JP5308630B2 - Polypeptide containing extracellular region of Fc receptor and method for producing the same - Google Patents

Description

  The present invention relates to a polypeptide containing an Fc receptor extracellular region, a method for producing the same, and a method for using the polypeptide.

Fc receptors are a group of molecules that bind to the Fc region of immunoglobulin molecules. Individual molecules recognize a single or the same group of immunoglobulin isotypes by a recognition domain on the Fc receptor by a recognition domain belonging to the immunoglobulin superfamily. This determines which accessory cells are driven in a certain immune response (Non-Patent Document 1). Fc receptors can be further classified into several subtypes, and the presence of FcγRI, FcγRIIa, FcγRIIb and FcγR3 has been reported as a receptor for IgG (immunoglobulin G) (Non-patent Document 1). Among the above receptors, it has been reported that FcγRI and IgG have high affinity and the equilibrium dissociation constant (Kd) is 10 ⁻⁸ M or less (Non-patent Document 2).

  FcγRI is a receptor for IgG (immunoglobulin G) and is constitutively expressed on monocytes and macrophages and inducibly expressed on neutrophils and eosinophils. FcγRI is divided into an extracellular region, a transmembrane region, and an intracytoplasmic region, and binding to IgG occurs in the Fc region of IgG and the extracellular region of FcγRI, and then a signal is transmitted to the cytoplasm. FcγRI is composed of two subunits, an α chain with a molecular weight of approximately 42,000 that is directly involved in binding to IgG and a γ chain, and the γ chain is shared via cysteine at the boundary between the cell membrane and the extracellular region. A homodimer is formed by binding (Non-patent Document 1).

In recent years, the unexpected immunosuppressive biological properties of Fc receptors have attracted attention as pharmaceuticals, particularly in the areas of autoimmune disease or autoimmune syndrome, transplant rejection and malignant lymphoproliferation. Further, the antibody adsorption ability, which is a function of FcγR1, can also be used as a capture function protein for various antibody purification chromatography gels.
The amino acid sequence and gene sequence of the FcγRIα chain were clarified by Janet et al. (Non-patent Document 2), and then by using E. coli (Non-patent Document, Patent Document 1) or animal cells by gene recombination technology, FcγRI expression was reported. However, in the expression system using Escherichia coli, the expression amount of the extracellular region protein of FcγRI is extremely low, and it is difficult to produce it on an industrial production scale. In addition, it has been reported that a system using animal cells can achieve a higher expression level than a system using Escherichia coli (Non-patent Document 3). However, since the culture takes time, productivity is not necessarily high.

JP-T-2004-530419 J. et al. V. Ravetch et al., Annu. Rev. Immunol. 9, 457, 1991 J. et al. M.M. Allen et al., Science, 243, 378, 1989 A. Paetz et al., Biochem. Biophys. Res. Commun. , 338, 1811, 2005

  An object of the present invention is to produce a microorganism that produces a polypeptide containing an Fc receptor extracellular region, particularly a polypeptide containing an FcγRI extracellular region, on an industrial production scale. Furthermore, another object of the present invention is to produce a polypeptide containing an FcγRI extracellular region using a production system for a polypeptide containing a novel FcγRI extracellular region to provide a polypeptide containing the FcγRI extracellular region.

As a result of intensive studies to solve the above problems, the inventors of the present application converted the sequence of the gene so that the codon of mRNA transcribed from the human FcγRI gene becomes a codon that is frequently used in the E. coli host. It has been found that the expression level of FcγRI in Escherichia coli is improved by introduction into a recombinant vector into which a receptor gene has been inserted.
That is, the present inventors have reviewed the gene sequence of FcγRI, which is a type of human Fc receptor, and converted the DNA sequence so that it corresponds to a codon that E. coli can easily translate to express the receptor in E. coli. Then, after synthesizing an oligonucleotide consisting of the corresponding conversion codon, a full-length FcγRI gene was prepared by PCR, and the expression plasmid vector into which the gene was inserted was introduced into Escherichia coli. As a result, the expression level of FcγRI extracellular region protein in the E. coli host Has been found to be dramatically improved, and the present invention has been completed.

That is, the present invention relates to a polynucleotide encoding a polypeptide containing the FcγRI extracellular region, the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2, or the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 2. A polynucleotide substantially homologous to a nucleotide is provided.
The present invention also relates to a polynucleotide encoding a region obtained by removing a signal sequence from a polypeptide containing an FcγRI extracellular region, the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3, or SEQ ID NO: 3 Provided is a polynucleotide that is substantially homologous to a polynucleotide comprising the described base sequence.
The present invention also relates to a polynucleotide encoding a polypeptide having a methionine added to the N-terminal side of a polypeptide comprising an FcγRI extracellular region, from which the signal sequence has been removed, the base described in SEQ ID NO: 4 A polynucleotide comprising a sequence or a polynucleotide substantially homologous to a polynucleotide comprising the base sequence set forth in SEQ ID NO: 4 is provided.

The present invention also provides a polypeptide encoded by a polynucleotide comprising the base sequence set forth in any one of SEQ ID NOs: 2 to 4.
The present invention also provides a polynucleotide in which an expression control sequence is functionally added to the polynucleotide comprising the base sequence described in any one of SEQ ID NOs: 2 to 4.
The present invention also provides a recombinant vector into which any of the polynucleotides described above is inserted.
The present invention also provides a transformant transformed with any of the above polynucleotides or the above recombinant vector.
The present invention also provides a method for producing a polypeptide containing the FcγRI extracellular region, comprising the step of culturing the transformant.
The present invention also provides an antibody purification adsorbent comprising the polypeptide obtained by the above production method immobilized on a carrier.
The present invention also provides a method of adsorbing an antibody to the antibody purification adsorbent.

  By making the gene codon encoding a polypeptide containing a human FcγRI extracellular region into an E. coli type, a large amount of the polypeptide containing the FcγRI extracellular region can be expressed in E. coli. This expression system is useful for the production of polypeptides comprising the FcγRI extracellular region on an industrial scale. Further, the polypeptide containing the FcγRI extracellular region thus obtained can be used as a capture group for an antibody affinity chromatography gel, that is, can be used as an adsorbent for antibody purification.

Hereinafter, the present invention will be described in detail.
First, the gene encoding the FcγRI protein will be described. The FcγRI gene is a DNA containing the base sequence of about 2.2K described in SEQ ID NO: 1. The functional structure of the protein encoded by this DNA is publicly known in the public database (European Bioinformatics Institute, URL: http://www.ebi.ac.uk/, access date: March 2, 2006). Yes, it is easy to determine a structural domain, or an amino acid sequence such as a signal sequence or a transmembrane region.

Next, the polynucleotide of the present invention will be described.
The polynucleotide of the present invention is a polynucleotide that encodes a polypeptide containing the human FcγRI extracellular region, and is a polynucleotide in which the codon of the genetic information is optimized for the codon usage of an appropriate host cell. That is, when a transformant obtained by transforming a host cell with a recombinant vector into which a polynucleotide encoding a polypeptide containing a human FcγRI extracellular region has been cultured to produce a polypeptide, The target polypeptide can be efficiently produced by changing the codon of the polynucleotide so that the host cell can correspond to the codon that can be easily translated. . In the present specification, when describing the present invention, the case where Escherichia coli is used as a host cell will be described. However, the present invention is not limited to the case where Escherichia coli is used as a host cell, but can be applied to the case of other host cells. Is done. That is, when another host cell is used, it can be determined by genome analysis of the optimal codon of the host cell and can be applied to other host cells.

The polynucleotide of the present invention is a polynucleotide represented by the following (a), (b) or (c).
(A) a polynucleotide encoding a polypeptide comprising the FcγRI extracellular region, which is substantially the same as a polynucleotide comprising the base sequence set forth in SEQ ID NO: 2 or a polynucleotide comprising the base sequence set forth in SEQ ID NO: 2 Homologous polynucleotide.
(B) a polynucleotide encoding a region obtained by removing a signal sequence from a polypeptide containing an FcγRI extracellular region, the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3, or the base set forth in SEQ ID NO: 3 A polynucleotide substantially homologous to a polynucleotide comprising the sequence.
(C) a polynucleotide encoding a polypeptide having a methionine added to the N-terminal side of a polypeptide including the extracellular region of FcγRI from which the signal sequence has been removed, comprising the base sequence set forth in SEQ ID NO: 4 A polynucleotide that is substantially homologous to a polynucleotide or a polynucleotide comprising the base sequence set forth in SEQ ID NO: 4.

The polynucleotide (a) will be described. The polynucleotide (a) is a gene that encodes a polypeptide containing the human FcγRI cell, and the codon of the genetic information is changed from a human type to an E. coli type codon, ie, E. coli codon usage. Polynucleotides optimized for frequency.
In other words, the polynucleotide (a) uses the E. coli rare codon present in the mRNA transcribed from the gene DNA encoding the human FcγRI extracellular region, while the encoded amino acid remains the same and is used as the E. coli codon. It is a polynucleotide sequence converted into a codon having a high frequency. Rare codons can be defined as those in which the frequency of codon usage in the host is low. The frequency of use in the host can be estimated by referring to the analysis result of the genomic gene. In E. coli, the amino acid Arg codons AGA, AGG, CGG, CGA, Ile codon AUA, Leu codon CUA, Gly codon GGA, and Pro codon CCC. Codon usage frequency can also be analyzed by using a public database (URL: http://www.kazusa.or.jp/codon/, access date: March 2, 2006).

Codon conversion is possible by changing the base sequence of the corresponding DNA sequence. In order to perform base substitution of a DNA sequence, for example, a known mutagenesis method such as Site-directed mutagenesis method can be used. Preferably, the DNAWorks method combining synthetic oligonucleotide and PCR can be exemplified (Nucleic Acid Res., 30, e43, 2002). According to this method, a full-length gene can be synthesized by synthesizing an oligonucleotide group consisting of several tens of bases from an amino acid sequence and assembling the synthetic oligonucleotide by PCR.
It is possible to convert rare codons of all genes, or partial conversion. The conversion ratio can be appropriately changed by quantifying the expression level of mRNA.

The polynucleotide (b) is a polynucleotide obtained by removing a portion encoding a signal sequence from the polynucleotide (a), that is, a polynucleotide encoding a region obtained by removing the signal sequence from a polypeptide containing the FcγRI extracellular region. It is a nucleotide.
The signal sequence is a region where a protein expressed in the cell passes through the cell membrane and is secreted outside the cell, and is usually present on the N-terminal side of the protein. Cleaved by the protease enzyme. Examples of the signal sequence include peptide sequences of amino acid numbers 1 to 15 or 1 to 20 of SEQ ID NO: 1. In addition, the FcγRI extracellular region is functionally sufficient even if it removes the signal sequence and only interacts with IgG.

  The polypeptide (c) is a polynucleotide that encodes a polypeptide in which methionine is added to the N-terminal side of the polypeptide including the FcγRI extracellular region from which the signal sequence has been removed.

  In the present invention, the polypeptide may be a naturally occurring polynucleotide or an artificially synthesized nucleotide.

The polynucleotides of (a), (b) and (c) are in addition to the polynucleotide comprising the base sequence described in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, for example, SEQ ID NO: 2 In the polypeptide encoded by the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 3 and SEQ ID NO: 4, within the amino acid sequence for the polypeptide chain within the range in which the identity of biological activity is not impaired A polypeptide that encodes a polypeptide comprising an amino acid sequence in which a part (preferably 1 to 20, more preferably about 1 to 10, most preferably 1 to 3) of amino acids is deleted, substituted or added. Also includes nucleotides. Again, the identity of biological activity means the binding of FcγRI to immunoglobulin. Here, the immunoglobulin is preferably IgG, more preferably IgG1.
A person skilled in the art can introduce substitution, deletion, and / or addition mutation into the polypeptides (a), (b), and (c) as appropriate using site-directed mutagenesis. it can.

  The polynucleotides (a), (b), and (c) also include polynucleotide sequences that are substantially homologous to the sequences (a), (b), and (c). Substantially homologous refers to 75% when two nucleic acids are aligned using one of the following sequence comparison algorithms or by manual alignment and visual test and aligned with maximum correspondence. Means a sequence or subsequence having a DNA identity greater than, preferably greater than 85%, more preferably greater than 95%. It goes without saying that this definition also means a sequence whose complement is hybridized to the test sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input to a computer, partial coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The parameters may use default values or specify alternative parameters. The sequence comparison algorithm then calculates the ratio of sequence identity of the test sequence to the reference sequence, based on the program parameters. Sequence alignment methods for comparison are known in the art. Optimal sequences for comparison are, for example, local homology algorithms (Smith et al., Adv. Appl. Math., 2, 482, 1981), homology alignment algorithms (Needlman et al., J. Mol. Biol. 48, 443 (1970), similarity search (Person et al., Proc. Natl. Acd. Sci. USA, 85, 2444, 1988).

  An example of a useful algorithm is the Pileup method, where multiple sequences can be created from related sequences using continuous, pairwise alignments to show relationships and percent sequence identity. Another example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm (Altschul et al., J. Mol. Iol., 215, 403, 1990).

  The polynucleotide of the present invention includes a DNA sequence that hybridizes with the polynucleotides (a), (b), and (c) above under stringent conditions. By hybridizing under stringent conditions is meant a condition in which a probe hybridizes to its target sequence in a mixture of nucleic acids but not to other nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences specifically hybridize at higher temperatures. In general, highly stringent conditions are selected to be about 5-10 ° C. lower than the thermal melting temperature of the specific sequence at a defined ionic strength and pH. Low stringency conditions are generally selected to be about 15-30 ° C. below the melting temperature. The melting temperature is the temperature at which 50% of the probe complementary to the target nucleic acid at a predetermined ionic strength, pH, and nucleic acid sequence occupies an equilibrium state.

  Specifically, the polynucleotide capable of hybridizing under stringent conditions means any at least 20, preferably at least 30, for example, 40, 60 or 100 consecutive sequences in SEQ ID NO: 1. The probe is used as a probe, for example, using ECL direct nucleic acid labeling and detection system (Amersham Pharmacia Biotech), conditions described in the manual (e.g., wash: 42 ° C., primary wash buffer containing 0.5 × SSC) Refers to a polynucleotide that hybridizes in More specific “stringent conditions” are, for example, usually 42 ° C., 2 × SSC, 0.1% SDS conditions, preferably 50 ° C., 2 × SSC, 0.1% SDS conditions, The conditions are preferably 65 ° C., 0.1 × SSC and 0.1% SDS, but are not particularly limited to these conditions. Factors affecting the stringency of hybridization may include multiple factors including temperature, salt concentration, probe concentration, probe length, and reaction time, and those skilled in the art can appropriately select these factors. Optimal stringency can be achieved. For details on the hybridization procedure, see `` Molecular Cloning, A Laboratory Manual 2nd ed. '' (Cold Spring Harbor Press (1989), especially Sections 9.47-9.58), `` Current Protocols in Molecular Biology '' (John Wiley & Sons (1987 -1997), especially Sections 6.3-6.4), "DNA Cloning 1: Core Techniques, A Practical Approach 2nd ed." (Oxford University (1995), especially Section 2.10).

  The present invention includes the polypeptides encoded by the polynucleotides (a), (b) and (c) described above. Examples of such a polypeptide include, for example, a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, and a part of the amino acid sequence (preferably 1 to 20, more preferably about 1 to 10, most preferably Preferred is a polypeptide having an amino acid sequence in which preferably 1 to 3 amino acids are deleted, substituted or added.

  The present invention provides a recombinant vector into which the polynucleotide of the present invention is inserted. By inserting the polynucleotide into a known expression vector, a vector (recombinant vector) for expressing a polypeptide containing the FcγRI extracellular region is provided. In addition, by culturing a transformant transformed with this recombinant vector, a polypeptide containing the FcγRI extracellular region can be obtained from the transformant. That is, the present invention includes culturing a transformant transformed with a recombinant vector into which the polynucleotide of the present invention is inserted, and recovering a polypeptide containing an FcγRI extracellular region from the culture. A method for producing a polypeptide comprising an FcγRI extracellular region is provided.

  The polynucleotide of the present invention may be used as it is and transformed into an appropriate cell, but is usually inserted into an appropriate known expression vector. Insertion into the expression vector may be performed by genetic engineering at an appropriate position of the expression vector. By appropriate position is meant a position that does not destroy the replication function of the expression vector, the desired antibiotic marker, or the region related to transmissibility. The polynucleotide of the present invention is preferably one in which an expression control sequence is functionally added. An expression control sequence is functionally added, for example, when a promoter, an enhancer, an operator, an inducer, or the like is added, and by adding such a sequence, the polypeptide is transformed into the host cell. Expression is possible.

  In the present invention, the host cell for transforming the recombinant vector is preferably a microorganism, and more preferably a prokaryotic cell. Among prokaryotic cells, Escherichia coli is preferable, but in the present invention, host cells are not limited, and all host cells used in genetic engineering are used. Examples of usable microorganisms include the following microorganisms.

E. coli (genus Escherichia)
Host vector systems such as Bacillus genus Pseudomonas genus Serratia genus Brevibacterium genus Corynebacterium genus Streptococcus genus Lactobacillus genus have been developed Bacterial Rhodococcus spp.Streptomyces sp. Yeastia spp Sporium (Cephalosporium) genus Toriko Luma (Trichoderma) mold that has been developed host vector system such as the genus

  Hereinafter, the case where E. coli is used as a host will be described. When inserting a polynucleotide into an expression vector, it is preferable to add a DNA sequence having a promoter function to the polynucleotide. A promoter means a DNA sequence capable of controlling the expression of a protein coding region or functional RNA. In the case of E. coli, examples include lac promoter, trc promoter, T7 promoter and the like. Among them, the T7 promoter having a strong transcription activity is preferable.

  As a plasmid vector (expression vector), any plasmid vector (expression vector) can be used without particular limitation as long as it is stably present in the cell to be transformed and has the property of replicating. Examples of such plasmid vectors include pUC, pBR, pET, and broad host range (Broad-Host-Range) plasmid vectors used for transformation of E. coli, and plasmid vectors that can replicate in target cells. Of the shuttle vector. For example, what is described in the book (Barbara E. Funnell, PLASMID BIOLOGY, pp. 545-566, ASM press) can be mentioned. Furthermore, a plasmid possessed by a cell to be transformed may be used. A vector as described later can also be used.

When using E. coli as the host cell, for example, the K12 JM109 strain is preferably used. More preferably, a BL21 (DE3) strain and the like that can use the T7 promoter sequence can be exemplified.
Moreover, as a host cell of the present invention, an E. coli mutant strain derived by mutation treatment of the above-mentioned E. coli or the like can be used. Mutation treatment may be performed using a mutating agent well known to those skilled in the art, such as nitrosoguanidine, ethyl methanesulfonate, ultraviolet light, and radiation.

  Procedures or methods for introducing and expressing foreign genes into microorganisms such as E. coli include those commonly used in the field of genetic engineering, other than those described below in the present invention. For example, it may be carried out according to “Plasmid transformation of Escherichia coli and other bacteria”, Method in Enzymology, 204, pages 63 to 113, 1991, Academic Press). Specific examples include a heat shock method and an electroporation method.

  The method for producing a polypeptide containing the FcγRI extracellular region of the present invention includes a step of culturing a transformant transformed with a recombinant vector into which the polypeptide of the present invention has been inserted. Specifically, the method includes culturing the transformant and recovering a polypeptide containing the FcγRI extracellular region from the culture.

  The transformant (E. coli) used to produce the FcγRI of the present invention is grown in a medium that is well known in the art and suitable for culturing selected host cells. An example of a suitable medium is LB (Luria broth) medium supplemented with necessary nutrients. In a preferred embodiment, in order to selectively allow the transformant to grow on the recombinant vector, it is preferable that the medium contains a selection agent based on the composition of the recombinant vector. For example, kanamycin is added to the medium for the growth of cells that express the kanamycin resistance gene. In addition to carbon, nitrogen and inorganic salt sources, a suitable nutrient source may be added to the medium. If desired, one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, and dithiothreitol may be included. The culture temperature in the growth of E. coli is 20 ° C. to 40 ° C., more preferably 25 ° C. to 35 ° C., and more preferably about 30 ° C. The pH of the medium is about 6.8 to 7.4 in E. coli culture, and more preferably 7.0.

  When an inducible promoter is used in the recombinant vector of the present invention, it is induced under conditions that allow good protein expression. An example of the inducer is IPTG. The turbidity (absorbance at 660 nm) of the culture solution is measured, and an appropriate amount of IPTG is added during the growth period of about 0.5 to 1.0, followed by culturing. Thus, protein expression can be induced. The addition concentration of IPTG is 0.1 to 1.0 mM, preferably about 0.5 mM. Various conditions for IPTG induction are well known in the art.

  In order to recover the polypeptide containing the FcγRI extracellular region of the present invention from the culture solution, an extraction method may be appropriately selected depending on the form of expression. When expressed in the culture supernatant, the cells are separated by centrifugation, and the polypeptide containing the FcγRI extracellular region may be extracted from the resulting culture supernatant. On the other hand, when a polypeptide containing an FcγRI extracellular region is expressed in the cell, the cells are collected by centrifugation, and the cells are disrupted by adding an enzyme treatment agent, a surfactant, etc., and FcγRI cells Polypeptides containing the outer region can be extracted. In order to separate and purify the polypeptide containing the FcγRI extracellular region from the extracted protein, methods known in the art can be used. For example, liquid chromatography can be used. Examples of liquid chromatography include ion exchange chromatography, hydrophobic interaction chromatography, gel filtration, affinity chromatography, and the like. By performing a purification operation by combining these chromatograms, a highly pure polypeptide containing the extracellular region of FcγRI can be prepared.

Next, the adsorbent for antibody purification of the present invention will be described.
The adsorbent for antibody purification of the present invention is obtained by immobilizing a polypeptide obtained by the above-described method for producing a polypeptide on a carrier.
Any carrier can be used without particular limitation as long as the polypeptide can be chemically or physically immobilized, and a chromatography carrier can be used. Examples of such a carrier include polysaccharides such as cellulose and agarose, glass, ceramics, and plastic materials such as polypropylene, vinyl chloride, and polystyrene. Such a carrier can be a commercially available one. For example, His / Bind Resin gel in the His / Bind purification kit (registered trademark) manufactured by Novagen, trade name “HiTrap NHS-Activated HP” (Amersham) Biosciences), trade name “Tosyl group activated Dynabeads M-280” (manufactured by Dynal) and the like can be used.

  It should be noted that when the polypeptide is immobilized on a carrier, it is preferable to use a polypeptide in which an appropriate spacer that can be used for competition with the carrier is inserted at the end of the peptide. As the spacer, for example, an amino acid, a peptide, a thread having a main chain of about 2 to 8 carbon atoms, preferably having a hydrophilic functional group such as a hydroxyl group (for example, polyvinyl alcohol having an active group at both ends) ), Sugar chains and the like. Examples of the amino acid include histidine, arginine, lysine, asparagine, glutamine, cysteine, aspartic acid, glutamic acid and the like, and polyhistidine in which a plurality of histidines (usually 2 to 10) are linked in succession is preferable. Used. When such a polyhistidine is used as a spacer, the carrier and the polypeptide are bound by affinity with the Ni chelate. When the amino acid is a spacer, the peptide into which the spacer is inserted can be synthesized as a single continuous polypeptide. When a polypeptide is produced by a genetic recombination method, a polynucleotide is inserted into a vector that adds a histidine tag to the end of the recombinant protein, and transformed into an appropriate host to produce the polypeptide. Can be made. As such a vector, a commercially available one can be used, and examples thereof include pET28b + manufactured by Novagen.

The present invention also provides a method of adsorbing an antibody to the antibody purification adsorbent. The above-mentioned adsorbent for antibody purification can be used for purification and separation of antibodies, for example. That is, the present invention provides an antibody purification method using the antibody purification adsorbent.
In order to adsorb the antibody to the adsorbent for antibody purification, for example, the adsorbent for antibody purification is packed in, for example, a column or the like, and a sample containing the antibody is introduced here under neutral conditions. As a result, the antibody in the sample reacts with and is adsorbed to the polypeptide immobilized on the antibody purification adsorbent.
In addition, when the antibody purification adsorbent is used for purification and separation of antibodies, it is carried out as follows. In order to purify and separate the antibody from the adsorbent for antibody purification on which the antibody has been adsorbed, a buffer solution containing a high salt concentration, for example, NaCl, KCl, Na ₂ SO ₄ , or the pH in the column is passed through the column. It is implemented by changing

Hereinafter, embodiments of the present invention will be described in detail. Needless to say, the present invention is not limited to these examples, and can be arbitrarily changed without departing from the scope of the invention.
(Example 1) Design of DNA sequence encoding Fc receptor Using DNAworks method (Nucleic Acid Res., 30, e43, 2002) based on the amino acid sequence of the extracellular region of FcγRI of SEQ ID NO: 1, The codon was converted to E. coli type. FIG. 1 shows the result of alignment of a DNA sequence in which codons have been converted by this method and a human codon. As shown in FIG. 1, the amino acid sequence was not changed, and DNA sequence conversion was performed, and the similarity between the DNA sequences was 75%.

(Example 2) Preparation of DNA sequence encoding Fc receptor 52 oligonucleotides were synthesized in order to prepare a DNA sequence converted from a human codon to an E. coli codon. The synthesized oligonucleotides are shown in SEQ ID NOs: 5 to 56. Subsequently, two-stage PCR was performed to prepare DNA encoding full-length FcγRI from the oligonucleotide. The reaction solution of the first stage PCR is as shown in Table 1, and the reaction conditions are 94 ° C., 5 minutes heat treatment, 94 ° C., 30 second first step, 62 ° C., 30 second second step, 72 A third step of 1 minute at 25 ° C. is performed 25 cycles, followed by a fourth step at 72 ° C. for 7 minutes. The DNA mix in Table 1 means a solution obtained by sampling and mixing 52 kinds of synthesized 50 pmol / μl oligonucleotides.

  The second stage PCR was carried out using the reaction liquid composition shown in Table 2 using the reaction liquid of the first stage PCR. As the sequence of the PCR primer, the oligonucleotide of SEQ ID NO: 5 (5′-ATGTGGGTTTCTGACCACGCTGTGTCTGTGGGGTGCCGGT-3 ′) and SEQ ID NO: 56 (5′-GGTCCGCCCCTGCGTCTCCTTACGATGCAC-3 ′) was used. The reaction conditions were 94 ° C., 5 minutes heat treatment, 25 cycles of 94 ° C., first step for 30 seconds, 65 ° C., second step for 30 seconds, 72 ° C., third step for 1 minute. The fourth step at 7 ° C. for 7 minutes. When the reaction was confirmed by 0.9% agarose electrophoresis, a DNA band of the designed size could be confirmed. Next, after extracting the target band (QIAquick Gel extraction kit: Qiagen), the 5 ′ end of the extracted DNA was phosphorylated (TaKaRa BKL Kit: Takara Bio Inc.) and inserted into the pUC19 plasmid vector digested with the restriction enzyme SmaI. Escherichia coli JM109 strain (Takara Bio Inc.) was transformed and prepared. This was designated as pECFcR. FIG. 2 shows the structure.

Example 3 Preparation of Fc Receptor Expression Plasmid Vector In order to express the extracellular region of FcγRI, SEQ ID NO: 57 (5′-C CCATGG CGTTGATTACGCTGCCAACACCCG-3 ′: underline part ( CCATGG ) is a restriction enzyme using pECFcR as a template. NcoI site) and SEQ ID NO: 58 (5′-CC AACCTT GACCGGGGTCGGCAGTTTGAAGACC-3 ′: underlined portion ( AACCTT ) is a restriction enzyme HindIII site) oligonucleotide is used as a primer to amplify DNA encoding the extracellular region did. The reaction conditions were 94 ° C., 5 minutes heat treatment, 25 cycles of 94 ° C., first step for 30 seconds, 65 ° C., second step for 30 seconds, 72 ° C., third step for 1 minute. The fourth step at 7 ° C. for 7 minutes. Table 3 shows the reaction solution composition.

When the reaction was confirmed by 0.9% agarose electrophoresis, a DNA band of the designed size could be confirmed. Subsequently, after extracting the target band using QIAquick Gel extraction kit (Qiagen), the amplified DNA was digested with restriction enzymes NcoI and XhoI, and digested in advance with these restriction enzymes pET28b + (Novagen) and Ligation Kit Ver2 (Takara Bio Inc.) was used for ligation. Next, E. coli JM109 was transformed and prepared using an LB agar medium supplemented with 50 μg / ml antibiotic kanamycin. This was designated as pETECFcR. FIG. 3 shows the structure. The obtained pETECFcR uses an E. coli type codon.

In parallel with the above, an expression vector using DNA encoding the extracellular region of FcγRI was also prepared. The insert was prepared by PCR, using Human cDNAclone TC119841 plasmid vector (Origene) as a template, SEQ ID NO: 59 (5′-C CCATGG CAGTGATCACTTTGCAGCCCTCC-3 ′: underline part ( CCATGG ) is the restriction enzyme NcoI site) and SEQ ID NO: 60 (5′-G CTCGAG GACAGGAGTTGGTAACTGGGAGGCC-3 ′: underlined portion ( CTCGAG ) is a restriction enzyme XhoI site) was used as a PCR primer. PCR was conducted at 94 ° C. for 5 minutes, followed by 25 cycles of 94 ° C., 30 second first step, 65 ° C., 30 second second step, 72 ° C., 1 minute third step, and finally 72 ° C. The fourth step of 7 minutes. Table 4 shows the reaction solution composition.

  When the reaction was confirmed by 0.9% agarose electrophoresis, a DNA band of the designed size could be confirmed. Subsequently, after extracting a target band using QIAquick Gel extraction kit (Qiagen), the amplified DNA was digested with restriction enzymes NcoI and XhoI, and pET28a + (Novagen) previously digested with these restriction enzymes and Ligation Kit Ver2 (Takara Bio Inc.) was used for ligation. Next, E. coli JM109 was transformed and prepared using an LB agar medium supplemented with 50 μg / ml antibiotic kanamycin. This was designated as pETHUFcR. FIG. 4 shows the structure.

(Example 4) Confirmation of Sequence The Big Dye Terminator Cycle Sequencing FS read Reaction kit (registered trademark) (PE Applied Biosystems) based on the chain terminator method was used for the DNA sequences inserted into the pETECFcR and pETHUFcR plasmid vectors prepared in Example 3. ) Was used for a cycle sequencing reaction and analyzed with a fully automatic DNA sequencer ABI Prism 3700 DNA analyzer (registered trademark) (PE Applied Biosystems). The oligonucleotide shown in SEQ ID NO: 61 (5′-TAATACGACTCACTATAGGGG-3 ′) was used as a sequencing primer. As a result of the analysis, it was confirmed that the base sequences of the DNA inserted into pETECFcR and pETHUFcR were as designed. The amino acid sequence of the FcγR1 protein encoded by the DNA inserted into pETECFcR is shown in SEQ ID NO: 62, and the amino acid sequence encoded by the DNA inserted into pETHUFcR is shown in SEQ ID NO: 63.

(Example 5) Expression induction of Fc receptor The pETECFcR plasmid vector was transformed into Escherichia coli BL21 (DE3) (Takara Bio Inc.) using an LB agar medium supplemented with 50 μg / ml antibiotic kanamycin. The resulting transformant was named BL21 (DE3) / pETECFcR. The same procedure was performed for the pETHUFcR plasmid vector to obtain a transformant, which was named BL21 (DE3) / pETUFUFR.
After the obtained transformant was cultured at 37 ° C. for 18 hours, an arbitrary colony that appeared was picked up and inoculated into an LB liquid medium supplemented with 50 μg / ml antibiotic kanamycin. After culturing at 37 ° C., when the value of OD600 nm reached about 0.5, 0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside, manufactured by Takara Bio Inc.) was added. The culture was continued even after the addition of IPTG, the medium was sampled every other hour, and the absorbance at 600 nm was measured. The results are shown in FIG. In FIG. 5, ■ indicates the results for BL21 (DE3) / pETECFcR, and □ indicates the results for BL21 (DE3) / pETHUFR. Alternatively, the X axis (horizontal axis) indicates time (unit is hr), and the Y axis (vertical axis) indicates absorbance at OD 600 nm (unit is arbitrary). As shown in FIG. 5, there was no particular difference in proliferation between the two.

  Next, a protein solution was prepared from each transformant for the medium whose absorbance was measured. The preparation was performed using BugBuster protein extraction Kit (registered trademark) (Novagen), and the prepared protein solution was subjected to SDS-PAGE. The result of the electrophoresis image is shown in FIG. A result shows the result 4 hours after IPTG addition. In FIG. 6, the lane a shows a result of transforming BL21 (DE3) / pET28a (vector only), the lane b shows the result for BL21 (DE3) / pETECFcR, and the lane c shows BL21 (DE3 ) / PETHUFcR results are shown. The position of the arrow indicates the migration position of the antibody receptor. As shown in FIG. 6, it was found that although the amino acid sequences of the expressed proteins are the same, the expression levels are different between the two. That is, the expression level of BL21 (DE3) / pETECFcR was higher than that of BL21 (DE3) / pETUFUCR, although the growth was similar. It shows that it succeeded in increasing the expression level significantly compared with the human type codon.

(Example 6) Refolding of Fc receptor and evaluation of antibody binding activity In Example 5, since FcγRI protein was obtained only as an E. coli insoluble fraction from BL21 (DE3) / pETECFcR, it was reactivated by refolding operation. went. Refolding was performed using Refolding CA kit (Takara Bio Inc.).
Antibody binding activity was performed by ELISA. First, 10 μg / ml of gamma globulin (manufactured by Chemo-Serum Therapy Laboratories), which is an antibody, was immobilized on a microplate (4 ° C., 18 hours) and blocked with StartingBlock Blocking Buffers (registered trademark) (PIERCE). . Subsequently, the protein solution prepared in Example 5 was serially diluted and subjected to an ELISA reaction. The reaction was carried out at 30 ° C. for 2 hours. Subsequently, it was washed with a Tris-HCl buffer solution (pH 8.0) containing 0.2% (w / v) Tween 20, 150 mM NaCl, and His-probe (H-15) HRP antibody (Santa Cruz Biotechnology) was added. did. After reacting at 30 ° C. for 2 hours, the plate was washed with the previous buffer, TMB Peroxidase Substrate (KPL) was added, and the absorbance at 450 nm was measured. The results are shown in FIG. In the figure, the X axis (horizontal axis) indicates the dilution factor of the sample, and the Y axis (vertical axis) indicates the absorbance at OD 450 nm (unit is arbitrary). As shown in FIG. 7, when the sample was diluted 6-fold, the decrease in absorbance was not so much observed, but when diluted 18-fold, the absorbance was considerably decreased.

  An ELISA reaction was also performed in which the antibody concentration at the time of immobilization was diluted stepwise from 50 μg / ml. Here, a certain amount of a protein preparation solution as an added sample was added and reacted. The results are shown in FIG. As shown in FIG. 8, the absorbance increased by increasing the immobilized antibody concentration. From these results, it was confirmed that the specific antibody binding activity was significantly expressed by refolding the polypeptide containing the extracellular region of FcγRI expressed in the E. coli insoluble fraction.

(Example 7) Evaluation of antibody-adsorbing ability of Fc receptor As shown in SEQ ID NO: 62 and SEQ ID NO: 63, the polypeptide containing FcγR1 extracellular region obtained in Example 6 has histidine at the C-terminus. Since it has six consecutive sequences, it can be bound to a carrier due to the affinity of the Ni chelate. 100 μg of the polypeptide obtained in Example 6 was immobilized on 500 μL of His / Bind Resin gel in Novagen's His / Bind Purification kit (registered trademark). Specifically, 100 μg of the polypeptide obtained in the example and 500 μL of the gel were packed in a column, washed with pure water, and then Ni was gelled using the Ni charge buffer solution (50 mM NiSO ₄ ) in the kit. It was made to carry on.

  Next, Tris-HCl buffer (pH 8.0) containing 20 mM imidazole, 500 mM NaCl, 10% glycerol, 0.001% surfactant (SB3-14) to prevent nonspecific adsorption of the antibody to the gel. Fully equilibrated. To the gel after completion of equilibration, 2.0 ml of 100 μg / ml gamma globulin diluted with equilibration buffer was added. After washing with equilibration buffer, elution was performed with Tris-HCl buffer (pH 8.0) containing 500 mM imidazole, 500 mM NaCl, 10% glycerol, and 0.001% surfactant (SB3-14).

  As a control experiment, an experiment was conducted in which the gel was not adsorbed with the polypeptide containing the FcγRI extracellular region, ie, the column was packed with only the carrier. The eluate eluted from the column was collected, and the amount of gamma globulin in the eluate was quantified using immunoglobulin as a standard protein using a protein assay kit manufactured by Bio-Rad. FIG. 9 shows the results using a gel on which a polypeptide containing the FcγRI extracellular region was immobilized, and FIG. 10 shows the results of a control experiment.

9 and 10, the X axis (horizontal axis) indicates the fraction number, the Y axis (vertical axis) indicates the concentration of the eluted protein (unit: μg / ml), and the position of the arrow indicates the eluate. The added position is indicated. As shown in FIG. 9, in the gel on which the polypeptide containing the FcγRI extracellular region was immobilized, elution of gamma globulin was not observed during washing with the equilibration buffer, and the elution fraction (fraction numbers 10 to 10 in FIG. 9). In 15), gamma globulin was detected. As shown in FIG. 10, in the control experiment, gamma globulin was not adsorbed to the gel but eluted in the flow-through fraction (fraction numbers 1 to 3 in FIG. 10).
From the above results, it is possible to obtain an antibody purification adsorbent for purifying an antibody using the polypeptide containing the Fc gamma RI extracellular region obtained by the method of the present invention. A method was provided for adsorbing antibodies to an adsorbent followed by purification.

It is the figure which compared the gene sequence between human and E. coli of antibody receptor FcγR1. In the figure, EcoFcGR1 is a sequence of a DNA sequence converted to an E. coli type codon, and HumanFcGR1 is a sequence of human FcγR1. It is the figure which compared the gene sequence between human and E. coli of antibody receptor FcγR1. In the figure, EcoFcGR1 is a sequence of a DNA sequence converted to an E. coli type codon, and HumanFcGR1 is a sequence of human FcγR1. It is a figure which shows the structure of plasmid vector pECFcR. It is a figure which shows the structure of plasmid vector pETECFcR. It is a figure which shows the structure of plasmid vector pETHUFcR. FIG. 5 is a diagram showing Fc receptor expression induction in Example 5. It is the electrophoresis image which compared the expression level of polypeptide containing an antibody receptor FcγR1 extracellular region. It is a figure which shows the antibody binding activity of the Fc receptor in Example 6. It is a figure which shows the antibody binding activity of the Fc receptor in Example 6. It is a figure which shows the antibody adsorption capacity of the Fc receptor in Example 7. FIG. 10 is a view showing a control experiment of the antibody adsorption ability of the Fc receptor in Example 7.

Claims (8)

A polynucleotide encoding a polypeptide comprising the FcγRI extracellular region, comprising the base sequence set forth in SEQ ID NO: 2. A polynucleotide encoding a region obtained by removing a signal sequence from a polypeptide containing an FcγRI extracellular region, comprising the nucleotide sequence set forth in SEQ ID NO: 3. A polynucleotide encoding a polypeptide having a methionine added to the N-terminal side of a polypeptide comprising an FcγRI extracellular region, from which the signal sequence has been removed, the polynucleotide comprising the base sequence set forth in SEQ ID NO: 4. A polynucleotide obtained by functionally adding an expression control sequence to a polynucleotide comprising the base sequence described in any one of SEQ ID NOs: 2 to 4. A recombinant vector into which the polynucleotide according to any one of claims 1 to 4 has been inserted. A transformant transformed with the polynucleotide according to any one of claims 1 to 4 or the recombinant vector according to claim 5 . The transformant according to claim 6 , wherein the transformant is a prokaryotic cell. A method for producing a polypeptide comprising an FcγRI extracellular region, comprising a step of culturing the transformant according to claim 6 or 7 .