JP3790810B2 - Method for producing protein variant, method for producing protein variant library, and method for producing library of cDNA encoding protein variant - Google Patents

Description

【００７１】
【配列表フリーテキスト】
配列番号７は、アミノ酸配列Asn-leu-Serをコードするヌクレオチド配列を示す。
配列番号８は、アミノ酸配列Ala-Thr-Pro-Ala-Proをコードするヌクレオチド配列を示す。
配列番号９は、アミノ酸配列Ser-Gly-Ser-Glyをコードするヌクレオチド配列を示す。
配列番号１０は、プロテオグリカンのコアタンパク質の部分配列をコードするヌクレオチド配列を示す。
【００７２】
配列番号１２は、#967プライマーのヌクレオチド配列を示す。
配列番号１３は、#630プライマーのヌクレオチド配列を示す。
配列番号１４は、#105プライマーのヌクレオチド配列を示す。
配列番号１５は、#124プライマーのヌクレオチド配列を示す。
配列番号１６は、#3n+1Fプライマーのヌクレオチド配列を示す。
配列番号１７は、#3n+1Rプライマーのヌクレオチド配列を示す。
【００７３】
配列番号１８は、#3n+2Fプライマーのヌクレオチド配列を示す。
配列番号１９は、#3n+2Rプライマーのヌクレオチド配列を示す。
配列番号２０は、#3nFプライマーのヌクレオチド配列を示す。
配列番号２１は、#3nRプライマーのヌクレオチド配列を示す。
配列番号２２は、#956プライマーのヌクレオチド配列を示す。
配列番号２３は、#957プライマーのヌクレオチド配列を示す。
【００７４】
配列番号２４は、FERM P-17181に導入されている変異型FGF-1遺伝子のヌクレオチド配列を示す。
配列番号２６は、FERM P-17182に導入されている変異型FGF-1遺伝子のヌクレオチド配列を示す。
配列番号２８は、FERM P-17183に導入されている変異型FGF-1遺伝子のヌクレオチド配列を示す。
配列番号３０は、FERM P-17184に導入されている変異型FGF-1遺伝子のヌクレオチド配列を示す。
配列番号３２は、FERM P-17185に導入されている変異型FGF-1遺伝子のヌクレオチド配列を示す。
配列番号３４は、FERM P-17186に導入されている変異型FGF-1遺伝子のヌクレオチド配列を示す。
【図面の簡単な説明】
【図１】図１は、代表的硫酸化多糖・グリコサミノグリカン糖鎖の構造を示す。
【図２】図２は、代表的N-型糖鎖の構造を示す。G はガラクトース、GNはN-アセチルグルコサミン、M はマンノース、F はフコース、Sia はシアル酸を示す。
【図３】図３は、代表的O-型糖鎖構造を示す。G はガラクトース、GalNAcはN-アセチルガラクトサミンを示す。
【図４】図４は、ペプチド挿入変異体secFGF-1 cDNAの構築を示す。
【図５】図５は、FERM P-17185、FERM P-17182、FERM P-17181、FERM P-17183、FERM P-17186およびFERM P-17184に導入された変異型FGF-1のcDNAの発現産物の未処理（−）、N-グリカナーゼによる処理後（＋）のイムノブロットを示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a mutant protein in which an arbitrary amino acid or peptide is introduced at a random site on the primary structure of the protein, a method for producing a library of the mutant protein, and live performance of cDNA encoding the mutant protein. The present invention relates to a method for manufacturing a rally.
[0002]
[Prior art]
In order to modify the primary structure of a protein that is a linear polymer of amino acids, a genetic engineering technique has been conventionally used in addition to a chemical technique. Modification of protein structure using genetic engineering techniques may result in amino acid substitution, deletion, or introduction by translating the base substitution, deletion, or introduction into the DNA structure. It has been done in general.
However, the introduction of such mutations can only introduce specific mutations at the intended specific site, and cannot be introduced at unintended random sites.
[0003]
In addition, in the prior art, in order to produce various mutant proteins, it was necessary to individually create and isolate cDNAs encoding the respective mutant proteins.
In recent years, elucidation of functional domains has progressed through the study of protein structure-activity relationships, and techniques have been developed to introduce heterogeneous peptides having various functions on the primary structure of any protein. In order to determine the introduction position, it had to be by trial and error.
[0004]
Furthermore, in recent years, with the understanding of the importance of sugar chains of glycoproteins on the function of the protein part, peptides having a sequence that can add sugar chains onto the primary structure of known proteins are introduced to introduce sugar chains. Although the technology for generating the amino acid was developed, the optimization of the introduction position of the glycosylated sequence amino acid was only by trial and error.
[0005]
[Problems to be solved by the invention]
An object of the present invention is to obtain a protein having no modified structure-activity relationship information that leads to functional modification or a protein having a modified function by modifying the structure of a protein for which functional modification cannot be predicted. The object is to provide a technique for producing a protein variant library in which an arbitrary amino acid or peptide is introduced at a site. It is another object of the present invention 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 a modified function by introducing a sugar chain modification into a protein having no structure-activity relationship information that leads to functional modification or a protein for which functional 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 sugar chain-modified amino acids or peptides are introduced at random sites. Furthermore, by selecting a glycoprotein that has changed to a desired function from this glycosylated mutant library, the glycosylated amino acid or peptide introduction site for obtaining the intended functionally modified glycoprotein is optimized. To provide technology.
[0007]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors cut a circular plasmid containing cDNA encoding a protein at a random position at one position, and DNA encoding an amino acid or peptide at this site. Through the introduction, a technique for introducing an amino acid or a peptide at an arbitrary position on the primary structure of the translated protein was completed. This makes it possible to construct a mutant library in which amino acids or peptides are introduced at various sites on the primary structure of the protein.
[0008]
The present inventors succeeded in producing secFGF-1 as a sugar chain-modified protein by applying the technique to the secreted simple protein secFGF-1 and introducing various glycosylated amino acids. In the present specification, “FGF” refers to fibroblast growth factor.
Furthermore, the present inventors also completed a technique for selecting a functionally modified secFGF-1 from the library, and as a result, completed a technique for completing a functional modification by introducing a mutation into a protein in a single step.
The present invention has been completed based on such findings.
[0009]
That is, the present invention is characterized in that a cDNA encoding a protein is incorporated into a circular plasmid, the circular plasmid is cleaved at one position at random positions, and a DNA encoding an amino acid or peptide is introduced into this cleavage site. The present invention provides a method for preparing a library of cDNAs encoding variants in which amino acids or peptides are introduced at random sites on the primary structure of proteins. In the present specification, “a library of cDNA encoding variants in which amino acids or peptides are introduced at random sites on the primary structure of a protein” refers to amino acids or random sites on the primary structure of a protein. A group of cDNAs encoding various mutants into which peptides have been introduced. These cDNAs are usually in a state of being incorporated into a vector such as a plasmid or a virus.
[0010]
The cDNA library production method of the present invention comprises the following steps:
(A) incorporating a cDNA encoding the protein into a circular plasmid;
(B) cleaving the circular plasmid at one position at random positions;
(C) DNA encoding an amino acid or peptide is introduced into the cleavage site of the plasmid, where an additional nucleotide is added to the 5 ′ end side and / or 3 ′ end side of the DNA encoding the amino acid or peptide. Making it possible to obtain a clone whose amino acid or peptide frame matches the frame of the protein into which it is introduced,
(D) introducing a plasmid into which a DNA encoding the amino acid or peptide is introduced into a host cell to obtain a transformant;
(E) recovering the plasmid from the transformant;
(F) a step of cutting out DNA in a region containing DNA encoding an amino acid or peptide and cDNA encoding a protein from the recovered plasmid;
(G) a step of incorporating the excised DNA into an expression vector;
(H) introducing the expression vector into a host cell; and
(I) a step of expressing a protein variant in the host cell
May be included. In step (c), DNA encoding an amino acid or peptide is introduced into the plasmid cleavage site in a state of being linked to the selection marker gene, and in step (d), based on the expression of the selection marker gene. A transformant is selected and the selection marker gene is removed in the step (f).
[0011]
The present invention also provides a library of cDNA encoding variants in which amino acids or peptides are introduced at random sites on the primary structure of the protein, and the protein mutations encoded by the cDNA in this library Provided is a method for producing a library of protein variants, characterized by expressing a body. In the present specification, the “protein variant library” refers to a group of various protein variants.
[0012]
Furthermore, the present invention creates a library of cDNAs encoding variants in which amino acids or peptides are introduced at random sites on the primary structure of the protein, and protein mutations encoded by the cDNAs in this library The present invention provides a method for producing a protein variant, characterized by selecting a cell expressing the body, expressing a cell expressing the target protein variant, and causing the cell to produce the protein variant. The protein variant production method of the present invention comprises the following steps:
(A) preparing a cell line showing a specific response to a protein;
(B) a step of preparing a cDNA library by introducing into the cell line an expression vector incorporating a cDNA encoding a variant having an amino acid or peptide introduced at a random site on the primary structure of the protein;
(C) detecting the response and selecting a cell that exhibits the response; and
(D) A step of culturing the selected cells and recovering the target protein variant from the culture solution and / or the cultured cells.
May be included.
[0013]
Any protein may be used as a protein to be mutated. Examples include factors belonging to the FGF family and related factors, factors belonging to the heparin-binding growth factor family, and related factors, and more specifically, the following (a) to (f ) Protein.
(A) A protein comprising the amino acid sequence of SEQ ID NO: 2 (FGF-1a).
(B) A protein having an FGF-1 activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted, added or modified in the amino acid sequence of SEQ ID NO: 2.
[0014]
(C) a protein comprising the amino acid sequence of SEQ ID NO: 4 (FGF-6 / 1a).
(D) A protein having an FGF-6 / 1a activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted, added or modified in the amino acid sequence of SEQ ID NO: 4.
(E) A protein comprising the amino acid sequence of SEQ ID NO: 6 (secFGF-1).
(F) A protein having a secFGF-1 activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted, added or modified in the amino acid sequence of SEQ ID NO: 6.
[0015]
The amino acid or peptide to be introduced may be any amino acid or any peptide consisting of two or more amino acids, and in particular, a functional peptide is preferable because it can be expected to alter the protein function.
For example, a glycosylation mutant can be created by introducing an amino acid or peptide capable of undergoing glycosylation.
Peptides that can undergo addition of sugar chains include Asn-X-Ser, Asn-X-Thr, Y-Thr-Pro-Z-Pro, Ser-Gly (X, Y, and Z are any amino acids), etc. And a wide variety of peptides containing these are included.
[0016]
When the sugar chain is a sulfated polysaccharide or glycosaminoglycan, the peptide capable of undergoing addition of the sugar chain may be a proteoglycan core protein or a part thereof. When the sugar chain is an N-type sugar chain (asparagine-linked sugar chain), the peptide capable of undergoing addition of the sugar chain 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 capable of undergoing addition of the sugar chain is preferably a peptide containing an O-type sugar chain-added amino acid sequence.
[0017]
Furthermore, the present invention provides a novel protein variant that can be produced by the above protein variant production method. Examples of protein mutants include mutants of factors belonging to the FGF family or related factors, factors belonging to the heparin-binding growth factor family, or mutants of similar factors. For example, mutants of the proteins (a) to (f) above, specifically, FGF-1 mutants containing the amino acid sequence of SEQ ID NO: 25, 27, 29, 31, 33, or 35 can be mentioned.
Hereinafter, the present invention will be described in detail.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, any protein may be used as a protein into which an amino acid or peptide is introduced. The amino acid or peptide to be introduced may be any amino acid or peptide.
Hereinafter, as a specific example, an example in which a peptide containing a sugar chain-binding sequence is introduced into heparin-binding growth factor FGF-1 is shown, but the present invention is not limited thereto.
As a protein into which a peptide is introduced, for example, any one of the following proteins (a) to (f) may be used.
[0019]
(A) A protein comprising the amino acid sequence of SEQ ID NO: 2 (FGF-1a).
(B) A protein having an FGF-1 activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted, added or modified in the amino acid sequence of SEQ ID NO: 2.
(C) a protein comprising the amino acid sequence of SEQ ID NO: 4 (FGF-6 / 1a).
(D) A protein having an FGF-6 / 1a activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted, added or modified in the amino acid sequence of SEQ ID NO: 4.
(E) A protein comprising the amino acid sequence of SEQ ID NO: 6 (secFGF-1).
(F) A protein having a secFGF-1 activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted, added or modified in the amino acid sequence of SEQ ID NO: 6.
[0020]
The proteins having the amino acid sequences of SEQ ID NOs: 2, 4, and 6 are encoded by the DNA sequences of SEQ ID NOs: 1, 3, and 5, respectively.
Some of these proteins include peptide sequences capable of undergoing addition of sugar chains and signal peptide sequences in addition to peptide sequences of factors belonging to the FGF family.
The heparin-binding protein and FGF referred to in the present specification are for the purpose of secretion called a signal peptide that is present at the amino terminus when secreted from cells, in addition to the protein that is primarily defined by the cDNA described in the sequence listing. It includes proteins in the form of truncated peptide sequences.
[0021]
Hereinafter, although the example which introduce | transduces the peptide containing a sugar_chain | carbohydrate coupling | bonding sequence is shown as a specific example, this invention is not limited to this.
Examples of sugar chains covalently bound to proteins include sulfated polysaccharides such as heparan sulfate and chondroitin sulfate, glycosaminoglycans, N-type sugar chains, and O-type sugar chains, but are not limited thereto. There is nothing.
The sulfated polysaccharide as used in the present specification refers to an extension starting from xylose bound to a serine residue existing in the primary structure of a protein, or to the non-reducing end side of an N-type sugar chain or an O-type sugar chain described later. It is a collective term for various sugar chain structures that extend or exist in a free state, many of which have a repeating structure of disaccharide units of amino sugar and uronic acid (or galactose), and several hydroxyl groups or amino groups Is substituted with a sulfate group.
[0022]
Further, glycosaminoglycans include those having the same structure but not substituted with sulfate groups. In the present specification, these are collectively referred to as sulfated polysaccharides and the like. The specific structure is described in “Fate of sugar chains in cells” (Nagai, Hakomori, Kiso, Kodansha Scientific), and a typical sugar chain sequence is shown in FIG.
The N-type sugar chain as used herein is a general term for various sugar chain structures extending from N-acetylglucosamine bound to an asparagine residue existing in the primary structure of a protein. The specific structure is described in “Fate of sugar chain in cells” (Nagai, Hakomori, Kiso, Kodansha Scientific), and a typical sugar chain sequence is shown in FIG.
[0023]
The O-type sugar chain as used herein is a general term for various sugar chain structures extending from N-acetylgalactosamine bound to a serine residue or threonine residue in the primary structure of a protein. The specific structure is described in “Fate of sugar chains in cells” (Nagai, Hakomori, Kiso, Kodansha Scientific), and a typical sugar chain sequence is shown in FIG.
These sulfated polysaccharides, such as N-type sugar chains and O-type sugar chains, may have additions, deletions, substitutions or modifications as part of their sugar chain sequences as long as they function. .
[0024]
In the present specification, “functional modification” means that the activity of a target protein is changed. Examples of functional modifications include not only simple modification of physiological activity (proliferation promoting activity, differentiation promoting activity, etc.), but also changes in the necessity of cofactors required for activity expression, resistance to heat, acid or alkali Examples include changes in physical stability such as changes, changes in biological stability indicated by changes in blood half-life, and the like.
An example of a method for producing a glycosylated FGF-1 by introducing a peptide containing a glycosylated sequence into secretory FGF-1 will be described below.
[0025]
For the cDNA of the protein into which the peptide is introduced, design appropriate primers from sequences registered in gene banks such as DDBJ (Japan DNA Data Bank), and perform RT-PCR (reverse transcription PCR) from the mRNA of the tissue of the animal. Can be obtained.
By ligating the obtained cDNA to an appropriate plasmid, the construction of the gene encoding the protein is completed. Any plasmid can be used as long as it can be replicated and maintained in the host. Examples include pBR322 and pUC18 derived from E. coli, and pET-3c constructed based on these plasmids. be able to.
[0026]
In addition, when the protein is a non-secretory protein that does not have a secretion signal, a secretory protein can be obtained by adding an appropriate secretion signal to the 5′-terminus, and the glycosylation pathway. In addition, it is possible to facilitate purification and activity confirmation. As an appropriate secretion signal, for example, the amino terminus of a typical secretory glycoprotein can be used, and specific examples include an amino acid having 40 residues from the N-terminus of mouse FGF-6.
[0027]
By subjecting the obtained plasmid to an appropriate treatment, the vector can be cleaved at one site at an arbitrary site. This treatment includes 10 mM Mg ²⁺ Examples include, but are not limited to, DNase I treatment and sonication in the presence. 10 mM Mg ²⁺ If DNase I treatment in the presence is 37 ° C within 5 minutes, the frequency of cleavage at two or more sites in the plasmid can be suppressed.
The cleaved vector can be separated from the uncut vector by, for example, subjecting it to agarose electrophoresis or the like.
[0028]
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 cDNA encoding a naturally occurring functional peptide by the method described above.
For example, amino acid sequences that undergo N-type glycosylation include Asn-X-Thr and Asn-X-Ser (wherein X is an arbitrary amino acid). Examples thereof include, but are not limited to, DNA containing the DNA sequence shown in SEQ ID NO: 7.
[0029]
For example, the amino acid sequence that undergoes O-type glycosylation includes X-Thr-Pro-X-Pro (wherein X is an arbitrary amino acid). Although the DNA containing the DNA sequence shown in SEQ ID NO: 8 is exemplified, it is not limited thereto.
For example, the amino acid sequence that undergoes glycosaminoglycan addition includes the Ser-Gly repeat sequence, and the DNA encoding this includes, for example, the DNA comprising the DNA sequence shown in SEQ ID NO: 9, It is not limited to.
[0030]
For example, another amino acid sequence that undergoes glycosaminoglycan addition includes a partial sequence of the core protein of proteoglycan, and examples of DNA encoding this include DNA containing the DNA sequence shown in SEQ ID NO: 10. However, it is not limited to these. The amino acid sequence encoded by the DNA sequence shown in SEQ ID NO: 10 is shown in SEQ ID NO: 11.
Selection of the introduced mutant can be facilitated by fusing a selection marker gene (for example, a drug resistance gene) to both ends, one end, or the inside of the DNA encoding the peptide. At this time, an appropriate adapter sequence can be used. When a selectable marker gene is fused inside, it can be designed to be a DNA sequence encoding the peptide by removing it and then performing self-ligation.
[0031]
Furthermore, by using a mixture of the fusion gene of DNA encoding the peptide and the selection marker gene, or a mixture obtained by adding a nucleotide having the number of bases of 3n (n is an integer) at one end, It is also possible to align the reading frame of protein genes.
By introducing a fragment of the fusion gene between the DNA encoding the peptide and the selectable marker gene with a vector cleaved at one place, the introduction of the cDNA encoding the peptide into the protein gene is completed. Examples of this binding method include the method described in T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, p. 239 (1982), and specifically, molecular biology such as a method using DNA ligase. It is possible to apply a method generally used in the above.
[0032]
After introducing the obtained vector into a suitable host and culturing in the presence of a selective drug, only E. coli containing the vector bound to the gene can be obtained as a living cell.
Examples of the host include E. coli DH5α strain. Examples of selectable marker genes include ampicillin resistance gene (Amp), chloramphenicol resistance gene (Cm), tetracycline resistance gene (Tc), kanamycin resistance gene (Km), and the like.
[0033]
The plasmid is recovered from the obtained mixture of transformants, the drug resistance marker gene is removed by restriction enzyme treatment, and self-ligation is caused to complete the construction of the peptide-introduced protein gene library.
From the plasmid library prepared as described above, a region containing a base sequence encoding a secretory signal, an introduced peptide and a protein to be introduced (hereinafter referred to as “region containing a base sequence encoding an introduced mutant protein”). Is cut out and ligated downstream of a promoter in a vector suitable for expression, whereby an expression vector library can be obtained.
[0034]
The region containing the base sequence encoding the introduced mutant protein has ATG as a translation initiation codon at the 5′-end, and TAA as a translation termination codon at the 3′-end.
, TGA or TAG.
Furthermore, in order to express the 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 suitable for the host used for gene expression. When the host to be transformed is Bacillus subtilis, examples include SP01 promoter, SP02 promoter, penP promoter, and when the host is yeast, examples include PHO5 promoter, PGK promoter, GAP promoter, ADH promoter and the like. When the host is an animal cell, examples include SV40-derived promoters and retrovirus promoters.
[0035]
By introducing a vector containing the above recombinant DNA into a host cell, a transformant carrying the vector is produced.
Any host cell may be used as long as it is suitable for the purpose. For example, when the purpose is to express a glycosylation mutant protein by introducing a glycosylated sequence peptide, those having a glycosylation pathway to the protein, such as yeast (for example, Pichia pasto ris, Saccharomyces cerevisiae), animals Examples include cells (for example, COS cells, CHO cells, BHK cells, NIH3T3 cells, BALB / c3T3 cells, HUVE cells, LEII cells), insect cells (for example, Sf-9 cells, Tn cells) and the like. However, it is not limited to these.
[0036]
The transformation is performed by a method generally performed for each host. Any method that can be applied may be used if it is not general. For example, if the host is yeast, a vector containing the recombinant DNA is introduced into a competent cell 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 recombinant DNA is introduced into cells in the growth phase or the like by the calcium phosphate method, lipofection method or electroporation method.
[0037]
A mutant protein is produced by culturing the transformant thus obtained in a medium.
When the transformant is cultured, a medium generally used for each host is used as the medium used for the culture. Or, if it is not general, it may be an applicable medium. As an example, if the host is yeast, YPD medium or the like is used. If the host is an animal cell, use Dulbecco's MEM supplemented with animal serum.
[0038]
Culturing is performed under conditions generally used for each host. Moreover, it is sufficient if the condition is applicable even if it is not general. As an example, if the host is yeast, it is carried out at about 25 to 37 ° C. for about 12 hours to 2 weeks, and if necessary, aeration or stirring can be added. If the host is an animal cell, at about 32-37 ° C, 5% CO ₂ It can be carried out under conditions of 100% humidity for about 24 hours to 2 weeks. If necessary, the gas phase conditions can be changed and stirring can be added.
[0039]
When selecting a function-modified mutant from the expression vector library, an appropriate method is selected based on the properties of the protein.
For example, when a peptide is introduced into the FGF-1 protein and a mutant having altered growth factor activity is selected, the following series of techniques are applied, but the present invention is not limited thereto.
First, a floating cell line exhibiting FGF-1-specific growth is obtained, and this is used as a host for gene transfer of an expression vector library, which is then subjected to the following function-modified mutant FGF-1 selection system.
[0040]
Examples of cell lines exhibiting FGF-1 specific proliferation include, but are not limited to, mouse (pro B cell line) Ba / F3 into which FGF receptor has been introduced.
When the cell line transfected with the above expression vector library is serially diluted and cultured in a liquid medium containing heparin and fetal calf serum, the function-modified mutant FGF-1 secreting clone is colonized by autocrine or paracrine. Form.
After selecting the clone, the structure of the function-modified FGF-1, that is, the optimal site of the introduced peptide can be determined by analyzing the gene sequence of the expressed FGF by RT-PCR or the like.
[0041]
In order to obtain the function-modified mutant FGF-1 from the obtained function-modified mutant FGF-1 secreting strain, one released in the culture medium can be directly recovered from the supernatant after centrifugation. In addition, when extracting from cultured cells or cells, the target protein is removed from the cells by culturing the cells or cells by homogenizer, French press, ultrasonic wave, lysozyme and / or freeze-thawing. And the protein can be obtained from the soluble fraction. In addition, when the target protein is contained in the insoluble fraction, a method in which the cells or cells are destroyed, the insoluble fraction is collected by centrifugation, and is made soluble by a buffer solution containing guanidine hydrochloride or the like and collected. In addition, there is a method in which cells or cells are directly destroyed with a buffer solution containing a protein denaturant such as guanidine hydrochloride, and the target protein is eluted outside the cells.
[0042]
In order to purify the function-modified mutant FGF-1 from the supernatant, known separation and purification methods can be combined appropriately. These known separation and purification methods include salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, SDS polyacrylamide gel electrophoresis, ion exchange chromatography, affinity chromatography, reverse phase high performance liquid chromatography, etc. Electric point electrophoresis or the like can be used. Furthermore, for many heparin-binding proteins, affinity chromatography methods using heparin sepharose as a carrier can be applied.
[0043]
The preparation thus obtained can be dialyzed and freeze-dried to obtain a dry powder as long as the activity of the function-modified mutant FGF-1 is not impaired. Furthermore, adding serum albumin or the like as a carrier and storing it is effective for preventing adsorption of the sample to the container.
Further, the coexistence of a trace amount of reducing agent in the purification process or storage process is suitable for preventing oxidation of the sample. Examples of the reducing agent include β-mercaptoethanol, dithiothreitol, glutatin and the like.
The function-modified mutant FGF-1 of the present invention can also be produced by introducing a peptide into a protein by a chemical method.
As described above, the present invention has been described taking the function-modified mutant FGF-1 as an example. However, amino acids or peptides can be introduced at random sites in proteins other than heparin-binding proteins.
[0044]
[Deposit of microorganisms]
The Escherichia coli DH5α strain containing the plasmid pMEXneo incorporating the gene encoding the function-modified mutant FGF-1 of the present invention (having the DNA sequences of SEQ ID NOs: 24, 26, 28, 30, 32 and 34, respectively) On January 28, 1999 at the Institute of Biotechnology, National Institute of Biotechnology, with deposit numbers FERM P-17181, FERM P-17182, FERM P-17183, FERM P-17184, FERM P-17185 and FERM P-17186 Deposited.
[0045]
【Example】
EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated concretely, this invention is not restrict | limited to the following Example.
〔Example〕
[1] Outline of the embodiment
Hereinafter, as an example, a method for introducing a sequence undergoing N-type glycosylation to a random position on the primary structure of the fibroblast growth factor FGF-1 will be described to specifically explain the present invention.
[0046]
[2] Construction of peptide insertion mutant secFGF-1 cDNA
1) Construction of secFGF-1 plasmid
1. Preparation of FGF-1a / pBluescript II (KS +) plasmid
Human FGF-1 cDNA was isolated from a human cDNA library according to J. Exp. Med. (1992), 175 (4), 1073-1080. Using 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 PCR reaction was performed using AAT TCT TTA ATC AGA AGA GAC TGG-3 ′) (SEQ ID NO: 13) as a primer. The specifically amplified 434 base pair band was separated by electrophoresis, extracted, and then double cut with EcoR I and Sal I. The resulting 422 base pair band was separated and extracted and inserted into pBluescript II (KS +) cloning vector (2934 base pairs, purchased from Stratagene) double-cut with EcoR I and Sal I. 1a / pBluescript II (KS +) was obtained.
FGF-1a / pBluescript II (KS +) was sequentially digested with Aor51H I and Sal I, and the resulting 2626 base pair band was separated and extracted and used in the ligation reaction shown below.
[0047]
2.Mouse FGF-6 <NQ> Creation of cDNA fragment
Mouse FGF-6 cDNA was isolated from a mouse cDNA library according to Oncogene (1990), 5 (6), 823-31. Using isolated mouse FGF-6 cDNA as a template, # 105 (5'-GCG TCG ACC CAC CAT GTC-3 ') (SEQ ID NO: 14) and # 124 (5'-GCG ATA TCC AGT AGC GTG CCT TGG GCG CG -3 ′) PCR reaction was performed using (SEQ ID NO: 15) as a primer. The specifically amplified 138 base pair band was separated by electrophoresis, extracted, and then double cut with EcoR V and Sal I. The resulting 130 base pair band was separated and extracted and used for the ligation reaction shown below.
[0048]
3. Creation of secFGF-1 gene
The mouse FGF-6 PCR product EcoR V / Sal I fragment and the FGF-1a / pBluescript II (KS +) Aor51H I / Sal I fragment were subjected to DNA ligation, and the secFGF-1 / pBluescript II (KS +) vector was used. Obtained. Furthermore, this was double-cut with EcoR I and Sal I, and the resulting band of 540 base pairs was separated and extracted. This was inserted into pMEXneo expression vector (5916 base pairs, Dr. M. Barbacid, Mol. Cell. Biol. (1989) 9 (1), 24-33) double-cut with EcoR I and Sal I, The final product of this process, secFGF-1 gene, was obtained. This expression vector includes the base sequence of SEQ ID NO: 5.
[0049]
2) One-point cutting with DNase I
10 ug of sec FGF-1 / pBluescript II (KS +) plasmid obtained as above was added to 10 mM MgCl ₂ The solution was treated with DNase I (400 pg) at 37 degrees for 5 minutes. The reaction products were separated by 1.4% agarose gel electrophoresis, and the DNA fragment migrated at the position of 3474 base pairs was recovered and purified, and used for the ligation shown below.
[0050]
3) Creation of NLS-Cm fragment
NLS-Cm fragments were created in three ways: frames 1, 2, and 3. This is because a large number of clones in which the frame of the introduced peptide matches the frame of the protein into which it is introduced are obtained.
[0051]
1. Creation of NLS-Cm frame 1
Using plasmid pCm containing the chloramphenicol resistance gene as a template, # 3n + 1F (5'-gcgcgcgtttaaacttaagcatcggcacgtaagaggttccaactt-3 ') (SEQ ID NO: 16) and # 3n + 1R (5'-atatatatcccgggcttaagttacgccccgccctgccactca-3') sequence PCR reaction was performed using 17) as a primer. The specifically amplified 741 base pair band was separated by electrophoresis, extracted, and then double-cut with Pme I and Sma I. The obtained band of 720 base pairs was separated and extracted to obtain NLS-Cm frame 1, which was used for the ligation reaction shown below.
[0052]
2. Creation of NLS-Cm frame 2
PCR reaction was performed using pCm as a template and # 3n + 2F (5'-atatatcgcgaacttaagcatcggcacgtaagaggttccaac-3 ') (SEQ ID NO: 18) and # 3n + 2R (5'-atatatatgccggcttaagttacgccccgccctgccactca-3') (SEQ ID NO: 19) as primers. It was. The specifically amplified 739 base pair band was separated by electrophoresis, extracted, and then double-cut with Nae I and Nru I. The obtained band of 720 base pairs was separated and extracted to obtain NLS-Cm frame 2, which was used for the ligation reaction shown below.
[0053]
3. Creation of NLS-Cm frame 3
PCR reaction was performed using pCm as a template and # 3nF (5'-gcgcgcgttaacttaagcatcggcacgtaagaggttccaactt-3 ') (SEQ ID NO: 20) and # 3nR (5'-atataagcgcttaagttacgccccgccctgccactca-3') (SEQ ID NO: 21) as primers. The specifically amplified 734 base pair band was separated by electrophoresis, extracted, and then double cut with Hinc II and Aor51H I. The obtained band of 717 base pairs was separated and extracted to obtain NLS-Cm frame 3, which was used for the ligation reaction shown below.
[0054]
4) Ligation
The DNase I-treated secFGF-1 / pBluescript II (KS +) plasmid and NLS-Cm frame 1, NLS-Cm frame 2, or NLS-Cm frame 3 were subjected to a DNA ligation reaction using DNA ligase.
The reaction product was used to transform E. coli DH5α, which was cultured for 18 hours in LB medium containing 30 ug / ml chloramphenicol and 50 ug / ml ampicillin.
[0055]
The plasmid was recovered from the grown Escherichia coli, purified, and cleaved with the restriction enzyme Afl II. Separation of reaction products by 1.4% agarose gel electrophoresis, recovery and purification of DNA fragments migrating to 4194 or 4191 base pairs, and self-ligation by DNA ligation using DNA ligase ) Using this, Escherichia coli DH5α was transformed again and cultured in LB medium containing 50 ug / ml ampicillin for 18 hours.
[0056]
Six types of Escherichia coli grown were selected, plasmid DNA (A, B, C, D, E and F) was collected from the E. coli and purified, and the nucleotide sequence was decoded by the cycle sequence method. As a result, the structure of secFGF-1 (having the DNA sequences of SEQ ID NOs: 24, 26, 28, 30, 32, and 34, respectively) into which DNA encoding a sequence that undergoes N-type glycosylation has been introduced is apparent. became. The glycosylated sequence-introduced secFGF-1 cDNA contained in these plasmids was used for incorporation into an expression vector as shown below. The amino acid sequences encoded by the DNA sequence 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.
[0057]
5) Incorporation into expression vector
The resulting plasmids A, B, C, D, E and F were double-cut with EcoR I and Sal I, and 552 or 549 base pair bands were separated and extracted on agarose gel electrophoresis. This was inserted into a pMEXneo expression vector (5916 base pairs) double-cut with EcoR I and Sal I to obtain G, H, I, J, K and L.
[0058]
[3] Confirmation of functionality of inserted sequence
It was confirmed by the following method that the sequence receiving N-type glycosylation introduced at random positions on the primary structure of secFGF-1 was functioning. Introducing secFGF-1 expression vectors G, H, I, J, K, and L, into which DNA encoding a sequence that undergoes N-type glycosylation has been introduced, into mouse pro B cell line Ba / F3 Transgenic cells were selected by culturing in the presence of single clones. These cell extracts were prepared, and a part of this extract containing the expressed protein was digested with N-glycanase, an enzyme that specifically cleaves N-type sugar chains. Untreated and digested samples were analyzed by SDS polyacrylamide gel electrophoresis and immunoblotting, respectively. As shown in FIG. 5, since all the proteins that are expression products of the six types of vectors were reduced in molecular weight by N-glycanase, it was confirmed that they were modified by N-type sugar chains. In FIG. 5, (−) indicates untreated, and (+) indicates an immunoblot of each function-modified mutant FGF-1 after enzyme treatment. The white triangle indicates the size of the protein backbone of secFGF-1. 1 is FERM P-17185, 2 is FERM P-17182, 3 is FERM P-17181, 4 is FERM P-17183, 5 is FERM P-17186, 6 is the expression product of the cDNA sequence of FERM P-17184. Since the molecular weight of the protein backbone of secFGF-1 was reduced by N-glycanase treatment, it was confirmed that all function-modified mutant FGF-1 was modified with N-glycans.
[0059]
[4] Selection of function-modified mutant FGF-1
1) Creation of FGF-specific target cell lines
An FGF receptor (subtype R1c) expression vector (provided by Dr. DM Ornitz, J. Biol. Chem. (1996) 271 (25), 15292-15297) was obtained from the mouse pro B cell line Ba / F3 (RIKEN) Gene-transfected cells (FGF-R1c / BaF3 cells) were selected by introducing the gene into Cell Development Bank RCB No. 0805) and culturing in the presence of geneticin. A single clone growing in RPMI 1640 medium containing 10 ng / ml FGF-1, 10 ug / ml heparin, 10% fetal calf serum in the absence of IL-3 is used for the following experiments.
[0060]
2) Integration into expression vectors
The obtained plasmid was double-cut 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.
[0061]
3) Gene transfer to the target cell line
The obtained NLS sec FGF-1 / pMEXneo vector is transfected into FGF-R1c / BaF3 cells by electroporation and cultured in RPMI 1640 medium containing 100 ug / ml heparin and 10% fetal bovine serum. After 14 days, a clone forming a colony is selected, and the function-modified mutant secFGF-1 produced and secreted by this clone is used for the following analysis.
[0062]
4) Acquisition of the gene of the function-modified mutant secFGF-1
The clone is cultured in a normal medium (RPM 1640 medium containing 10% WEHI culture supernatant and 10% fetal calf serum as an IL-3 source), and its total RNA is recovered by the guanidine isothiocyanate method. This was subjected to a reverse transcription reaction using random hexamers as primers, and the resulting cDNA was used as a template for # 956 (5'-TCTTCCgATAgACTgCgTCg-3 ') (SEQ ID NO: 22) and # 957 (5'-CATTCTAgTTgTggTTTgTCC-3') PCR reaction is performed using (SEQ ID NO: 23) as a primer. This reaction product was double-cut with EcoR I and Sal I and inserted into a pMEXneo vector (5916 base pairs) double-cut with EcoR I and Sal I to obtain NLS-secFGF-1 / pMEXneo. The DNA sequence was decoded by cycle sequencing, and the structure of the function-modified mutant FGF-1 was obtained. The DNA sequences were shown in SEQ ID NOs: 30 and 32.
[0063]
[3] Production of function-modified mutant FGF-1
1) Gene transfer and mass production
An expression vector containing the DNA sequences shown in SEQ ID NOs: 30 and 32 is introduced into CHO-K1 cells (Chinese hamster ovary cell K1 sub-line, RIKEN Cell Development Bank RCB 0285) by lipofection and cultured in the presence of geneticin. By doing so, transgenic cells were selected. The obtained cells were increased until the culture dish was almost full, and the substance production was increased by replacing the medium with a serum-free medium. The medium was changed every two days, and the conditioned medium obtained was centrifuged at low speed, and the supernatant was stored at 4 ° C.
[0064]
2) Purification of function-modified mutant FGF-1
Heparin sepharose beads were added to the conditioned medium of the functionally modified mutant FGF-1 secreting cells and stirred at 4 ° C. for 2 hours or more. The beads that settled by low-speed centrifugation were collected, washed thoroughly with physiological phosphate buffer (PBS: phosphate buffered saline, pH 7.4), and then 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 then the sample was again subjected to high performance liquid chromatography packed with heparin affinity affinity beads, and the function-modified mutant FGF-1 was eluted with a NaCl concentration gradient. did.
[0065]
[Test Example 1]
DNA synthesis promoting activity against FGF receptor-expressing cell line FGF-R1c / BaF3
Immediately after removing IL-3 from the culture solution of FGF-R1c / BaF3, a medium containing a function-modified mutant FGF-1, 10 ug / ml heparin, and 10% fetal bovine serum is added. After culturing for 36 hours, radiolabeled thymidine is incorporated for 4 hours, and the amount of DNA newly synthesized by the radioactivity incorporated into DNA during this period is obtained.
[0066]
[Test Example 2]
Heparin dependence in promoting proliferation of human vascular endothelial cells
HUVECs (human umbilical cord-derived vascular endothelial cells) stop 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. ) After adding the functionally-modified mutant FGF-1, the radiolabeled thymidine is incorporated for 6 hours, and the amount of DNA newly synthesized by the radioactivity incorporated into the DNA during this period is obtained.
[0067]
[Test Example 3]
Cell surface lectin binding activity
The binding activity to cell surface membrane proteins that recognize sugar chains was evaluated. Macrophage lectin (ML) cDNA expression vector that recognizes non-reducing terminal galactose (isolated from mouse peritoneal macrophage cDNA library according to J. Biochem., (1992) 111, 331-336 and incorporated into pMEXneo vector) Was introduced into Ba / F3 cells without FGF receptor and cultured in the presence of geneticin to select transgenic cells (ML / BaF3 cells). The ML / BaF3 cells are mixed with labeled functionally modified mutant FGF-1, and after incubation, the cells are collected by centrifugation, washed with medium, and the amount of functionally 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 function-modified mutant FGF-1 has a significant amount of binding to the cell surface as an effect of the sugar chain.
[0068]
[Test Example 4]
Binding activity to hepatocytes, intracellular translocation activity and cell growth promoting activity
Binding activity, intracellular translocation activity, and growth promoting activity for cells having a membrane protein having a property of recognizing a sugar chain on the cell surface were evaluated. Hepatocytes have an FGF receptor and an asialoglycoprotein receptor that recognizes non-reducing terminal galactose on the cell surface. Function-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, it was shown that the amount of the function-modified mutant FGF-1 bound to the cell surface was significantly large as an effect of the sugar chain. The amount taken up into the cells was also measured. As a result, it was shown that the amount of function-modified mutant FGF-1 incorporated into cells as a sugar chain effect was significantly large. In addition, the cells were mixed with unlabeled function-modified mutant FGF-1, and 18 hours later, the amount of DNA synthesis representing cell proliferation was evaluated by incorporation of radiolabeled thymidine. As a result, it was shown that the cell growth promoting activity of the function-modified mutant FGF-1 significantly changed as an effect of the sugar chain.
[0069]
【The invention's effect】
According to the present invention, a technique for inserting a peptide at a random position on a protein primary 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 systematic mutants without trial and error.
[0070]
[Sequence Listing]

[0071]
[Sequence Listing Free Text]
SEQ ID NO: 7 shows the nucleotide sequence encoding the amino acid sequence Asn-leu-Ser.
SEQ ID NO: 8 shows the 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 the core protein of proteoglycan.
[0072]
SEQ ID NO: 12 shows the nucleotide sequence of # 967 primer.
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 shows the nucleotide sequence of # 124 primer.
SEQ ID NO: 16 shows the nucleotide sequence of # 3n + 1F primer.
SEQ ID NO: 17 shows the nucleotide sequence of # 3n + 1R primer.
[0073]
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 shows the nucleotide sequence of # 3nR primer.
SEQ ID NO: 22 shows the nucleotide sequence of # 956 primer.
SEQ ID NO: 23 shows the nucleotide sequence of the # 957 primer.
[0074]
SEQ ID NO: 24 shows the nucleotide sequence of the mutant FGF-1 gene introduced into FERM P-17181.
SEQ ID NO: 26 shows the nucleotide sequence of the mutant FGF-1 gene introduced into FERM P-17182.
SEQ ID NO: 28 shows the nucleotide sequence of the mutant FGF-1 gene introduced into FERM P-17183.
SEQ ID NO: 30 shows the nucleotide sequence of the mutant FGF-1 gene introduced into FERM P-17184.
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 representative sulfated polysaccharide / glycosaminoglycan sugar chain.
FIG. 2 shows the structure of a typical N-type sugar chain. G represents galactose, GN represents N-acetylglucosamine, M represents mannose, F represents fucose, and Sia represents sialic acid.
FIG. 3 shows a representative O-type sugar chain structure. G represents galactose, and GalNAc represents N-acetylgalactosamine.
FIG. 4 shows the construction of peptide insertion mutant secFGF-1 cDNA.
FIG. 5 shows the expression of cDNA of mutant FGF-1 introduced into FERM P-17185, FERM P-17182, FERM P-17181, FERM P-17183, FERM P-17186 and FERM P-17184. Shown are immunoblots of the product untreated (-) and after treatment with N-glycanase (+).

Claims (1)

The cDNA encoding the protein is incorporated into a circular plasmid, and this circular plasmid is treated with DNaseI treatment in the presence of 10 mM Mg ⁺² at 37 ° C for 5 minutes or less , cutting at random positions and cutting only at one location. At least one selected from the group consisting of Asn-X-Ser, Asn-X-Thr, Y-Thr-Pro-Z-Pro and Ser-Gly (X, Y and Z are arbitrary amino acids) When introducing a DNA fragment encoding one sugar chain-binding amino acid sequence, (1) 3n nucleotides on the 5 ′ end side of the DNA encoding the sugar chain-binding amino acid sequence, DNA fragment with 3n nucleotides added to (2), 3n + 1 nucleotides added to the 5 'end of the DNA encoding the sugar chain-binding amino acid sequence, and 3n + 2 nucleotides added to the 3' end (3) 3n + 2 nucleotides at the 5 ′ end of the DNA encoding the sugar chain-binding amino acid sequence, and the 3 ′ end By using a mixture of DNA fragments added with 3n + 1 nucleotides on the end side ( where n is an integer in (1) to (3) above), the frame of the sugar chain binding site of the protein into which it is introduced A method for preparing a library of cDNAs encoding variants in which a sugar chain-binding site has been introduced at one random place on the primary structure of a protein, characterized in that clones matching the frame can be obtained .

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