by changes in blood half-life, and the like. FGF- secreted peptide containing glycosylation sequence
An example of the method for producing sugar chain-added FGF-1 introduced into the above-described method is described below.

The cDNA of the protein into which the peptide is introduced is DDBJ
It can be obtained by designing appropriate primers from sequences registered in a gene bank such as Japan DNA Data Bank and performing RT-PCR (reverse transcription PCR) from mRNA of the tissue of the animal. By ligating the obtained cDNA to an appropriate plasmid, the construction of the gene encoding the protein is completed. As a plasmid incorporating the cDNA,
Any of those that can be replicated and maintained in the host can be used.For example, pBR322 and pUC18 derived from Escherichia coli can be used.
And pET-3c constructed on the basis of these.

When the protein is a non-secretory protein having no secretory signal, a secretory protein can be obtained by adding an appropriate secretory signal to its 5′-terminal. It can be placed on a chain addition pathway, and can be easily purified and confirmed for its activity. As an appropriate secretion signal, for example, the amino terminus of a typical secretory glycoprotein can be used, and specific examples include amino acids of the mouse FGF-6, which are 40 residues from the N-terminus.

By subjecting the obtained plasmid to an appropriate treatment, the vector can be cut at one site at an arbitrary site. Examples of this treatment include DNase I treatment and ultrasonic treatment in the presence of 10 mM Mg2 ⁺ .
It is not limited to these. When the DNase I treatment in the presence of 10 mM Mg ²⁺ is performed at 37 ° C. for 5 minutes or less, the frequency of cleavage at two or more sites in the plasmid can be suppressed. The cut vector can be separated from the uncut vector by subjecting it to, for example, agarose electrophoresis.

The DNA encoding the peptide to be introduced can be obtained by arbitrary synthesis based on the amino acid translation codon table. Alternatively, it is also possible to obtain a cDNA encoding a naturally occurring functional peptide by the method described above. For example, amino acid sequences that undergo N-glycosylation include Asn-X-Thr and Asn-X-Ser (in the sequence, X is any amino acid). For example, D shown in SEQ ID NO: 7
Examples include, but are not limited to, DNAs containing NA sequences.

For example, the amino acid sequence subjected to O-glycosylation includes X-Thr-Pro-X-Pro (in the sequence, X is any amino acid). Examples include, but are not limited to, DNAs containing the DNA sequence shown in SEQ ID NO: 8. For example, the amino acid sequence subjected to glycosaminoglycan addition includes a Ser-Gly repeating sequence, and the DNA encoding the same includes, for example, a DNA containing the DNA sequence shown in SEQ ID NO: 9. However, the present invention is not limited to this.

For example, another amino acid sequence subjected to glycosaminoglycan addition includes a partial sequence of a core protein of proteoglycan, and the DN
A includes, for example, the DNA sequence shown in SEQ ID NO: 10.
DNA includes, but is not limited to. The amino acid sequence encoded by the DNA sequence shown in SEQ ID NO: 10 is shown in SEQ ID NO: 11. Encodes the peptide
By fusing a selectable marker gene (for example, a drug resistance gene) to both ends, one end, or the inside of DNA, selection of the introduced mutant can be facilitated. At this time, an appropriate adapter sequence can be used. When a selectable marker gene is fused therein, self-ligation can be performed after removal of the selectable marker gene to design a DNA sequence encoding the peptide.

Furthermore, a mixture of a fusion gene of a DNA encoding the peptide and a selectable marker gene, to which both ends or one end thereof are added nucleotides whose total number of bases is 3n (n is an integer) is used. This makes it possible to align the reading frames of the gene of the protein. The fragment of the fusion gene of the DNA encoding the peptide and the selectable marker gene is ligated to the vector cut at one site, thereby completing the introduction of the cDNA encoding the peptide into the protein gene. For example, T. Maniatis et al., Molecular Cloning,
The method described in Cold Spring Harbor Laboratory, p. 239 (1982) can be mentioned. Specifically, a method generally used in molecular biology, such as a method using DNA ligase, can be applied.

The resulting vector is introduced into a suitable host, and then cultured in the presence of a selective drug, whereby only Escherichia coli containing the vector linked to the gene is obtained as a viable cell. Examples of the host include E. coli DH5α strain. Selectable marker genes include ampicillin resistance gene (Amp), chloramphenicol resistance gene
(Cm), tetracycline resistance gene (Tc), kanamycin resistance gene (Km) and the like.

The plasmid is recovered from the resulting mixture of the transformants, and the drug resistance marker gene is removed by treatment with a restriction enzyme, followed by self-ligation to complete the construction of the peptide-introduced protein gene library. From the plasmid library prepared as described above, a region containing a nucleotide sequence encoding a secretion signal, an introduced peptide and a protein to be introduced (hereinafter, referred to as a “region containing a nucleotide sequence encoding an introduced mutant protein”). Is excised and ligated downstream of a promoter in a vector suitable for expression, whereby an expression type vector library can be obtained.

The region containing the nucleotide sequence encoding the introduced mutant protein has ATG as a translation initiation codon at its 5'-terminal, and TAA, TGA or TGA as a translation termination codon at its 3'-terminal. May have a TAG. Further, to express a protein encoded by the coding region, a promoter is connected upstream thereof. The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. If the host to be transformed is Bacillus subtilis, SP
When the host is yeast, such as 01 promoter, SP02 promoter, penP promoter, PHO5 promoter,
PGK promoter, GAP promoter, ADH promoter and the like. When the host is an animal cell, examples include an SV40-derived promoter and a retrovirus promoter.

By introducing a vector containing the above-mentioned recombinant DNA into a host cell, a transformant carrying the vector is produced. Any host cell may be used as long as it is appropriate for the purpose. For example, when the objective is to express a glycosylated mutant protein by introducing a glycosylated sequence peptide, a protein having a glycosylated pathway to a protein, such as yeast (for example, Pichia pasto ris, Sacc
haromyces cerevisiae), animal cells (eg, COS cell,
CHOcell, BHK cell, NIH3T3 cell, BALB / c3T3 cell, HU
VE cell, LEII cell), insect cell (for example, Sf-9 cell
, Tn cell) and the like. But,
It is not limited to these.

The above transformation is carried out by a method generally used for each host. In addition, any method that is not general but can be applied may be used. For example, if the host is yeast, a vector containing the recombinant DNA is introduced into competent cells prepared by the lithium method or other methods by a temperature shock method or an electroporation method. If the host is an animal cell, a vector containing the recombinant DNA is introduced into cells in the growth phase or the like by the calcium phosphate method, lipofection method or electroporation method.

By culturing the thus obtained transformant in a medium, a mutant protein is produced.
When culturing a transformant, a medium generally used for each host is used as a culture medium. Alternatively, any medium that is not general but can be used may be used. As an example, if the host is yeast, a YPD medium or the like is used. If the host is an animal cell, Dulbecco's
For example, MEM to which animal serum is added is used.

The cultivation is performed under conditions generally used for each host. In addition, it is sufficient if the condition is applicable even if it is not general. For example, if the host is yeast, the reaction is performed at about 25 to 37 ° C. for about 12 hours to 2 weeks, and aeration and stirring can be added as necessary. Host of about 32 to 37 ° C. If animal cells, under conditions of 5% CO _2, 100% humidity for about 24 hours to
It is carried out for 2 weeks, and the gas phase conditions can be changed and stirring can be added as necessary.

When selecting a function-modified mutant from the expression type vector library, an appropriate method is selected based on the properties of the protein. For example, when introducing a peptide into the FGF-1 protein and selecting a mutant having altered growth factor activity, a series of techniques shown below are applied,
It is not limited to this. First, a floating cell line exhibiting FGF-1-specific growth is obtained, an expression type vector library is transfected with the obtained cell line, and the resulting gene is subjected to the following function-modified mutant FGF-1 selection system.

Cell lines showing FGF-1-specific proliferation include, but are not limited to, mouse (pro B cell line) Ba / F3 transfected with FGF receptor gene. When the cell line into which the above-mentioned expression type vector library is transfected is subjected to limiting dilution and cultured in a liquid medium containing heparin and fetal bovine serum, a functionally modified mutant is obtained.
FGF-1 secreting clones form colonies by autocrine or paracrine. After selecting the clone, by analyzing the gene sequence of the expressed FGF by RT-PCR or the like, the structure of the function-modified FGF-1, that is, the optimal site of the introduced peptide can be determined.

In order to obtain a function-modified mutant FGF-1 from the obtained function-modified mutant FGF-1 secreting strain, those released into the culture can be directly recovered from the supernatant after centrifugation.
When extracting from cultured cells or cells, after culturing, the cells or cells are destroyed by homogenizer, French press, ultrasonic waves, lysozyme and / or freeze-thawing to remove the target protein from the cells. Can be eluted to obtain the protein from the soluble fraction. When the target protein is contained in the insoluble fraction, a method in which cells or cells are disrupted, the insoluble fraction is recovered by centrifugation, and the recovered protein is made soluble with a buffer containing guanidine hydrochloride or the like, and then recovered. In addition, there is a method in which cells or cells are directly destroyed by a buffer containing a protein denaturant such as guanidine hydrochloride, and a target protein is eluted outside the cells.

In order to purify the function-modified mutant FGF-1 from the supernatant, known separation and purification methods can be appropriately combined. These known separation and purification methods include salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, S
DS polyacrylamide gel electrophoresis, ion exchange chromatography, affinity chromatography,
Reversed-phase high-performance liquid chromatography, isoelectric focusing and the like can be used. Furthermore, affinity chromatography using heparin sepharose as a carrier can be applied to many heparin-binding proteins.

The preparation thus obtained can be dialyzed and freeze-dried to a dry powder as long as the activity of the function-modified mutant FGF-1 is not impaired. further,
Preservation by adding serum albumin or the like as a carrier is effective for preventing adsorption of the sample to the container. Also,
The coexistence of a trace amount of a reducing agent in the purification step or the storage step is suitable for preventing oxidation of the sample. Examples of the reducing agent include β-mercaptoethanol, dithiothreitol, and glutatin. Function-modified mutant FG of the present invention
F-1 can also be produced by introducing a peptide into a protein by a chemical method. As described above, the present invention has been described using the function-modified mutant FGF-1 as an example. However, amino acids or peptides can be introduced into random sites in proteins other than heparin-binding proteins.

[0044]

[Deposit of microorganism] The gene encoding the function-modified mutant FGF-1 of the present invention (SEQ ID NOS: 24, 26, 28, 30, 32)
And 34 DNA sequences, respectively. ) Containing E. coli DH5α strain containing plasmid pMEXneo was deposited with the National Institute of Advanced Industrial Science and Technology under the deposit numbers FERM P-17181, FE
RM P-17182, FERM P-17183, FERM P-17184, FERM P-171
85 and FERM P-17186 were deposited on January 28, 1999.

[0045]

〔Example〕

[1] Outline of Examples Hereinafter, as an example, a method for introducing a sequence that undergoes N-glycosylation at a random position on the primary structure of fibroblast growth factor FGF-1 will be described. Is specifically described.

[2] Peptide insertion mutant secFGF-1 cDNA
1) Construction of the secFGF-1 plasmid 1. Preparation of FGF-1a / pBluescript II (KS +) plasmid The human FGF-1 cDNA was isolated from J. Exp. Med. (1992), 175 (4), 1
It was isolated from a human cDNA library according to 073-1080.
Using the isolated human FGF-1 cDNA as a template, # 967 (5'-GCG
TCG ACA GCG CTA ATT ACA AGA AGC CCA AAC TC-3 ') (SEQ ID NO: 12) and # 630 (5'-CCG AAT TCG AAT TCT T
PCR reaction was performed using TA ATC AGA AGA GAC TGG-3 ′) (SEQ ID NO: 13) as a primer. 434 specifically amplified
The base pair band was separated by electrophoresis, extracted, and double-cut with EcoR I and Sal I. Obtained
A 422 base pair band was separated and extracted, which was used for EcoR I, Sa
Insertion into the pBluescript II (KS +) cloning vector (2934 base pairs, purchased from Stratagene) double digested with lI yielded FGF-1a / pBluescript II (KS +). FGF-1a / p
Bluescript II (KS +) was sequentially digested with Aor51H I and Sal I, and the resulting 2626 base pair band was separated and extracted, and used for the ligation reaction described below.

2. Preparation of Mouse FGF-6 <NQ> cDNA Fragment Mouse FGF-6 cDNA was prepared from Oncogene (1990), 5 (6), 823-31.
Was isolated from a mouse cDNA library. Using the isolated mouse FGF-6 cDNA as a template, # 105 (5'-GCG TCG A
CC CAC CAT GTC-3 ') (SEQ ID NO: 14) and # 124 (5'
PCR was carried out using -GCG ATA TCC AGT AGC GTG CCT TGG GCG CG-3 ') (SEQ ID NO: 15) as a primer. The specifically amplified 138 bp band was separated by electrophoresis, extracted, and double-cut with EcoR V and Sal I. The resulting 130 base pair band was separated and extracted and used for the ligation reaction described below.

3. Creation of secFGF-1 gene [0048] EcoR V / Sal I fragment of PCR product of mouse FGF-6 and F
Aor51H I / Sal I fragment of GF-1a / pBluescript II (KS +)
The DNA was subjected to a DNA ligation reaction to obtain a secFGF-1 / pBluescript II (KS +) vector. This was further cut with EcoR I and Sal I, and the resulting 540 bp band was separated and extracted. This was pMEXneo expression vector (5916 base pairs, Dr. M. Barbacid, Mol. Cel) double-cut with EcoR I and Sal I.
l. Biol. (1989) 9 (1), 24-33) to obtain the end product of this step, the secFGF-1 gene. This expression type vector contains the nucleotide sequence of SEQ ID NO: 5.

2) One-point cleavage with DNase I sec FGF-1 / pBluescript II (KS +) obtained as described above
The 10 ug plasmid in a solution containing 10 mM MgCl _2, DNas
The mixture was treated with eI (400 pg) at 37 ° C. for 5 minutes. The reaction product
The DNA fragment separated by 1.4% agarose gel electrophoresis and electrophoresed at the position of 3474 base pairs was recovered and purified, and used for the ligation reaction described below.

3) Preparation of NLS-Cm Fragments NLS-Cm fragments were prepared in three types, frames 1, 2, and 3.
This is to obtain a large number of clones in which the frame of the introduced peptide matches the frame of the protein into which it is introduced.

1. Construction of NLS-Cm frame 1 Plasmid pCm containing chloramphenicol resistance gene
As a template, # 3n + 1F (5'-gcgcgcgtttaaacttaagcatcg
gcacgtaagaggttccaactt-3 ') (SEQ ID NO: 16), and #
3n + 1R (5'-atatatatcccgggcttaagttacgccccgccctgccac
PCR reaction was performed using tca-3 ′) (SEQ ID NO: 17) as a primer. The 741 bp band that was specifically amplified was separated by electrophoresis and extracted, followed by Pme I and Sm
Double cut with aI. The obtained band of 720 base pairs was separated and extracted to obtain NLS-Cm frame 1, which was used in the ligation reaction shown below.

2. Preparation of NLS-Cm frame 2 Using pCm as a template, # 3n + 2F (5'-atatatcgcgaacttaagca
tcggcacgtaagaggttccaac-3 ′) (SEQ ID NO: 18), and
# 3n + 2R (5'-atatatatgccggcttaagttacgccccgccctgccac
PCR reaction was performed using tca-3 ′) (SEQ ID NO: 19) as a primer. The specifically amplified 739 bp band was separated by electrophoresis, extracted, and extracted with NaeI and Nru.
Double cut with I. The obtained 720 base pair band was separated and extracted to obtain NLS-Cm frame 2, which was used in the ligation reaction described below.

3. Preparation of NLS-Cm frame 3 Using pCm as a template, # 3nF (5'-gcgcgcgttaacttaagcatcg
gcacgtaagaggttccaactt-3 ′) (SEQ ID NO: 20), and #
3nR (5'-atataagcgcttaagttacgccccgccctgccactca-3 ')
(SEQ ID NO: 21) was used as a primer to carry out a PCR reaction. The specifically amplified 734 bp band was separated by electrophoresis and extracted, followed by Hinc II and Aor51H.
Double cut at I. The obtained 717 base pair band was separated and extracted to obtain NLS-Cm frame 3, which was used for the ligation reaction shown below.

4) Ligation Using DNase I-treated secFGF-1 / pBluescript II (KS +) plasmid and NLS-Cm frame 1, NLS-Cm frame 2, or NLS-Cm frame 3 using DNA ligase.
It was subjected to a DNA ligation reaction. Using the reaction product, E. coli D
Transform H5α and transform it in LB medium containing 30 ug / ml chloramphenicol and 50 ug / ml ampicillin.
Cultured for 18 hours.

The plasmid was recovered from the grown Escherichia coli, purified, and cut with the restriction enzyme Afl II. Reaction products 1.4
% Agarose gel electrophoresis, 4194 or
The DNA fragment migrated at the position of 4191 base pairs was recovered and purified, and self-ligated (self-ligated) by performing a DNA ligation reaction using DNA ligase. Using this, E. coli DH5α was transformed again and cultured for 18 hours in LB medium containing 50 ug / ml ampicillin.

Six types of E. coli were selected from the grown E. coli, plasmid DNAs (A, B, C, D, E and F) were recovered and purified from the E. coli, and the nucleotide sequence was decoded by a cycle sequence method. As a result, the structure of secFGF-1 (SEQ ID NO: 24, 26, 28, 30, 32, and 34) into which DNA encoding a sequence that undergoes N-glycosylation was introduced.
Each having a DNA sequence of ) Became clear. Introduction of glycosylated sequence contained in these plasmids secFGF-1
The cDNA was used for integration into an expression vector as shown below. The amino acid sequences encoded by the DNA sequences of SEQ ID NOS: 24, 26, 28, 30, 32 and 34 are shown in SEQ ID NOs: 25, 27, 29, 31, 33 and 35, respectively. The outline of the above operation is shown in FIG.

5) Integration into Expression Vector The obtained plasmids A, B, C, D, E and F were double-digested with EcoR I and Sal I and subjected to agarose gel electrophoresis.
Bands of 2 or 549 base pairs were separated and extracted. this,
PMEXneo expression vector double-cut with EcoR I and Sal I
(5916 base pairs) to give G, H, I, J, K and L.

[3] Confirmation of Functionality of Insertion Sequence It is confirmed by the following method that the sequence which is introduced at a random position on the primary structure of secFGF-1 and undergoes N-glycosylation is functioning. did. SecFGF-1 expression vector G, H, I, into which a DNA encoding a sequence to undergo N-glycosylation has been introduced
J, K and L were transfected into mouse pro B cell line Ba / F3, and the transfected cells were selected by culturing in the presence of geneticin to obtain a single clone. These cell extracts were prepared, and a part of the extract containing the expressed protein was digested with N-glycanase, an enzyme that specifically cleaves N-glycans. Untreated and digested respectively
Analysis was performed by SDS polyacrylamide gel electrophoresis and immunoblot. As shown in FIG. 5, since the respective proteins, which are the expression products of the six types of vectors, were all reduced in molecular weight by N-glycanase, it was confirmed that the proteins were modified by N-glycans. In FIG. 5, (-) is untreated, (+)
Shows an immunoblot of each function-modified mutant FGF-1 after enzyme treatment. Open triangles indicate the size of the protein backbone of secFGF-1. 1 is FERMP-17185, 2 is FERM P-17182, 3 is FERM
P-17181, 4 is FERM P-17183, 5 is FERMP-17186, 6 is FE
Expression product of the cDNA sequence of RM P-17184. Since the molecular weight of secFGF-1 was reduced to the size of the protein skeleton by N-glycanase treatment, all the function-modified mutant FGF-1
It was confirmed that the sugar chain had been modified.

[4] Selection of Function-Modified Mutant FGF-1 1) Preparation of FGF-specific Target Cell Line Expression vector of FGF receptor (subtype R1c) (Dr.
Provided by DM Ornitz, J. Biol. Chem. (1996) 271 (2
5), 15292-15297) into a mouse pro B cell line Ba / F3 (RIKEN Cell Development Bank RCB No. 0805), and cultured in the presence of geneticin to produce a transfected cell (FGF-R1c / BaF3 cells). The following experiments are performed using a single clone grown in RPMI 1640 medium containing 10 ng / ml FGF-1, 10 ug / ml heparin, and 10% fetal bovine serum in the absence of IL-3.

2) Integration into Expression Vector The obtained plasmid was double-digested with EcoRI and SalI, and a band of 552 or 549 base pairs was separated and extracted on agarose gel electrophoresis. This was inserted into a pMEXneo expression vector (5916 base pairs) double-cut with EcoRI and SalI to obtain NLS secFGF-1 / pMEXneo.

3) Gene Transfer into the Target Cell Line The obtained NLS sec FGF-1 / pMEXneo vector was transfected into FGF-R1c / BaF3 cells by the electroporation method, and 100 ug / ml heparin, 10% bovine RPM with fetal serum
Culture in I 1640 medium. After 14 days, a clone that formed a colony was selected, and the function-modified mutant secFGF-1 produced and secreted by this clone was used for the following analysis.

4) Acquisition of a Gene for a Function-Modified Mutant secFGF-1 The clone was cultured in a normal medium (RPI 1640 medium containing 10% WEHI culture supernatant and 10% fetal bovine serum as IL-3 source). By guanidine isocyanate method, the total
Collect the RNA. This was subjected to a reverse transcription reaction using a random hexamer as a primer, and # 956 (5′-TCTTCCgATAgACTgCgTCg-3 ′) (SEQ ID NO: 2) using the obtained cDNA as a template.
PCR reaction is performed using 2) and # 957 (5′-CATTCTAgTTgTggTTTgTCC-3 ′) (SEQ ID NO: 23) as primers. This reaction product is double-cut with EcoR I and Sal I, and EcoR I and Sa
NLS-secFGF-1 / pMEXneo was obtained by insertion into the pMEXneo vector (5916 base pairs) double-cut with lI. The DNA sequence was decoded by the cycle sequencing method, and the structure of the function-modified mutant FGF-1 was obtained. The DNA sequence is shown in SEQ ID NOs: 30 and 32.

[3] Production of Function-Modified Mutant FGF-1 1) Gene Transfer and Mass Production The expression vector containing the DNA sequence shown in SEQ ID NOs: 30 and 32 was lipofected into CHO-K1 cells (Chinese hamster ovary cell K1 subunit). The gene was introduced into a strain, RIKEN Cell Development Bank RCB 0285), and the cells were cultured in the presence of geneticin to select the transfected cells. The resulting cells were expanded until the culture dish was almost full, and the medium production was increased by replacing the medium with a serum-free medium. The medium was changed every two days, and the resulting conditioned medium was centrifuged at low speed, and the supernatant was stored at 4 ° C.

2) Purification of Function-Modified Mutant FGF-1 Heparin Sepharose beads were added to a conditioned medium of function-modified mutant FGF-1 secreting cells, and the mixture was stirred at 4 ° C. for 2 hours or more.
The precipitated beads are collected by low-speed centrifugation, and the solution is added to a physiological phosphate buffered saline (PBS: phosphate buffered saline, pH 7.
After washing sufficiently in 4), the protein bound to the heparin-immobilized beads was eluted with PBS containing 2.5 M NaCl. Distilled water was added to the eluate to reduce the salt concentration, and the solution was subjected to high-performance liquid chromatography packed with heparin affinity affinity beads again to elute the functionally modified mutant FGF-1 by the NaCl concentration gradient. did.

[Test Example 1] DNA synthesis promoting activity against FGF receptor-expressing cell line FGF-R1c / BaF3 Immediately after removing IL-3 from the culture of FGF-R1c / BaF3, the functionally modified mutant FGF-1 and 10 Add medium containing ug / ml heparin, 10% fetal calf serum. After culturing for 36 hours, radiolabeled thymidine is incorporated for 4 hours, and the amount of DNA newly synthesized by the radioactivity incorporated into the DNA during this period is determined.

Test Example 2 Heparin Dependence on the Promotion of Human Vascular Endothelial Cell Proliferation HUVEC (human umbilical cord-derived vascular endothelial cell) arrests the cell cycle when growth factors such as FGF are deficient even in the presence of 15% serum. HUVEC (Exp.Cell Res.
(1987) 172 , 92-100. )), 18 hours later, radiolabeled thymidine is incorporated for 6 hours, and the amount of DNA newly synthesized by radioactivity incorporated into the DNA during this period is determined.

[Test Example 3] Cell surface lectin binding activity The binding activity to cell surface membrane proteins recognizing sugar chains was evaluated. Macrophage lectin (ML) cDNA expression vector that recognizes non-reducing terminal galactose (J. B
According to iochem., (1992) 111, 331-336, it was isolated from a mouse peritoneal macrophage cDNA library and incorporated into a pMEXneo vector. Ba) without FGF receptor
/ F3 cells were transfected and transfected cells were selected by culturing in the presence of geneticin (ML / BaF3
cell). This ML / BaF3 cell is labeled with a function-modified variant
After mixing with FGF-1 and incubation, the cells were collected by centrifugation, washed with a medium, and the amount of the function-modified mutant FGF-1 bound to the cells was measured. The amount specifically bound was evaluated as the amount dissociated by the medium containing lactose. As a result, it was shown that the amount of the function-modified mutant FGF-1 binding to the cell surface was significantly large as an effect of the sugar chain.

[Test Example 4] Binding activity to hepatocytes, intracellular translocation activity and cell growth promoting activity Activity was evaluated. Hepatocytes have an FGF receptor and an asialoglycoprotein receptor that recognizes non-reducing terminal galactose on the cell surface. Functionally modified mutant FGF-1 was added to hepatocytes and their related cells, and the amount of binding was measured in the same manner as in Test Example 3. As a result, the function-modified mutant FGF-1
It was shown that the amount of sugar chain binding to the cell surface was significantly large. In addition, the amount taken into the cells was measured. As a result, it was shown that the amount of the function-modified mutant FGF-1 was significantly increased into cells as an effect of the sugar chain. In addition, a function-modified mutant FGF-1 in which cells are not labeled
After 18 hours, the amount of DNA synthesis, which indicates cell proliferation, was evaluated by incorporation of radiolabeled thymidine.
As a result, it was shown that the function-modified mutant FGF-1 significantly changed the cell growth promoting activity as an effect of the sugar chain.

[0069]

According to the present invention, a technique for inserting a peptide into a random position on a primary protein sequence is provided. This technique is an effective means for optimizing the introduction site when artificially introducing a mutation into a protein. This makes it possible to create a systematic mutant without performing trial-and-error mutagenesis.

[0070]

[Sequence List] SEQUENCE LISTING <110> Director-General of Agency of Industrial Science and Technology <120> A process for producing a mutant protein, a process for preparing a mutant protein library and a process for preparing a mutant protein-encoding cDNA library <130> 11900243 <160> 35 <170> PatentIn Ver. 2.0 <210> 1 <211> 408 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (408) <400> 1 atg gct aat tac aag aag ccc aaa ctc ctc tac tgt agc aac ggg ggc 48 Met Ala Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly 1 5 10 15 cac ttc ctg agg atc ctt ccg gat ggc aca gtg gat ggg aca agg gac 96 His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp 20 25 30 agg agc gac cag cac att cag ctg cag ctc agt gcg gaa agc gtg ggg 144 Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly 35 40 45 gag gtg tat ata aag agt acc gag act ggc cag tac ttg gcc atg gac 192 Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp 50 55 60 acc gac ggg ctt tta tac ggc tca ca g aca cca aat gag gaa tgt ttg 240 Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu 65 70 75 80 ttc ctg gaa agg ctg gag gag aac cat tac aac acc tat ata tcc aag 288 Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys 85 90 95 aag cat gca gag aag aat tgg ttt gtt ggc ctc aag aag aat ggg agc 336 Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser 100 105 110 tgc aaa cgc ggt cct cgg act cac tat ggc cag aaa gca atc ttg ttt 384 Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe 115 120 125 ctc ccc ctg cca gtc tct tct gat 408 Leu Pro Leu Pro Val Ser Ser Asp 130 135 <210> 2 <211> 136 <212> PRT <213> Homo sapiens <400> 2 Met Ala Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly 1 5 10 15 His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly Thr Arg Asp 20 25 30 Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly 35 40 45 Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp 50 55 60 Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu 65 70 75 80 Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys 85 90 95 Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser 100 105 110 Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe 115 120 125 Leu Pro Leu Pro Val Ser Ser Asp 130 135 <210> 3 <211> 525 <212> DNA <213> Artificial Sequence <220> <221 > CDS <222> (1) .. (525) <220> <223> Description of Artificial Sequence: artificial chimera (Mus musculus / Homo sapiens) <400> 3 atg tcc cgg gga gca gga cgt gtt cag ggc acg ctg cag gct ctc gtc 48 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 ttc tta ggc gtc cta gtg ggc atg gtg gtg ccc tca cct gcc ggc gcc 96 Phe Leu Gly Val Leu Val Gly Met Val Pro Ser Pro Ala Gly Ala 20 25 30 cgc gcc aac ggc acg cta ctg gac gct aat tac aag aag ccc aaa ctc 144 Arg Ala Asa Gly Thr Leu Leu Leu Asp Ala Asn Tyr Lys Lys Pro Lys Leu 35 40 45 ctc tac tgt agc aac ggg ggc cac ttc ctg agg atc ctt ccg gat ggc 192 Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly 50 55 60 aca gtg gat ggg aca agg gac agg agc gac cag cac att cag ctg cag 240 Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln 65 70 75 80 ctc agt gcg gaa agc gtg ggg gag gtg tat ata aag agt acc gag act 288 Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr 85 90 95 ggc cag tac ttg gcc atg gac acc gac ggg ctt tta tac ggc tca cag 336 Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln 100 105 110 aca cca aat gag gaa tgt ttg ttc ctg gaa agg ctg gag gag aac cat 384 Thr Pro Asn Glu Glu Cys Leu Phe Glu Arg Leu Glu Glu Asn His 115 120 125 tac aac acc tat ata tcc aag aag cat gca gag aag aat tgg ttt gtt 432 Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val 130 135 140 ggc ctc aag aag aat ggg agc tgc aaa cgc ggt cct cgg act cac tat 480 Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr 145 150 155 160 ggc cag aaa gca atc ttg ttt ctc ccc ctg cca gtc tct tct gat Gln Lys A la Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 165 170 175 <210> 4 <211> 175 <212> PRT <213> Artificial Sequence <400> 4 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 Phe Leu Gly Val Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Ala 20 25 30 Arg Ala Asn Gly Thr Leu Leu Asp Ala Asn Tyr Lys Lys Pro Lys Leu 35 40 45 Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly 50 55 60 Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln 65 70 75 80 Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr 85 90 95 Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln 100 105 110 Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His 115 120 125 Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val 130 135 140 Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr 145 150 155 160 Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 165 170 175 <210> 5 <211> 525 <212> DNA <213> Artificial Sequence <2 20> <221> CDS <222> (1) .. (525) <220> <223> Description of Artificial Sequence: artificial chimera (Mus musculus / Homo sapiens) <400> 5 atg tcc cgg gga gca gga cgt gtt cag ggc acg ctg cag gct ctc gtc 48 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 ttc tta ggc gtc cta gtg ggc atg gtg gtg ccc tca cct gcc ggc gcc 96 Lehe Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Ala 20 25 30 cgc gcc caa ggc acg cta ctg gac gct aat tac aag aag ccc aaa ctc 144 Arg Ala Gln Gly Thr Leu Leu Asp Ala Asn Tyr Lys Lys Pro Lys Leu 35 40 45 ctc tac tgt agc aac ggg ggc cac ttc ctg agg atc ctt ccg gat ggc 192 Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly 50 55 60 aca gtg gat ggg aca agg gac agg agc gac cg ctg cag 240 Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln 65 70 75 80 ctc agt gcg gaa agc gtg ggg gag gtg tat ata aag agt acc gag act 288 Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr 85 90 95 ggc cag tac ttg gc catg gac acc gac ggg ctt tta tac ggc tca cag 336 Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln 100 105 110 aca cca aat gag gaa tgt ttg ttc ctg gaa agg ctg gag gag aac cat 384 Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His 115 120 125 tac aac acc tat ata tcc aag aag cat gca gag aag aat tgg ttt gtt 432 Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val 130 135 140 ggc ctc aag aag aat ggg agc tgc aaa cgc ggt cct cgg act cac tat 480 Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr 145 150 155 160 ggc cag aaa gca atc ttg ttt ctc ccc ctg cca gtc tct tct gat 525 Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 165 170 175 <210> 6 <211> 175 <212> PRT <213> Artificial Sequence <400> 6 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 Phe Leu Gly Val Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Ala 20 25 30 Arg Ala Gln Gly Thr Leu Leu Asp Ala Asn Tyr Lys Lys Pro Lys Leu 35 40 45 Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly 50 55 60 Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln 65 70 75 80 Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr 85 90 95 Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln 100 105 110 Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Aslu His 115 120 125 Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val 130 135 140 Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr 145 150 155 160 Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 165 170 175 <210 > 7 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: a nucleotide sequence encoding amino acid sequence Asn-Leu-Ser <400> 7 aacttaagc 9 <210> 8 <211 > 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: a nucleotide sequence encoding amino acid sequence Ala-Thr-Pro-Ala-Pro <400> 8 gcaactccgg cgcca 15 <210> 9 < 211> 12 <212> DNA <213> Art ificial Sequence <220> <223> Description of Artificial Sequence: a nucleotide sequence encoding amino acid sequence Ser-Gly-Ser-Gly <400> 9 tctggctctg ga 12 <210> 10 <211> 258 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: a nucleotide sequence encoding a partial sequence of the core protein in a proteoglycan <220> <221> CDS <222> (1) .. (258) <400> 10 atg gcc ccc gcc cgt ctg ttc gcg ctg ctg ctg ttc ttc gta ggc gga 48 Met Ala Pro Ala Arg Leu Phe Ala Leu Leu Leu Phe Phe Val Gly Gly 1 5 10 15 gtc gcc gag tcg atc cga gag act gag ccc atg gac ctc 96 Val Ala Glu Ser Ile Arg Glu Thr Glu Val Ile Asp Pro Gln Asp Leu 20 25 30 cta gaa ggc cga tac ttc tcc gga gcc cta cca gac gat gag gat gta 144 Leu Glu Gly Arg Tyr Phe Ser Gly Ala Leu Pro Asp Asp Glu Asp Val 35 40 45 gtg ggg ccc ggg cag gaa tct gat gac ttt gag ctg tct ggc tct gga 192 Val Gly Pro Gly Gln Glu Ser Asp Asp Phe Glu Leu Ser Gly Ser Gly 50 55 60 gat ctg gat gac ttg gaa gac tcc atg atc ggc cct g aa gtt gtc cat 240 Asp Leu Asp Asp Leu Glu Asp Ser Met Ile Gly Pro Glu Val Val His 65 70 75 80 ccc ttg gtg cct cta gat 258 Pro Leu Val Pro Leu Asp 85 <210> 11 <211> 86 <212> PRT <213> Artificial Sequence <400> 11 Met Ala Pro Ala Arg Leu Phe Ala Leu Leu Leu Phe Phe Val Gly Gly 1 5 10 15 Val Ala Glu Ser Ile Arg Glu Thr Glu Val Ile Asp Pro Gln Asp Leu 20 25 30 Leu Glu Gly Arg Tyr Phe Ser Gly Ala Leu Pro Asp Asp Glu Asp Val 35 40 45 Val Gly Pro Gly Gln Glu Ser Asp Asp Phe Glu Leu Ser Gly Ser Gly 50 55 60 Asp Leu Asp Asp Leu Glu Asp Ser Met Ile Gly Pro Glu Val Val His 65 70 75 80 Pro Leu Val Pro Leu Asp 85 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: # 967 primer <400> 12 gcgtcgacag cgctaattac aagaagccca aactc 35 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: # 630 primer <400> 13 ccgaattcga attctttaat cagaagagac tgg 33 <210> 14 <211 > 18 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: # 105 primer <400> 14 gcgtcgaccc accatgtc 18 <210> 15 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: # 124 primer <400> 15 gcgatatcca gtagcgtgcc ttgggcgcg 29 <210> 16 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: # 3n + 1F primer <400> 16 gcgcgcgttt aaacttaagc atcggcacgt aagaggttcc aactt 45 <210 > 17 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: # 3n + 1R primer <400> 17 atatatatcc cgggcttaag ttacgccccg ccctgccact ca 42 <210> 18 <211> 42 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: # 3n + 2F primer <400> 18 atatatcgcg aacttaagca tcggcacgta agaggttcca ac 42 <210> 19 <211> 41 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: # 3n + 2R primer <400> 19 atatatatgc cggcttaagt tacgccccgc cctgccactc a 41 <210> 20 <211> 43 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: # 3nF primer <400> 20 gcgcgcgtta acttaagcat cggcacgtaa gaggttccaa ctt 43 <210> 21 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: # 3nR primer <400> 21 atataagcgc ttaagttacg ccccgccctg ccactca 37 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: # 956 primer <400> 22 tcttccgata gactgcgtcg 20 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: # 957 primer <400> 23 cattctagtt gtggtttgtc c 21 <210> 24 <211> 540 <212> DNA <213 > Artificial Sequence <220> <223> Description of Artificial Sequence: mutant FGF-1 gene contained in FERM P-17181 <220> <221> CDS <222> (1) .. (540) <400> 24 atg tcc cgg gga gca gga cgt gtt cag ggc acg ctg cag gct ctc gtc 48 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 ttc tta ggc gtc cta gtg ggc atg gtg gtg ccc tca ccc 96 Phe Leu Gly Val Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Ala 20 25 30 cgc gcc caa ggc acg cta ctg gac gct aat tac aag aag ccc aaa ctc 144 Arg Ala Gla Gln Gly Thr Leu Leu Asp Ala Asn Tyr Lys Lys Pro Lys Leu 35 40 45 ctc tac tgt agc aac ggg ggc cac ttc ctg agg atc ctt ccg gat ggc 192 Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly 50 55 60 aca gtg gat ggg aca agg gac agg agc gac cag caa aac ttag Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln Gln Asn Leu Ser Pro 65 70 75 80 att cag ctg cag ctc agt gcg gaa agc gtg ggg gag gtg tat ata aag 288 Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 85 90 95 agt acc gag act ggc cag tac ttg gcc atg gac acc gac ggg ctt tta 336 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 100 105 110 tac ggc tca cag aca cca aat gag gaa tgt ttg ttc ctg gaa agg ctg 384 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 115 120 125 gag gag aac cat tac aac acc tat ata tcc aag aag cat gca gag aag 432 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 130 135 140 aat tgg ttt gtt ggc ctc aag aag aat ggg agc tgc aaa cgc ggt cct 480 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro 145 150 155 160 cgg act cac tat ggc cag aaa gca atc ttg ttt ctc ccc ctg cca gtc 528 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 165 170 175 tct tct gat taa 540 Ser Ser Asp 180 <210> 25 <211> 179 <212> PRT <213> Artificial Sequence <400> 25 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 Phe Leu Gly Val Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Ala 20 25 30 Arg Ala Gln Gly Thr Leu Leu Asp Ala Asn Tyr Lys Lys Pro Lys Leu 35 40 45 Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly 50 55 60 Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln Gln Asn Leu Ser Pro 65 70 75 80 Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 85 90 95 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 100 105 110 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Le u Glu Arg Leu 115 120 125 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 130 135 140 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro 145 150 155 160 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 165 170 175 Ser Ser Asp <210> 26 <211> 540 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: mutant FGF-1 gene contained in FERM P-17182 <220> <221> CDS <222> (1) .. (540) <400> 26 atg tcc cgg gga gca gga cgt gtt cag ggc acg ctg cag gct ctc gtc 48 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 ttc tta ggc gtc cta gtg ggc atg gtg gtg ccc tca cct gcc ggc gcc 96 Phe Leu Gly Val Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Ala 20 25 30 cgc gcc caa ggc acg cta ctg gac gct aat tac aag aag ccc aaa ctc 144 Arg Ala Gln Gly Thr Leu Leu Asp Ala Asn Tyr Lys Lys Pro Lys Leu 35 40 45 ctc tac tgt agc aac ggg ggc cac ttc ctg ctt ccg gat ggc 192 Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly 50 55 60 aca gtg gat ggg aca agg gac acg aac tta agc cgg agc gac cag cac 240 Thr Val Asp Gly Thr Arg Asp Thr Asn Leu Ser Arg Ser Asp Gln His 65 70 75 80 att cag ctg cag ctc agt gcg gaa agc gtg ggg gag gtg tat ata aag 288 Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 85 90 95 agt acc gag act ggc cag tac ttg gcc atg gac acc gac ggg ctt tta 336 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 100 105 110 tac ggc tca cag aca cca aat gag gaa tgt ttg ttc ctg gaa agg ctg 384 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 115 125 gag gag aac cat tac aac acc tat ata tcc aag aag cat gca gag aag 432 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 130 135 140 aat tgg ttt gtt ggc ctc aag aag aat ggg agc tgc aaa cgc ggt cct 480 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro 145 150 155 160 cgg act cac tat ggc cag aaa gca atc ttg ttt ctc ccc ctg cca gtc 528 Arg Thr His Tyr Gly Gln Lys Ala Leu Phe Le u Pro Leu Pro Val 165 170 175 tct tct gat taa 540 Ser Ser Asp 180 <210> 27 <211> 179 <212> PRT <213> Artificial Sequence <400> 27 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 Phe Leu Gly Val Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Ala 20 25 30 Arg Ala Gln Gly Thr Leu Leu Asp Ala Asn Tyr Lys Lys Pro Lys Leu 35 40 45 Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly 50 55 60 Thr Val Asp Gly Thr Arg Asp Thr Asn Leu Ser Arg Ser Asp Gln His 65 70 75 80 Ile Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys 85 90 95 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 100 105 110 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 115 120 125 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 130 135 140 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro 145 150 155 160 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 165 170 175 Ser Ser Asp <210> 28 <211> 540 <212> DNA <21 3> Artificial Sequence <220> <223> Description of Artificial Sequence: mutant FGF-1 gene contained in FERM P-17183 <220> <221> CDS <222> (1) .. (540) <400> 28 atg tcc cgg gga gca gga cgt gtt cag ggc acg ctg cag gct ctc gtc 48 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 ttc tta ggc gtc cta gtg ggc atg gtg gtg ccc tc cct gcc 96 Phe Leu Gly Val Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Ala 20 25 30 cgc gcc caa ggc acg cta ctg gac gct aat tac aag aag ccc aaa ctc 144 Arg Ala Gln Gly Thr Leu Leu Asp Ala Asn Tyr Lys Lys Pro Lys Leu 35 40 45 ctc tac tgt agc aac ggg ggc cac ttc ctg agg atc ctt ccg gat ggc 192 Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly 50 55 60 aca gtg gat ggg aca agg agg agc gac cag cac att cag ctg cag 240 Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln 65 70 75 80 ctc agt gcg gaa agc gtg ggg gag gcg aac tta agc ctg tat ata aag Ala Alu Seru Glu Ser Val Gly Glu Ala Asn Leu Ser Leu Tyr Ile Lys 85 9 0 95 agt acc gag act ggc cag tac ttg gcc atg gac acc gac ggg ctt tta 336 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 100 105 110 tac ggc tca cag aca cca aat gag gaa tgt ttg ttc ctg gaa agg ctg 384 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 115 120 125 gag gag aac cat tac aac acc tat ata tcc aag aag cat gca gag aag 432 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 130 135 140 aat tgg ttt gtt ggc ctc aag aag aat ggg agc tgc aaa cgc ggt cct 480 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro 145 150 155 160 cgg act cac tat ggc cag aaa gca atc ttg ttt ctc ccc ctg cca gtc 528 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 165 170 175 tct tct gat taa 540 Ser Ser Asp 180 <210> 29 <211> 179 <212> PRT <213> Artificial Sequence <400> 29 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 Phe Leu Gly Val Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Ala 20 25 30 Arg Ala Gln Gly Thr Leu Leu Asp Ala Asn Tyr Lys Lys Pro Lys Leu 35 40 45 Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly 50 55 60 Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln 65 70 75 80 Leu Ser Ala Glu Ser Val Gly Glu Ala Asn Leu Ser Leu Tyr Ile Lys 85 90 95 Ser Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu 100 105 110 Tyr Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu 115 120 125 Glu Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 130 135 140 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro 145 150 155 160 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 165 170 175 Ser Ser Asp <210> 30 <211> 537 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: mutant FGF -1 gene contained in FERM P-17184 <220> <221> CDS <222> (1) .. (537) <400> 30 atg tcc cgg gga gca gga cgt gtt cag ggc acg ctg cag gct ctc gtc 48 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 ttc tta ggc gtc cta gtg ggc atg gtg gtg ccc tca cct gcc ggc gcc 96 Phe Leu Gly Val Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Ala 20 25 30 cgc gcc caa ggc acg cta ctg gac gct aat tac aag aag ccc ctc 144 Arg Ala Gln Gly Thr Leu Leu Asp Ala Asn Tyr Lys Lys Pro Lys Leu 35 40 45 ctc tac tgt agc aac ggg ggc cac ttc ctg agg atc ctt ccg gat ggc 192 Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Leu Pro Asp Gly 50 55 60 aca gtg gat ggg aca agg gac agg agc gac cag cac att cag ctg cag 240 Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln 65 70 75 80 ctc agt gcg gaa agc gtg ggg gag gtg tat ata aag agt acc gag act 288 Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr 85 90 95 ggc cag tac ttg gcc atg gac acc gac ggg ctt tta tac ggc tca cag 336 Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln 100 105 110 aca cca aat gag gaa tgt ttg ttc ctg gaa agg ctg gag gag aac cat 384 Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His 115 120 125 tac aac acc tat ata tcc aag aag cat gca gag aag aat tgg ttt gtt 432 Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val 130 135 140 ggc ctc aag aag aat ggg agc tgc aaa cgc ggt cct cgg act cac tat 480 Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr 145 150 155 160 ggc cag aaa gca atc ttg ttt ctc ccc ctg cca gtc tct tct gat aac 528 Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp Asn 165 170 175 tta agc taa 537 Leu Ser <210> 31 <211> 178 <212> PRT <213> Artificial Sequence <400> 31 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 Phe Leu Gly Val Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Ala 20 25 30 Arg Ala Gln Gly Thr Leu Leu Asp Ala Asn Tyr Lys Lys Pro Lys Leu 35 40 45 Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly 50 55 60 Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln 65 70 75 80 Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr 85 90 95 Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Le u Leu Tyr Gly Ser Gln 100 105 110 Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His 115 120 125 Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val 130 135 140 Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr 145 150 155 160 Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp Asn 165 170 175 Leu Ser <210> 32 <211> 537 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: mutant FGF-1 gene contained in FERM P-17185 <220> <221> CDS <222> (1) .. (537) <400> 32 atg tcc cgg gga gca gga cgt gtt cag ggc acg ctg cag gct ctc gtc 48 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 ttc tta ggc gtc cta gtg ggc atg gtg ccc tca ggc gcc 96 Phe Leu Gly Val Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Ala 20 25 30 cgc gcc caa ggc acg cta ctg gac aac tta agc gct aat tac aag aag 144 Arg Ala Gln Gly Thr Leu Leu Asp Asn Leu Ser Ala Asn Tyr Lys Lys 35 40 45 ccc aaa ctc ctc tac tgt agc aac ggg ggc cac ttc ctg agg atc ctt 192 Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu 50 55 60 ccg gat ggc aca gtg gat ggg aca agg gac agg agc gac cag cac att 240 Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile 65 70 75 80 cag ctg cag ctc agt gcg gaa agc gtg ggg gag gtg tat ata aag agt 288 Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser 85 90 95 acc gag act ggc cag tac ttg gcc atg gac acc gac ggg ctt tta tac 336 Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr 100 105 110 ggc tca cag aca cca aat gag gaa tgt ttg ttc ctg gaa ag gag 384 Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu 115 120 125 gag aac cat tac aac acc tat ata tcc aag aag cat gca gag aag aat 432 Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn 130 135 140 tgg ttt gtt ggc ctc aag aag aat ggg agc tgc aaa cgc ggt cct cgg 480 Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg 145 150 155 160 act cac tat ggc cag aaa gca atc t tg ttt ctc ccc ctg cca gtc tct 528 Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser 165 170 175 tct gat taa 537 Ser Asp <210> 33 <211> 178 <212> PRT <213> Artificial Sequence <400> 33 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 Phe Leu Gly Val Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Ala 20 25 30 Arg Ala Gln Gly Thr Leu Leu Asp Asn Leu Ser Ala Asn Tyr Lys Lys 35 40 45 Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu 50 55 60 Pro Asp Gly Thr Val Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile 65 70 75 80 Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser 85 90 95 Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr 100 105 110 Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu 115 120 125 Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn 130 135 140 Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg 145 150 155 160 Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser 165 170 175 Ser Asp <210> 34 <211> 540 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: mutant FGF-1 gene contained in FERM P-17186 <220> <221> CDS <222> (1) .. (540) <400> 34 atg tcc cgg gga gca gga cgt gtt cag ggc acg ctg cag gct ctc gtc 48 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 ttc tta ggc gtc cta gtg ggc atg gtg gtg ccc tca cct gcc ggc gcc 96 Phe Leu Gly Val Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Ala 20 25 30 cgc gcc caa ggc acg cta ctg gac gct aat tac aag aag ccc aaa ctc 144 Arg Ala Gln Gly Thr Leu Leu Asp Ala Asn Tyr Lys Lys Pro Lys Leu 35 40 45 ctc tac tgt agc aac ggg ggc cac ttc ctg agg atc ctt ccg gat ggc 192 Leu Tyr Cy Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly 50 55 60 aca gtg gat ggg aca agg gac agg agc gac cag cac att cag ctg cag 240 Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln 65 70 75 80 ctc agt gcg gaa agc gtg ggg gag gtg tat ata aag agt acc gag act 288 Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr 85 90 95 ggc cag tac ttg gcc atg gac acc gac ggg ctt tta tac ggc tca cag 336 Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln 100 105 110 aca cca aat gag gaa tgt ttg ttc ctg gaa agg ctg gag gcg aac tta 384 Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Ala Asn Leu 115 120 125 agc cag aac cat tac aac acc tat atacc aag aag cat gca gag aag 432 Ser Gln Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 130 135 140 aat tgg ttt gtt ggc ctc aag aag aag aat ggg agc tgc aaa cgc ggt cct 480 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro 145 150 155 160 cgg act cac tat ggc cag aaa gca atc ttg ttt ctc ccc ctg cca gtc 528 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 165 170 175 tct tct gat taa 540 Ser Ser Asp 180 <210> 35 <211> 179 <212> PRT <213> Artificial Sequence <400> 35 Met Ser Arg Gly Ala Gly Arg Val Gln Gly Thr Leu Gln Ala Leu Val 1 5 10 15 Phe Leu Gly Val Leu Val Gly Met Val Val Pro Ser Pro Ala Gly Ala 20 25 30 Arg Ala Gln Gly Thr Leu Leu Asp Ala Asn Tyr Lys Lys Pro Lys Leu 35 40 45 Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly 50 55 60 Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln 65 70 75 80 Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr 85 90 95 Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln 100 105 110 Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu Ala Asn Leu 115 120 125 Ser Gln Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys 130 135 140 Asn Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro 145 150 155 160 Arg Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val 165 170 175 Ser Ser Asp

[0071]

[Sequence listing free text] SEQ ID NO: 7 is an amino acid sequence
1 shows a nucleotide sequence encoding Asn-leu-Ser. SEQ ID NO: 8 shows a nucleotide sequence encoding the amino acid sequence Ala-Thr-Pro-Ala-Pro. SEQ ID NO: 9 shows the nucleotide sequence encoding the amino acid sequence Ser-Gly-Ser-Gly. SEQ ID NO: 10 shows a nucleotide sequence encoding a partial sequence of a proteoglycan core protein.

SEQ ID NO: 12 shows the nucleotide sequence of primer # 967. SEQ ID NO: 13 shows the nucleotide sequence of # 630 primer. SEQ ID NO: 14 shows the nucleotide sequence of # 105 primer. SEQ ID NO: 15 is # 124
Shows the nucleotide sequence of the primer. SEQ ID NO: 16
Indicates the nucleotide sequence of the # 3n + 1F primer. SEQ ID NO: 17 shows the nucleotide sequence of the # 3n + 1R primer.

SEQ ID NO: 18 shows the nucleotide sequence of # 3n + 2F primer. SEQ ID NO: 19 shows the nucleotide sequence of # 3n + 2R primer. SEQ ID NO: 20 shows the nucleotide sequence of # 3nF primer. SEQ ID NO: 21
2 shows the nucleotide sequence of the # 3nR primer. SEQ ID NO: 2
2 shows the nucleotide sequence of the # 956 primer. SEQ ID NO: 23 shows the nucleotide sequence of # 957 primer.

SEQ ID NO: 24 shows the nucleotide sequence of the mutant FGF-1 gene introduced into FERM P-17181.
SEQ ID NO: 26 is a mutant introduced into FERM P-17182
1 shows the nucleotide sequence of the FGF-1 gene. SEQ ID NO: 28
Shows the nucleotide sequence of the mutant FGF-1 gene introduced into FERM P-17183. SEQ ID NO: 30 is FERM P-1
7184 shows the nucleotide sequence of the mutant FGF-1 gene introduced into 7184. SEQ ID NO: 32 shows the nucleotide sequence of the mutant FGF-1 gene introduced into FERM P-17185. SEQ ID NO: 34 shows the nucleotide sequence of the mutant FGF-1 gene introduced into FERM P-17186.

[Brief description of the drawings]

FIG. 1 shows the structure of a typical sulfated polysaccharide / glycosaminoglycan sugar chain.

FIG. 2 shows the structure of a typical N-type sugar chain. G indicates galactose, GN indicates N-acetylglucosamine, M indicates mannose, F indicates fucose, and Sia indicates sialic acid.

FIG. 3 shows a representative O-type sugar chain structure. G indicates galactose, and GalNAc indicates N-acetylgalactosamine.

FIG. 4 shows the construction of a peptide insertion mutant secFGF-1 cDNA.

FIG. 5 shows FERM P-17185, FERM P-17182, and FERM P
-17181, FERM P-17183, FERM P-17186 and FERM P-171
The immunoblot of the untreated (-) and the (+) of the expression product of the cDNA of the mutant FGF-1 introduced at 84 after treatment with N-glycanase is shown.

[Procedure amendment]

[Submission date] January 24, 2000 (2000.1.2
4)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12R 1:91) (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) (72 Inventor Osamu Suzuki 1-3-1 Higashi, Tsukuba, Ibaraki Pref., Institute of Biotechnology, Institute of Industrial Technology (72) Inventor Shuichi Oka 1-3-1, Higashi, Tsukuba, Ibaraki Pref. ) Inventor Atsuko Yoneda 2418 B201 Inari-mae, Inari-mae, Tsukuba-shi, Ibaraki 4B024 AA03 AA20 BA21 BA80 CA04 CA06 DA02 EA04 FA10 GA13 GA14 HA01 HA09 4B064 AG02 CA10 CA19 CC24 DA16 DA20 4B065 AA77X AA80 ABA ABA ABA ABA ABA ABA 91 CA60 4H045 AA10 AA20 BA10 BA53 CA40 DA01 EA20 FA72 FA74 HA05

Claims (12)

[Claims] 1. A method comprising incorporating a cDNA encoding a protein into a circular plasmid, cutting the circular plasmid at one random position, and introducing a DNA encoding an amino acid or a peptide into the cleavage site. A method of preparing a library of cDNAs encoding mutants in which amino acids or peptides are introduced at random sites on the primary structure of a protein. 2. The following steps: (a) a step of incorporating a cDNA encoding a protein into a circular plasmid, (b) a step of cleaving the circular plasmid at a random position, and (c) a step of encoding an amino acid or peptide. Is introduced into the cleavage site of the plasmid, wherein the DNA encoding the amino acid or peptide
Adding additional nucleotides to the 5 'end and / or 3' end of to obtain a clone whose amino acid or peptide frame matches the frame of the protein into which it is introduced, ( d) a step of introducing a plasmid into which the DNA encoding the amino acid or peptide has been introduced into a host cell to obtain a transformant; (e) a step of recovering the plasmid from the transformant; and (f) a step of recovering the plasmid. DNA encoding amino acids or peptides and cDNA encoding proteins
And (g) cutting out the DNA in the region containing
2. The method according to claim 1, comprising the steps of incorporating NA into an expression vector, (h) introducing the expression vector into a host cell, and (i) expressing a protein variant in the host cell. 3. In step (c), a DNA encoding an amino acid or a peptide is introduced into a cleavage site of the plasmid while being linked to the selection marker gene, and in step (d), expression of the selection marker gene is performed. 3. The method according to claim 2, wherein the transformant is selected based on the above, and the selection marker gene is removed in the step (f). 4. A variant in which an amino acid or peptide is introduced at a random site on the primary structure of a protein.
Create a library of cDNAs and include the cD
A method for producing a library of protein variants, comprising expressing a protein variant encoded by NA. 5. A variant in which an amino acid or peptide is introduced at a random site on the primary structure of a protein.
Create a library of cDNAs and include the cD
A method for producing a protein variant, comprising expressing a protein variant encoded by NA, selecting cells expressing the protein variant of interest, and causing the cells to produce the protein variant. . 6. The following steps: (a) a step of preparing a cell line showing a specific response to the protein, and (b) a mutant having an amino acid or peptide introduced at a random site on the primary structure of the protein. CDNA encoding
A step of preparing a cDNA library by introducing an expression vector incorporating the above into the cell line, (c) a step of detecting the response and selecting a cell showing a response, and (d) a step of: The method according to claim 5, comprising a step of culturing and recovering the target protein mutant from the culture solution and / or the cultured cells. 7. The method according to claim 5, wherein the amino acid or peptide to be introduced is capable of undergoing addition of a sugar chain. 8. The amino acid or peptide to be introduced, which can undergo addition of any of sulfated polysaccharide, glycosaminoglycan, N-type sugar chain, O-type sugar chain or a combination thereof. Item 8. The method according to any one of Items 5 to 7. 9. The method according to claim 5, wherein the protein is a factor belonging to the FGF family or a closely related factor. 10. The method according to claim 5, wherein the protein is a factor belonging to the heparin-binding growth factor family or a factor closely related thereto. 11. The method according to claim 5, wherein the protein is any one of the following proteins (a) to (f): (A) a protein consisting of the amino acid sequence of SEQ ID NO: 2 (FGF-1a); (B) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, added or modified in the amino acid sequence of SEQ ID NO: 2, and having FGF-1 activity; (C) a protein consisting of the amino acid sequence of SEQ ID NO: 4 (FGF-6 / 1a); (D) a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, added or modified in the amino acid sequence of SEQ ID NO: 4, and having FGF-6 / 1a activity. (E) a protein consisting of the amino acid sequence of SEQ ID NO: 6 (secFGF-1); (F) a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted, added or modified in the amino acid sequence of SEQ ID NO: 6, and having secFGF-1 activity. 12. A novel protein mutant which can be produced by the method according to claim 5.