JP2021114975A - Recombinant protein and method for producing the same - Google Patents

Abstract

To provide a recombinant protein having functions as a canine basic fibroblast growth factor, from which harmful substances such as endotoxin are removed.SOLUTION: The present disclosure provides a method for producing a recombinant protein that comprises a specific amino acid sequence and has a growth stimulation action on a canine fibroblast, by a wheat germ acellular protein synthesis system. There is also provided a recombinant protein obtained by the method.SELECTED DRAWING: None

Description

The present invention relates to a recombinant protein and a method for producing the same.

Basic fibroblast growth factor (bFGF, FGF-2) is an angiogenic factor that belongs to the FGF family and is widely distributed in the human body. bFGF has a function of promoting angiogenesis and arteriogenesis, and is known to be involved in the formation of nerves and bones. Furthermore, in recent years, it has become clear that bFGF is a factor necessary for maintaining the undifferentiated state of pluripotent stem cells such as human induced pluripotent stem cells.

In bone regenerative medicine, treatment using transgenic human bFGF (rh-bFGF) has been carried out for the purpose of promoting bone union, and the effectiveness of rh-bFGF has also been shown in dogs as well. (See, for example, Non-Patent Documents 1 and 2). However, it is considered desirable to use recombinant canine bFGF (rc-bFGF) when developing treatment with bFGF in dogs. Therefore, the production of rc-bFGF has been attempted at many research facilities in the world, but most of them are methods using Escherichia coli.

Nakamura T et al., “Recombinant Human Basic Fibroblast Growth Factor Accelerates Fracture Healing by Enhancing Callus Remodeling in Experimental Dog Tibial Fracture.”, JOURNAL OF BONE AND MINERAL RESEARCH, Vol. 13, No. 6, pp. 942-949, 1998 .. Honnami M, et al., “Bone Regeneration by the Combined Use of Tetrapod-Shaped Calcium Phosphate Granules with Basic Fibroblast Growth Factor-Binding Ion Complex Gel in Canine Segmental Radial Defects.”, J. Vet. Med. Sci., Vol. 76, No.7, pp. 955-961, 2014.

However, in order to clinically apply cytokine therapy using rc-bFGF, it is difficult to completely eliminate harmful substances such as endotoxin from the obtained recombinant protein by the conventional method using Escherichia coli, and further, as bFGF. It is often difficult to purify a product that retains the various functions of.

The present invention has been made in view of the above circumstances, and provides a recombinant protein having a function as a canine basic fibroblast growth factor and a method for producing the same.

That is, the present invention includes the following aspects.
(1) Recombinant consisting of any of the following amino acid sequences (a) to (c), having a growth-promoting ability for canine fibroblasts, and being a compound by a wheat germ cell-free protein synthesis system. protein.
(A) Amino acid sequence shown in SEQ ID NO: 1;
(B) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1;
(C) Amino acid sequence having 98% or more identity with the amino acid sequence shown in SEQ ID NO: 1 (2) Proliferation against canine fibroblasts consisting of any of the following amino acid sequences (a) to (c) A method for producing a recombinant protein having a facilitating ability.
A production method for synthesizing the recombinant protein using a wheat germ cell-free protein synthesis system.
(A) Amino acid sequence shown in SEQ ID NO: 1;
(B) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1;
(C) Amino acid sequence having 98% or more identity with the amino acid sequence shown in SEQ ID NO: 1 (3) The synthesized recombinant protein has a histidine tag attached to its N-terminal or C-terminal. The production method according to (2), wherein the synthesized recombinant protein is purified using the histidine tag.

According to the recombinant protein of the above aspect and the method for producing the same, it is possible to provide the recombinant protein having a function as a canine basic fibroblast growth factor and the method for producing the same. The obtained recombinant protein can be used for canine regenerative medicine such as cytokine therapy.

It is an image which shows the result of confirming the expression of pErk in the HEK293 cell which added the recombinant dog bFGF in Example 1. It is an image which shows the state of the fibroblast which was cultured by adding the recombinant dog bFGF in Example 1. The scale bar indicates 500 μm. 3 is a graph showing changes in the number of cells in the recombinant dog bFGF-added group or the non-added group in Example 1.

Hereinafter, the recombinant protein according to one embodiment of the present invention (hereinafter, abbreviated as “the present embodiment”) and the method for producing the same will be described in detail.

≪Recombinant protein≫
The recombinant protein of the present embodiment comprises any of the following amino acid sequences (a) to (c), has a proliferative ability for canine fibroblasts, and is synthesized by a wheat germ cell-free protein synthesis system. It is a thing.
(A) Amino acid sequence shown in SEQ ID NO: 1;
(B) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1;
(C) An amino acid sequence having 98% or more identity with the amino acid sequence shown in SEQ ID NO: 1.

It is difficult to completely eliminate harmful substances and impurities derived from genetically modified organisms by the conventional synthetic method using genetically modified organisms such as Escherichia coli, and the recombination has a sufficient function as a canine bFGF. Synthesis of canine bFGF was difficult.
On the other hand, the recombinant protein of the present embodiment is a recombinant dog bFGF that retains each function as a dog bFGF such as a growth promoting ability for canine fibroblasts by using a wheat germ cell-free protein synthesis system. Obtainable. The "proliferative ability of canine fibroblasts" as used herein means the ability of canine fibroblasts to promote cell proliferation. Specifically, for example, as shown in Examples described later ^{, the cell proliferation rate (cells after culture) when 1.5 × 10 4} cells of canine fibroblasts were cultured for 5 days under the condition of no addition of canine bFGF. ^{The cell proliferation rate when 1.5 × 10 4} cells of canine fibroblasts were cultured for 5 days under the condition of adding canine bFGF to the number / number of cells before culture × 100) was 1.1 times or more, preferably 1. When it is 2 times or more, more preferably 1.5 times or more, it can be evaluated that the dog bFGF has a proliferation promoting ability for dog fibroblasts.
In addition to the ability to promote proliferation of canine fibroblasts, the functions of canine bFGF include functions known as bFGF such as angiogenesis and angiogenesis and ERK activation ability (ERK phosphorylation ability). Can be mentioned.

The recombinant protein of the present embodiment consists of the following amino acid sequence (a).
(A) Amino acid sequence shown in SEQ ID NO: 1.

The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of canine bFGF and is disclosed as Genbank accession number: XP_003432529.

The recombinant protein of the present embodiment may be a protein having the following amino acid sequence (b) or (c) as a protein functionally equivalent to the protein having the amino acid sequence of (a).
(B) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1;
(C) An amino acid sequence having 98% or more identity with the amino acid sequence shown in SEQ ID NO: 1.

In the amino acid sequence of (b), the number of amino acids that may be deleted, substituted, or added is preferably 1 or more and 3 or less, more preferably 1 or more and 2 or less, and even more preferably 1. ..

The "substitution" referred to here is preferably substituted with another amino acid residue having a chemically similar side chain. A group of amino acid residues having chemically similar amino acid side chains is well known in the art to which the recombinant protein of this embodiment belongs. For example, acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine and histidine), neutral amino acids, amino acids having a hydrocarbon chain (glycine, alanine, valine, leucine, isoleucine and proline), hydroxy groups. Amino acids with (serine and threonine), amino acids containing sulfur (cysteine and methionine), amino acids with amide groups (asparagin and glutamine), amino acids with imino groups (proline), amino acids with aromatic groups (phenylalanine, tyrosine and It can be classified by tryptophan) and the like. Commonly possible amino acid substitutions include, for example, alanine / serine, valine / isoleucine, aspartic acid / glutamic acid, threonine / serine, alanine / glycine, alanin / threonine, serine / asparagine, alanin / valine, serine / glycine, Examples thereof include tyrosine / phenylalanine, alanine / proline, lysine / arginine, aspartic acid / aspartic acid, leucine / isoleucine, leucine / valine, alanine / glutamic acid, aspartic acid / glycine and the like.

The protein consisting of the amino acid sequence of (c) has 98% or more sequence identity in order to be a protein functionally equivalent to the protein consisting of the amino acid sequence of (a). The sequence identity is preferably 98.5% or more, more preferably 99% or more, further preferably 99.5% or more, particularly preferably 99.7% or more, and most preferably 99.9% or more.

Here, the sequence identity of the target amino acid sequence with respect to the reference amino acid sequence can be obtained, for example, as follows. First, the reference amino acid sequence and the target amino acid sequence are aligned. Here, each amino acid sequence may include a gap so as to maximize the sequence identity. Subsequently, the number of matching amino acids in the reference amino acid sequence and the target amino acid sequence can be calculated, and the sequence identity can be determined according to the following formula.

"Sequence identity (%)" = [Number of matched amino acids] / [Total number of amino acids in the target amino acid sequence] x 100

The protein consisting of the amino acid sequence of (b) or (c) is required to have a proliferative ability for canine fibroblasts.

≪Production method of recombinant protein≫
The recombinant protein of the present embodiment is a protein consisting of any of the amino acid sequences (a) to (c) above, and is synthesized by a wheat germ cell-free protein synthesis system. That is, in the method for producing a recombinant protein of the present embodiment (hereinafter, may be simply referred to as "the method for producing the present embodiment"), the recombinant protein is synthesized using a wheat germ cell-free protein synthesis system.

Next, the production method of the present embodiment using the wheat germ cell-free protein synthesis system will be described in detail below.

The manufacturing method of the present embodiment includes, for example, the following steps 1) to 3) (hereinafter, may be referred to as "step 1)", "step 2)", and "step 3)", respectively).
1) Step of preparing a transfer template;
2) A step of mixing a transfer reaction solution and a transfer template to carry out a transfer reaction;
3) A transcription solution containing mRNA, which is a transcription reaction product, is added to a cell extract derived from wheat germ for protein synthesis to prepare a translation reaction solution, and then the translation reaction substrate solution is layered on the translation reaction solution. The process of performing a translation reaction.

<Step 1)>
As used herein, the term "transcription template" means a nucleic acid that can be used as a template molecule for an in vitro transcription reaction, and has at least a base sequence encoding the recombinant protein downstream of an appropriate promoter sequence. The appropriate promoter sequence refers to a promoter sequence that can be recognized by RNA polymerase used in the transcription reaction, and examples thereof include SP6 promoter and T7 promoter. Examples of the base sequence encoding the recombinant protein include the base sequence shown in SEQ ID NO: 2. The base sequence shown in SEQ ID NO: 2 encodes a protein consisting of "(a) the amino acid sequence shown in SEQ ID NO: 1".

The transcription template preferably has a base sequence (translation enhancer) having an activity of controlling translation efficiency between the promoter sequence and the base sequence encoding the target protein, and for example, an RNA such as an Ω sequence derived from tobacco mosaic virus. A fully artificial sequence E01 selected from a 5'untranslated region derived from a virus, a Kozak sequence, a random nucleic acid sequence pool, or the like can be used. Further, the transcription template preferably contains a 3'untranslated region including a transcription termination region and the like downstream of the base sequence encoding the recombinant protein. As the 3'untranslated region, about 1.0 kb or more and about 3.0 kb or less downstream of the stop codon is preferably used. For the 3'untranslated region, for example, that of the natural canine bFGF gene can be used, but that of another gene may be used.

The DNA encoding the recombinant protein can be used as it is as a transcription template without purifying the reaction product amplified and synthesized by the PCR method.
The transcription template DNA obtained as described above may be purified by chloroform extraction or alcohol precipitation and then subjected to a transcription reaction, but in the production method of the present embodiment, the reaction solution after the PCR reaction is used as it is as a transcription template solution. It is possible to do.

Further, the DNA encoding the recombinant protein may be, for example, in a form inserted into an expression vector. The expression vector used here can be appropriately selected depending on the host to be used, the purpose, and the like, and examples thereof include a plasmid, a phage vector, and a viral vector. For example, when the host is Escherichia coli, a plasmid vector such as pEU, which is an expression vector optimized for maximizing the synthesis efficiency in a wheat cell-free protein synthesis system, can be used.

The expression vector may appropriately have factors such as a promoter capable of inducing expression, a gene encoding a signal sequence, a marker gene for selection, and a terminator. In addition, a sequence expressed as a fusion protein with thioredoxin, histidine (His) tag, GST (glutathione S-transferase), etc. may be added so as to facilitate isolation and purification. Above all, it is preferable that the expression vector is added with a sequence expressed as a fusion protein with a histidine (His) tag.

<Step 2)>
From the transcription template DNA prepared in step 1), mRNA, which is a translation template, is generated by an in vitro transcription reaction. In step 2), the solution containing the transfer template provided in the reaction system (for example, a commercially available container such as a 96-well titer plate), RNA polymerase compatible with the promoter in the transfer template (for example, SP6 RNA polymerase, etc.) and the like. After mixing with a solution containing components necessary for transcription reaction (also referred to as "transcription reaction solution") such as a substrate for RNA synthesis (4 types of ribonucleoside triphosphate), about 20 ° C. or higher and 60 ° C. or lower. It is preferably carried out by incubating the mixed solution at about 30 ° C. or higher and 42 ° C. or lower for about 30 minutes or more and 16 hours or less, preferably about 2 hours or more and 10 hours or less.

<Step 3)>
The transcription solution obtained in step 2) is directly added to the cell extract derived from wheat germ for protein synthesis. Here, direct addition means addition to a cell extract derived from wheat germ for protein synthesis without adding any purification step to the transcription solution containing mRNA of the transcription reaction product. Further, the cell extract derived from wheat germ for protein synthesis used here may be any cell extract as long as it can translate a translation template to produce a protein encoded by the template. Specifically, a commercially available product may be used, and a known method, specifically, [Madin K et al., “”, Proc Natl Acad Sci USA, Vol. 97, Issue 2, pp. 559- 564, 2000.] (Reference 1) and International Publication No. 00/68412 (Reference 2) can be used for preparation.
As a commercially available cell extract derived from wheat germ for protein synthesis, it is attached to "WEPRO (registered trademark, hereinafter the description of" registered trademark "is omitted) 7240H Expression Kit" (trade name) (manufactured by CellFree Sciences). ("WEPRO7240H" (trade name)) and the like. Further, in the case of the prepared cell extract derived from wheat germ for protein synthesis, the wheat seed germ extract from which the endosperm component and the low molecular weight protein synthesis inhibitor mixed in the preparation step have been substantially removed is preferable. Is. This is because the components and substances involved in the inhibition of protein synthesis in the extract are reduced as compared with the conventional wheat seed germ extract.

Next, in a mixed solution of a transfer solution and a cell extract derived from wheat germ for protein synthesis (hereinafter, may be referred to as "translation reaction solution"), an amino acid as a substrate, an energy source, various ions, a buffer solution, and ATP are added. A solution containing components necessary or suitable for a translation reaction, such as a regeneration system, a nucleolytic enzyme inhibitor, tRNA, a reducing agent, polyethylene glycol, 3', 5'-cAMP, a folate, and an antibacterial agent ("Translation reaction substrate"). The translation reaction can be carried out by adding a solution) and incubating at a temperature suitable for the translation reaction for an appropriate time.

The amino acids used as substrates are usually 20 kinds of natural amino acids constituting proteins, but analogs and isomers thereof can also be used depending on the purpose. Moreover, as an energy source, ATP, GTP and the like can be mentioned. Examples of various ions include acetates such as potassium acetate, magnesium acetate and ammonium acetate, glutamate and the like. As the buffer solution, Hepes-KOH, Tris-acetic acid and the like are used. Examples of the ATP regeneration system include a combination of phosphoenolpyruvate and pyruvate kinase, a combination of creatine phosphate (creatine phosphate) and creatine kinase, and the like. Examples of the nucleolytic enzyme inhibitor include ribonuclease inhibitors and nuclease inhibitors. Among these, as a specific example of the ribonuclease inhibitor, a human placenta-derived RNase inhibitor (manufactured by TOYOBO, etc.) or the like is used. Commercially available tRNA can be used. Examples of the reducing agent include dithiothreitol and the like. Examples of the antibacterial agent include sodium azide, ampicillin and the like. The amount of these additions can be appropriately selected within a range that can be usually used in cell-free protein synthesis.

The mode of addition of the translation reaction substrate solution to the translation reaction solution may be any system known per se that can be applied to the wheat germ cell-free protein synthesis method, and for example, a batch method, amino acids, energy sources, etc. are continuously used. Examples thereof include a continuous cell-free protein synthesis method, a dialysis method, and a multi-layer method, which are supplied to the reaction system. Furthermore, it is prepared by a discontinuous gel filtration method in which template RNA, amino acids, energy sources, etc. are supplied to the synthetic reaction system when necessary and the synthetics and decomposition products are discharged when necessary, and a carrier capable of molecular sieving the synthetic reaction tank. , The above-mentioned synthetic material or the like is developed using the carrier as a mobile phase, a synthetic reaction is carried out during the development, and a method capable of recovering the synthesized protein as a result can be used. Among them, the batch method or the multi-layer method is preferable from the viewpoint of simplifying the structure of the synthetic system, saving space, low cost, and providing a multi-sample simultaneous synthesis system applicable to high-throughput analysis, and a relatively large amount of protein can be obtained. The multi-layer method is particularly preferable in that it can be used.

When the translation reaction is carried out by the batch method, the translation reaction substrate solution may be added to the translation reaction solution and mixed. Alternatively, when the components contained in the translation reaction substrate solution are mixed with the cell extract derived from wheat germ for protein synthesis in advance, the addition of the translation reaction substrate solution can be omitted. The composition of the mixed solution of the translation reaction solution and the translation reaction substrate solution is, for example, HEPES-KOH (pH 7.8) of 10 mM or more and 50 mM or less, potassium acetate of 55 mM or more and 120 mM or less, 1 mM or more and magnesium acetate of 5 mM or less, 0. .1 mM or more and 0.6 mM or less spermidine, L-amino acid (each amino acid concentration is 0.025 mM or more and 1 mM or less), 20 μM or more and 70 μM or less, preferably 30 μM or more and 50 μM or less DTT, 1 mM or more and 1.5 mM or less ATP, GTP of 0.2 mM or more and 0.5 mM or less, creatine phosphate of 10 mM or more and 20 mM or less, ribonuclease inhibitor of 0.5 units / μL or more and 1.0 units / μL or less, protein disulfide isomerase of 0.01 μM or more and 10 μM or less, and 24v / Those containing a cell extract derived from wheat germ of w% or more and 75 v / w% or less are used. When such a translation reaction solution is used, the pre-incubation is about 10 ° C. or higher and 40 ° C. or lower for about 5 minutes or more and 10 minutes or less, and the incubation in this reaction (translation reaction) is also about 10 ° C. or higher and 40 ° C. or lower, preferably about 10 ° C. or higher. At 18 ° C. or higher and 30 ° C. or lower, more preferably 20 ° C. or higher and 26 ° C. or lower, the batch method is usually carried out for about 10 minutes or longer and 7 hours or shorter until the reaction is stopped.

Further, the substrate and the energy source supply solution (supply phase) are continuously or discontinuously supplied to the translation reaction system of the reaction phase by a free drop or a liquid feed pump, and high magnesium ions derived from the transfer solution are supplied. Cell-free protein synthesis method by feed batch method (hereinafter referred to as feed batch method) that prolongs the duration of the synthetic reaction and enhances the efficiency of the synthetic reaction by diluting the by-products generated in the reaction phase and the nucleotides. Can also be adopted.
In the feed batch method, the size and morphology of the reaction vessel are not restricted by the liquid feed from the feed phase, and the mixing rate of both liquids, which is an important rate-determining parameter of the protein synthesis rate, is freely controlled. The synthesis reaction can be controlled, and highly efficient large-scale protein production becomes possible. Furthermore, the efficiency of the synthetic reaction can be further enhanced by using the feed phase to which mRNA is added. The specific rate of addition of the feed solution is such that the same amount of feed solution as the reaction phase at the start of the reaction is continuously or discontinuously added to the reaction phase in about 5 minutes or more and 15 hours or less, preferably about 10 minutes or more and 10 hours or less. The flow velocity within the supply range is good.

When the translation reaction is carried out by the multi-layer method, it can be carried out by using the method described in International Publication No. 02/24939 (Reference 3). Specifically, protein synthesis is performed by layering a translation reaction solution on the translation reaction solution so as not to disturb the interface. More specifically, for example, a cell extract derived from wheat germ for protein synthesis, which has been pre-incubated for an appropriate time as needed, is added to a transcription solution containing mRNA as a translation template and mixed to prepare a reaction phase. The translation reaction solution (supply phase) is layered on the upper layer of this reaction phase so as not to disturb the interface, and the reaction is carried out. The interface between the two phases does not necessarily have to be formed in a horizontal plane by multiple layers, and it is also possible to form a horizontal plane by centrifuging the mixed solution containing both phases. When the diameter of the circular interface between the two phases is 7 mm, the volume ratio between the reaction phase and the supply phase is preferably 1: 4 or more and 1: 8 or less, but 1: 5 is preferable. The larger the interface area consisting of both phases, the higher the substance exchange rate due to diffusion, and the higher the protein synthesis efficiency. Therefore, the capacitance ratio of both phases changes depending on the interface area of both phases. The translation reaction is carried out, for example, under static conditions at about 10 ° C. or higher and 40 ° C. or lower, preferably about 13 ° C. or higher and 30 ° C. or lower, more preferably about 13 ° C. or higher and 26 ° C. or lower, and usually about 10 hours or longer and 20 hours or shorter. be able to.

Further, (1) a step of mixing and treating the supply phase and the reaction phase before and after the protein synthesis rate is substantially reduced, before and after the synthesis reaction is substantially stopped, or in the middle of them, and (2) the start of synthesis using a concentrated membrane or the like. The steps of centrifugally concentrating to about the amount of liquid, (3) the step of superimposing the feed liquid to be the supply phase on the reaction phase and restarting the synthesis, and (4) the above steps (1) to (3) are repeated a plurality of times. A cell-free protein synthesis method (hereinafter, repeated layering method) by a layered repeating method including a repeating step can also be adopted.

<Step 4)>
The recombinant protein obtained in the above step 3) may be used as it is, but it can be purified and used. That is, the production method of the present embodiment is referred to as a step of purifying the obtained recombinant protein after step 3) in addition to the steps 1) to 3) (hereinafter, "step 4)". In some cases) may be further included.

Examples of the method for purifying the recombinant protein include salting out, ion exchange chromatography, adsorption chromatography, affinity chromatography, gel filtration chromatography and the like. When the recombinant protein is expressed as a fusion protein with His tag, GST, etc., it can be isolated and purified by a purification method utilizing the properties of these fusion proteins and tags. Specifically, for example, when it is expressed as a fusion protein of the recombinant protein and His tag, it can be purified using a support or a carrier to which nickel (Ni) is bound. For example, when expressed as a fusion protein of the above recombinant protein and GST, it can be purified using a support or carrier to which glutathione is bound. As the support and the carrier, for example, those made of materials such as resin, glass, and magnetic material and having shapes such as columns, beads, and chips can be used.
In addition, the production method of the present embodiment further includes a step of removing the tag using an enzyme or the like after the step 4) when the recombinant protein is a fusion protein with various tags. Can be done.

The manufacturing method of the present embodiment can be performed using an apparatus that automates each step from the above steps 1) to the above steps 3) (if necessary, the above steps 4)). Here, "automation" means that the experimenter does not directly operate the reaction system (reaction vessel) during a series of steps. Therefore, when the experimenter manually operates the predetermined operation buttons, switches, etc. provided in the automatic synthesizer used when executing each process, the requirement of "automatic" in the present specification is impaired. is not it.
As a commercially available automatic synthesizer, for example, "Protemist (registered trademark) DT II" (trade name) (manufactured by Cellfree Science Co., Ltd.) can be used.

Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1]
(Manufacture of rc-bFGF using wheat germ cell-free protein synthesis system)
The rc-bFGF is manufactured using a fully automatic protein synthesizer / purifier (“Protemist® DT II” (trademark)) using the cell-free protein synthesis kit “WEPRO7240H Expression Kit” (trade name) manufactured by CellFree Sciences. Name), manufactured by CellFree Sciences).

Specifically, first, a cDNA encoding canine bFGF (SEQ ID NO: 2) was inserted into the multicloning site of the pEU-E01-MCS vector to obtain a vector [pEU-E01-His-TEV-DogbFGF]. The vector [pEU-E01-His-TEV-DogbFGF] was transformed into Escherichia coli and cultured. The plasmid DNA was extracted and purified from Escherichia coli using the plasmid Midi Kit (manufactured by QIAGEN), and then the concentration and purity of the purified plasmid DNA were confirmed so that the plasmid DNA concentration was 1.0 μg / μL. An appropriate amount of TE buffer was added to obtain a plasmid DNA solution. It was confirmed by sequence analysis that the DNA of canine bFGF was inserted into the multi-cloning site of the vector.

To the transcription reaction solution (5 × Transscription Buffer LM), then 25 μL of the template DNA solution (1.0 μg / μL) was added and gently pipetting was performed. Then, the reaction was carried out at 37 ° C. for 6 hours. After the transcription reaction, 1 μL of the transcript was electroinduced on an agarose gel, and mRNA synthesis was confirmed. Furthermore, RT-PCR was performed on the obtained mRNA to obtain cDNA of canine bFGF. When this cDNA was subjected to agarose gel electrophoresis together with the cDNA of dog bFGF derived from Escherichia coli as a control, a band was observed at the same position.

Then, a translation reaction was carried out using the obtained mRNA. For translation, a wheat germ extract (“WEPRO® 7240H” (trade name), manufactured by Cellfree Science) optimized for the synthesis and purification of a histidine (His) -tagged protein was used. .. After lowering the temperature of the transcription reaction solution containing mRNA to room temperature and gently pipetting and suspending it, add 250 μL of the suspended transcription reaction solution to 250 μL of wheat germ extract and 1 μL of creatine kinase, and quietly so as not to foam. (This mixed solution may be referred to as "translation reaction solution" below). Then, the entire amount of the translation reaction solution (501 μL) is carefully poured into the bottom of the translation reaction substrate solution (“SUB-AMIX® SGC” (trade name)) (5.5 mL) to form a layer, and the layer reaction is performed. Was done. The translation reaction was carried out by keeping the temperature at 15 ° C. for 20 hours.

After the translation reaction, the reaction solution was gently pipetted to purify the synthetic protein solution. For purification, a His-tagged recombinant canine bFGF (rc-bFGF) is adsorbed using a nickel resin, and the resin is washed with a washing buffer (20 mM Na-Phosphate (pH 7.5), 300 mM NaCl, 20 mM Imidazole). Then, the target protein was eluted with half the usual amount of elution buffer (20 mM Na-Phosphate (pH 7.5), 300 mM NaCl, 500 mM Imidazole). At this time, by reducing the amount of the elution buffer used to half the normal amount, the concentration operation after elution becomes unnecessary, and as a result, the loss of rc-bFGF due to the concentration operation is eliminated, and the rc-bFGF concentration is as efficient as 1 mg / mL. Realized production.

Western blotting analysis was performed on the obtained purified protein together with the canine bFGF protein derived from Escherichia coli as a control, and a band was observed at the same position. Therefore, it was confirmed that rc-bFGF was obtained.

In addition, the obtained rc-bFGF was added to HEK cells, and the expression of pErk was confirmed by Western blotting analysis (see FIG. 1). As a result, the expression of pErk was confirmed.

Further, the obtained rc-bFGF was added to canine fibroblasts and cultured (see FIGS. 2 and 3). As a result, an increase in the number of cells was confirmed in the rc-bFGF-added group as compared with the rc-bFGF-free group.

From the above, it was confirmed that the obtained protein is recombinant canine bFGF (rc-bFGF) and has a function as canine bFGF.

According to the recombinant protein of the present embodiment and the method for producing the same, it is possible to provide the recombinant protein having a function as a canine basic fibroblast growth factor and the method for producing the same. The obtained recombinant protein can be used for regenerative medicine such as cytokine therapy.

Claims (3)

A recombinant protein consisting of any of the following amino acid sequences (a) to (c), having a proliferative ability for canine fibroblasts, and being a compound obtained by a wheat germ cell-free protein synthesis system.
(A) Amino acid sequence shown in SEQ ID NO: 1;
(B) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1;
(C) An amino acid sequence having 98% or more identity with the amino acid sequence shown in SEQ ID NO: 1. A method for producing a recombinant protein consisting of any of the following amino acid sequences (a) to (c) and having a proliferative ability for canine fibroblasts.
A production method for synthesizing the recombinant protein using a wheat germ cell-free protein synthesis system.
(A) Amino acid sequence shown in SEQ ID NO: 1;
(B) An amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1;
(C) An amino acid sequence having 98% or more identity with the amino acid sequence shown in SEQ ID NO: 1. The recombinant protein after synthesis has a histidine tag attached to its N-terminal or C-terminal.
The production method according to claim 2, wherein the recombinant protein after synthesis is purified using the histidine tag.

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