Patent Application
20200172576
This application claims priority from prior Japanese Patent Application No. 2018-223373, filed on Nov. 29, 2018, entitled “Fusion polypeptide, method for producing fusion polypeptide, and DNA encoding fusion polypeptide”, the entire contents of which are incorporated herein by reference.
The present disclosure includes a fusion polypeptide, and a method for producing a fusion polypeptide.
K. Stubenraucha, Bachmannb, Rudolpha, H. Lilie; Journal of Chromatography B, 737 (2000) 77-841 describes that, when recombinant VP1 protein is expressed in E. coli and purified by ion exchange chromatography according to a conventional protein production protocol, the yield of the recombinant VP1 protein is deteriorated. Then, Non-Patent Document 1 also describes that a sequence containing oligoglutamate sequences (E6, E8, E10) immediately after cysteine is inserted near a HI-loop of the VP1 protein to prevent aggregation of the recombinant VP1 protein to form virus-like particles.
There is a problem that, when producing a polypeptide by gene recombination, a free thiol group contained in cysteine of the expressed polypeptide is unstable and binds to other thiol groups, resulting that it is easy to form a multimer of the produced polypeptide.
The scope of the present invention is defined solely by the appended claims, and is not affected to any degree by the statements within this summary.
An embodiment of the present disclosure is a fusion polypeptide containing a target polypeptide and a tag polypeptide, in which the tag polypeptide is present on a N-terminal side and/or a C-terminal side of the target polypeptide and has a structure represented by Xaa-Cys-Zaa. In this fusion polypeptide, (1) Xaa is Asp or Glu, and Zaa is Asp or Glu, or (2) Xaa is Lys or Arg, and Zaa is Lys or Arg. According to this embodiment, a fusion polypeptide in which a thiol group of cysteine of a tag polypeptide portion is protected can be provided, and the protected thiol group can easily bind a label or the like.
One embodiment relates to a method for producing a fusion polypeptide, the method including steps of: producing a fusion polypeptide using a DNA encoding the fusion polypeptide, and recovering an expressed fusion polypeptide. One embodiment of the present disclosure relates to a DNA encoding a fusion polypeptide. According to these embodiments, a fusion polypeptide in which a thiol group of cysteine of a tag polypeptide portion is protected can be produced.
A first embodiment of the present disclosure relates to a fusion polypeptide.
As shown in
The target polypeptide intends a polypeptide corresponding to an intended use of the fusion polypeptide. The number of amino acid residues constituting the target polypeptide is not particularly limited. For example, it may be an oligopeptide of several residues, or a protein of hundreds to tens of thousands of residues.
The target polypeptide is not particularly limited, and examples thereof include antibodies, antibody fragments, antigens, enzymes, receptor proteins, ligand proteins, and the like. Specific examples include immunoglobulins constituting antibodies, cell surface antigens, intracellular proteins, nuclear proteins, and the like. It may also be a protein (generally called a blocking agent) for blocking a surface of a solid phase (plate, bead, membrane, or the like) used in an immunoassay or the like. Examples thereof include albumin, casein, gelatin, and the like. Examples of the antibody fragments can include Fab of the antibody, Fab′ of the antibody, a heavy chain or light chain of the antibody. The heavy chain or light chain may be a part of a heavy chain and/or light chain containing at least a portion of a variable region. At least a portion of the variable region contains at least one, preferably at least two, and more preferably three complementarity determining regions (CDRs). In addition, at least a portion of the variable region may contain a framework region (FR) adjacent to the CDR. A part of the heavy chain and light chain containing at least a portion of the variable region may also include, for example, like scFv (single chain Fv), an embodiment of so-called single chain antibody, containing at least one of the heavy chain CDRs and at least one of the light chain CDRs in one polypeptide. More preferably, examples of the target polypeptide can include Fab of the antibody, Fab′ of the antibody, and a Fab portion or Fab′ portion of the heavy chain. The Fab of the antibody intends a fragment in which the Fab portion of the heavy chain and the light chain are bound to each other. The Fab′ of the antibody intends a fragment in which the Fab′ portion of the heavy chain and the light chain are bound to each other. The Fab portion of the heavy chain intends a heavy chain fragment that is an antigen-binding region that does not contain two cysteines that serve as a hinge portion of the antibody and is not bound to the light chain. The Fab′ portion of the heavy chain intends a heavy chain fragment that is an antigen-binding region that contains two cysteines that serve as a hinge portion of the antibody and is not bound to the light chain.
A constant region of the heavy chain may be any of α chain, δ chain, ε chain, γ chain, and μ chain. Isotypes of α chain and γ chain are not limited. Also, a constant region of the light chain may be either κ chain or λ chain. Examples of origins of the heavy chain or light chain can include human, mouse, rat, chicken, dog, cat, sheep, guinea pig, goat, pig, and the like. Further, the heavy chain or light chain may be obtained by modifying an amino acid sequence derived from an animal species other than human into a human amino acid sequence. The heavy chain or light chain may be a chimera having amino acid sequences of two or more heterogeneous animals.
The tag polypeptide has a structure represented by Xaa-Cys (cysteine)-Zaa. Xaa and Zaa each represent one amino acid. “Zaa” is distinguished from “Z” representing “glutamic acid or glutamine” in the one-letter code of amino acid. Xaa intends the N-terminal side, and Zaa intends the C-terminal side. In one embodiment, both Xaa and Zaa are acidic amino acids. In this embodiment, Xaa can be Asp or Glu, and Zaa can be Asp or Glu. In another embodiment, both Xaa and Zaa are basic amino acids. In this embodiment, Xaa can be Lys or Arg, and Zaa can be Lys or Arg. In the present disclosure, an amino acid is sometimes represented by a one-letter code, in addition to a three-letter code. In one-letter code, cysteine is represented by “C”, aspartic acid is represented by “D”, glutamic acid is represented by “E”, lysine is represented by “K”, arginine is represented by “R”, and alanine is represented by “A”.
Preferably, the tag polypeptide has a structure represented by (Paa)m-Xaa-Cys-Zaa-(Qaa)n. “Paa” represents one amino acid. “Paa” is distinguished from “P” representing “proline” in the one-letter code of amino acid. “Qaa” represents one amino acid. “Qaa” is distinguished from “Q” representing “glutamine” in the one-letter code of amino acid. X and Z each represent an amino acid, and m and n each independently represent the number of amino acids selected from an integer of 0 to 20, and preferably 1 to 15. More preferably, m and n each independently represent the number of amino acids selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.
(Paa)m-Xaa and Zaa-(Qaa)n preferably have the same electrical polarity. (Paa)m-Xaa and Zaa-(Qaa)n have the same electrical polarity, whereby they are expected to repel each other and prevent a thiol group of the sandwiched cysteine from binding to other groups. Therefore, the electrical polarity of (Paa)m-Xaa and Zaa-(Qaa)n may be negative or positive.
Based on a pKa value of a side chain of each amino acid contained in (Paa)m-Xaa or Zaa-(Qaa)n, the electrical polarity can be obtained by calculating an effective charge of (Paa)m-Xaa or Zaa-(Qaa)n at a pH of a solution in which the tag polypeptide is present. When the effective charge is a positive value, the electrical polarity is positive, and when the effective charge is a negative value, the electrical polarity is negative. The effective charge of (Paa)m-Xaa and the effective charge of Zaa-(Qaa)n may not be equal.
When the electrical polarity of (Paa)m-Xaa and Zaa-(Qaa)n is negative, Xaa is Asp or Glu, m is 1 or more, and the amino acid present immediately before Xaa, that is, the amino acid adjacent to the N-terminal side of Xaa is preferably Asp or Glu. Moreover, when the electrical polarity of (Paa)m-Xaa and Zaa-(Qaa)n is negative, Zaa is Asp or Glu, n is 1 or more, and the amino acid present immediately after Zaa, that is, the amino acid adjacent to the C-terminal side of Zaa is preferably Asp or Glu. Further, when the electrical polarity of (Paa)m-Xaa and Zaa-(Qaa)n is negative, it is more preferable that (Paa)m and/or (Qaa)n does not contain a basic amino acid.
When the electrical polarity of (Paa)m-Xaa and Zaa-(Qaa)n is positive, Xaa is Lys or Arg, m is 1 or more, and the amino acid present immediately before Xaa, that is, the amino acid adjacent to the N-terminal side of Xaa is preferably Lys or Arg. Moreover, when the electrical polarity of (Paa)m-Xaa and Zaa-(Qaa)n is positive, Zaa is Lys or Arg, n is 1 or more, and the amino acid present immediately after Zaa, that is, the amino acid adjacent to the C-terminal side of Zaa is preferably Lys or Arg. Further, when the electrical polarity of (Paa)m-Xaa and Zaa-(Qaa)n is positive, it is more preferable that (Paa)m and/or (Qaa)n does not contain an acidic amino acid.
The polarity of amino acids is as follows. Acidic amino acids and basic amino acids are sometimes collectively referred to as polar amino acids.
Acidic amino acids: Asp (aspartic acid), Glu (glutamic acid)
Basic amino acids: Arg (arginine), Trp (tryptophan),
His (histidine), Lys (lysine)
(Paa)m and/or (Qaa)n preferably does not contain Cys.
In the fusion polypeptide, the tag polypeptide may be present on the N-terminal side or the C-terminal side of the target polypeptide.
The tag polypeptide and the target polypeptide may be directly bonded. The tag polypeptide and the target polypeptide may be bonded via a linker as shown in
The fusion polypeptide may consist of a single peptide unit as shown in
The fusion polypeptide may contain sequences other than the target polypeptide and tag polypeptide on the N-terminal side or the C-terminal side.
In one embodiment, the fusion polypeptide contains a first target polypeptide, a tag polypeptide, and a second target polypeptide. That is, in this fusion polypeptide, the tag polypeptide is sandwiched between two types of target polypeptides (
The fusion polypeptide may contain sugar chain modification.
Cysteine having a structure represented by Xaa-Cys-Zaa in the tag polypeptide contained in the fusion polypeptide may be labeled with a labeling substance as shown in
The labeling substance can be bound to the fusion polypeptide via a thiol group of cysteine. A method for binding a labeling substance to a thiol group is publicly known as follows.
The thiol group of cysteine can be bound to a labeling substance by cross-linking with a compound having a maleimide group or a bromo (or iodo) acetamide group as a reactive group (see the figure below). For example, when a thiol group of cysteine of a tag polypeptide and a labeling substance having a thiol group are cross-linked, a cross-linking reagent or linker having maleimide at both ends may be used.
The above cross-linking reaction can be performed under normal temperature and normal pressure. The solvent used in the reaction is not particularly limited as long as it is inert to the above reaction and each compound to be subjected to the reaction can be lysed or dispersed. Examples of the solvent include aromatic hydrocarbons such as benzene, toluene and xylene, ethers such as diethyl ether, tetrahydrofuran, ethylene glycol dimethyl ether and 1,4-dioxane, and amides such as N,N-dimethylformamide, sulfoxides such as dimethyl sulfoxide, halogenated hydrocarbons such as dichloromethane and chloroform, and the like. These solvents can be used alone or as a mixture.
The second embodiment of the present disclosure relates to a method for producing a fusion polypeptide described in the first embodiment. The method for producing a fusion polypeptide is not limited as long as the desired fusion polypeptide is obtained. For example, production of a fusion polypeptide can be performed using genetic engineering techniques. The production of a fusion polypeptide can be performed with a cell-free system or cells using a DNA encoding the fusion polypeptide. The method for producing a fusion polypeptide at least includes steps of producing a fusion polypeptide using a DNA encoding the fusion polypeptide, and recovering the expressed fusion polypeptide.
First, a nucleic acid (DNA) encoding an amino acid sequence of the fusion polypeptide is prepared. The nucleic acid encoding the fusion polypeptide inserts, for example, a DNA encoding a complementary strand sequence of a cDNA sequence for expressing a target polypeptide (hereinafter simply referred to as “encoding a target polypeptide”) and a DNA encoding a tag sequence into an expression vector to construct an expression vector that expresses the fusion polypeptide. When the tag polypeptide is present on the C-terminal side of the fusion polypeptide, as shown in
The sequence encoding a target polypeptide and the sequence encoding a tag polypeptide may be inserted into an expression vector separately. The sequence encoding a target polypeptide and the sequence encoding a tag polypeptide may be inserted as a nucleic acid in which the sequence encoding a target polypeptide and the sequence encoding a tag polypeptide are connected.
The sequence encoding a linker may be a sequence included in the expression vector in advance, or the sequence encoding a linker may be added to the sequence encoding a tag polypeptide or the sequence encoding a target polypeptide. Alternatively, it may be inserted as a nucleic acid in which the sequence encoding a target polypeptide, the sequence encoding a linker, and the sequence encoding a tag polypeptide are connected.
As the expression vector, a known one can be selected. Specific examples of the vector can include plasmid vectors, cosmid vectors, phage vectors, viral vectors (for example, adenoviral vectors, adeno-associated viral vectors, retroviral vectors (including lentiviral vectors), herpes viral vectors, and the like) and the like.
Examples of cell-free expression systems and vectors when a host is E. coli include plasmid vectors such as pBlueScript, pBR322 or variants thereof (for example, pB325, pAT153, pUC8, and the like), pUC19, pGEM-T, pCR-Blunt, pTA2, and pET, M13 phage or variants thereof, λ phage or variants thereof, and the like. Preferably, the vector contains an auxotrophic marker, a drug resistance marker, an expression promoter sequence other than a promoter that expresses a fusion polypeptide, and an expression terminator sequence. More preferably, the vector contains an ampicillin resistance gene derived from pBlueScript, and a lactose promoter. It is also possible to use a vector suitable for self-cloning.
When yeast is used as a host, plasmid vectors such as pYepSec1, pMFa, and pYES2 can be used. When insect cells are used as hosts, for example, plasmid vectors such as pAc and pVL can be used. When mammalian cells are used as hosts, for example, plasmid vectors such as pCDM8 and pMT2PC can be used, but are not limited thereto.
The method for constructing an expression vector for expressing the fusion polypeptide described above is publicly known.
When the fusion polypeptide is expressed using a cell-free expression system, first, an RNA encoding a fusion polypeptide is obtained. The RNA can be obtained by cleaving a vector containing a DNA encoding the fusion polypeptide with a restriction enzyme downstream from the 3′ end of the fusion polypeptide sequence to linearize the vector, and performing a transcription reaction using a transcriptase that can translate from a promoter present on the 5′ end side of the fusion polypeptide. In this case, T7 promoter, T3 promoter, SP6 promoter or the like can be used as a promoter. Transcriptases corresponding to the above promoters are T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase. RNA obtained by transcription reaction is mixed with cell-free extracts (containing ribosome, translation factors, tRNA, and the like) that maintain a translation function extracted from E. coli, rabbit reticulocyte lysate (RRL), wheat germ extract, insect cells (such as SF9 or SF21), and the like, an energy source (ATP) and an amino acid, and the mixture is incubated at a predetermined temperature for a predetermined time to produce a fusion polypeptide.
Examples of host cells into which an expression vector for expressing a fusion polypeptide is introduced can include E. coli, yeast, insect cells, and mammalian cells. The method for introducing an expression vector into these host cells and a method for culturing host cells are publicly known.
A method for inducing expression of a fusion polypeptide inserted into an expression vector in the host cells into which the expression vector has been introduced is also publicly known. For example, when the host has a DE3 sequence in a genome, it has a Lac operator sequence as a promoter for expressing T7 RNA polymerase or T3 RNA polymerase, so that expression of T7 RNA polymerase or T3 RNA polymerase can be induced by isopropyl-β-thiogalactopyranoside. In such host cells, the expression of the fusion polypeptide can be controlled by the presence or absence of addition of isopropyl-β-thiogalactopyranoside.
The expressed fusion polypeptide can be recovered according to a known method. The recovered fusion polypeptide can be refolded if necessary. Refolding methods are also publicly known.
When the expressed fusion polypeptide is present in extracellular fractions such as the medium, the extracellular fractions and the host cells are separated by centrifugation or the like, and then the extracellular fractions containing the fusion polypeptide are recovered as a fusion polypeptide roughly purified solution.
When E. coli is used as the host cell, the expressed fusion polypeptide is sometimes present in a periplasmic fraction. In this case, for example, the recovered host cells are suspended in a periplasmic extraction buffer such as sucrose buffer including sucrose, Tris-HCl buffer, EDTA and the like, and an osmotic shock with magnesium sulfate is applied, whereby the periplasmic fraction can be extracted. The extract of the periplasmic fraction can be recovered as a roughly purified solution of the fusion polypeptide.
When the expressed fusion polypeptide is present in soluble fractions of the host cells, the host cells are suspended in a cell lysis buffer containing a surfactant or the like, and the suspension is crushed with ultrasound or the like, if necessary, and a supernatant obtained by centrifugation or the like can be recovered as a roughly purified solution of the fusion polypeptide.
When the expressed fusion polypeptide is present in insoluble fractions (solid fraction or inclusion body after lysing total cells with a cell lysis buffer or the like, and the like) of the host cells, the insoluble fractions are suspended and lysed in a denaturing buffer such as urea or guanidine hydrochloride or the like, then the lysate is dialyzed to remove a denaturant component, whereby a roughly purified solution of the fusion polypeptide can be obtained.
Purification of the fusion polypeptide from the recovered roughly purified solution of the fusion polypeptide can be performed by gel filtration, ion exchange chromatography or the like. When the fusion polypeptide has a linker and the above-described second tag sequence is present in the linker, the fusion polypeptide can be purified by performing affinity purification according to the second tag sequence.
Further, when the purified fusion polypeptide is not correctly folded, refolding can be performed. Refolding can be performed by, for example, lysing the purified fusion polypeptide in a denaturing buffer including urea, guanidine hydrochloride or the like or adding a denaturant such as urea or guanidine hydrochloride to the roughly purified solution of the fusion polypeptide to lyse aggregation of the fusion polypeptide, and then performing, for example, dilution refolding, dialysis refolding, solid phase refolding, size exclusion chromatography refolding, surfactant refolding, or the like, described in Arakawa and Ejima (Antibodies 2014, 3, 232-241; doi: 10.3390/antib3020232). Dialysis refolding is preferable.
For example, in order to produce a Fab portion and Fab′ portion of the antibody, a fusion polypeptide having a Fab portion (or Fab′ portion) of a heavy chain of the antibody as a target polypeptide and a light chain of the antibody are each expressed in E. coli in different flasks, E. coli is recovered from each flask, and the periplasmic fraction of each E. coli is lysed in a denaturing buffer (preferably, a 6 M guanidine hydrochloride buffer). The obtained denaturing buffer lysates are mixed so that the expressed fusion polypeptide and the light chain of the antibody are almost the same by molar ratio, and the mixed lysate is dialyzed according to the dialysis refolding described in, for example, Arakawa and Ejima, whereby the Fab portion (or Fab′ portion) of an antibody in which the Fab portion (or Fab′ portion) of the heavy chain and the light chain bound to each other contained in the fusion polypeptide can be recovered.
The dialysis conditions can be appropriately changed according to a purification scale and the type of the target polypeptide. Dialysis for refolding the Fab portion (or Fab′ portion) of the antibody can be performed under conditions of dialysate 1 [3 M to 3.5 M guanidine hydrochloride; about 50 mM Tris-HCl buffer (pH 7.8 to 8.2); about 1 mM EDTA] for about 6 to 10 hours, dialysate 2 [2 M to 2.5 M guanidine hydrochloride; about 50 mM Tris-HCl buffer (pH 7.8 to 8.2); about 1 mM EDTA] for about 10 to 14 hours, dialysate 3 [1 M to 1.5 M guanidine hydrochloride; 0.3 M to 0.5 M arginine hydrochloride; about 50 mM Tris-HCl buffer (pH 7.8 to 8.2); about 1 mM EDTA; disulfide exchange reagent (1 mM GSH/1 mM GSSG)] for about 10 to 14 hours, dialysate 4 [0.3 M to 0.75 M guanidine hydrochloride; 0.3 M to 0.5 M arginine hydrochloride; about 50 mM Tris-HCl buffer (pH 7.8 to 8.2); about 1 mM EDTA; disulfide exchange reagent (1 mM GSH/1 mM GSSG)] for about 10 to 14 hours, and dialysate 5 [about 50 mM Tris-HCl buffer (pH 7.8 to 8.2); about 1 mM EDTA] for about 10 to 14 hours.
Furthermore, the method for producing a fusion polypeptide may include a step of binding Cys in the Xaa-Cys-Zaa contained in the tag polypeptide and a labeling substance. Regarding the “labeling substance” and the binding of the labeling substance, description in the section of [Fusion Polypeptide] is incorporated herein.
Hereinafter, embodiments of the present disclosure will be described in more detail by way of examples, but the embodiments of the present disclosure are not to be construed as being limited to the examples.
A fusion polypeptide in which tag polypeptides of SEQ ID NOs: 1 to 5 shown in Table 1 were connected to the C-terminal side of a target polypeptide maltose binding protein (MBP) via 6 histidine polyhistidine tags was expressed using pMAL-p5x E. coli expression system. In this system, MBP does not contain cysteine, so that aggregation properties of the fusion polypeptide will depend on cysteine in the tag polypeptides.
A periplasmic fraction containing the expressed fusion polypeptide was extracted by osmotic shock under ice-cooling using a sucrose buffer (30 mM Tris HCl, 20% sucrose, pH=8, 1 mM EDTA) and 5 mM magnesium sulfate. The fusion polypeptide was purified from the extracted periplasmic fraction using amylose resin and an elution buffer (20 mM Tris-Cl, 200 mM NaCl, 1 mM EDTA, pH 7.4). The purified fusion polypeptide was analyzed by SDS-PAGE without reduction. As a control that does not form a disulfide bond, MBP combined with 15 amino acid peptide tag (Sib) designed by SibTech was used. As a control for forming a disulfide bond, Mouse Hinge (VPRDCGG: SEQ ID NO: 14) Heavy chain inter-chain disulphide sequence (M. Hinge) was used.
The results are shown in
As to the results shown in
It was shown that, as compared with M. Hinge, the D3CD3, D6CD2, D4CD4, D4CD2 or D5CD tag polypeptide had an improved Monomer ratio/M. Hinge, indicating that aggregation was suppressed.
Next, when MBP was used as the target polypeptide as in Example 1 and amino acids other than cysteine contained in the tag polypeptide were changed to basic amino acids, the number and position of aspartic acids was changed, and the monomer ratio was investigated by Western blotting. The investigation was performed by Western blotting. Anti-6-His Rabbit Polyclonal antibody was used as a primary antibody for Western blotting. Polyclonal Goat Anti Rabbit Immunoglobulin/HRP conjugated was used as a secondary antibody.
The sequence of the tag polypeptide used in Example 2 is shown in Table 3.
For the results shown in
As shown in the left figure of
In terms of the yield of the fusion polypeptide, comparing SEQ ID NOs: 6, 7, and 8 where two polar amino acids are present on both sides of cysteine, the fusion polypeptide containing the tag polypeptide of SEQ ID NO: 8 containing acidic amino acids showed a band more strongly in
Next, a Fab′ portion of a HBs85 monoclonal antibody was constructed in vitro. As a target polypeptide, a fusion polypeptide in which glycine was sandwiched as a linker and the tag polypeptides shown in SEQ ID NOs: 1, 2, and 5 were connected to the C-terminal side of the Fab′ portion of a heavy chain of the HBs85 monoclonal antibody was expressed in DE3-positive E. coli using a pET system. In addition, a light chain of the HBs85 monoclonal antibody was expressed in another E. coli. A periplasmic fraction of E. coli expressing the fusion polypeptide and a periplasmic fraction of E. coli expressing the light chain were extracted and each lysed in a denaturing buffer containing 6 M guanidine hydrochloride to prepare lysates. The obtained lysates were mixed so that the fusion polypeptide and the light chain were almost the same by molar ratio, and the mixed lysate was subjected to dialysis under the following conditions to bind the fusion polypeptide and the light chain while refolding. Dialysate 1 [3 M guanidine hydrochloride; 50 mM Tris-HCl buffer (pH 8.0); 1 mM EDTA] for 8 hours, dialysate 2 [2 M guanidine hydrochloride; 50 mM Tris-HCl buffer (pH 8.0); 1 mM EDTA] For 12 hours, dialysate 3 [1 M guanidine hydrochloride; 0.4 M arginine hydrochloride; 50 mM Tris-HCl buffer (pH 8.0); 1 mM EDTA; disulfide exchange reagent (1 mM GSH/1 mM GSSG)] for 12 hours, dialysate 4 [0.3 M guanidine hydrochloride; 0.4 M arginine hydrochloride; 50 mM Tris-HCl buffer (pH 8.0); 1 mM EDTA; disulfide exchange reagent (1 mM GSH/1 mM GSSG)] for 12 hours, and dialysate 5 [50 mM Tris-HCl buffer (pH 8.0); 1 mM EDTA] for 12 hours.
Dialysis was performed at 4° C.
The Fab′ portion of the refolded HBs85 monoclonal antibody was evaluated by Western blotting under non-reducing conditions and reducing conditions. As an antibody, an HRP-labeled anti-mouse immunoglobulin antibody was used. As a control for forming a disulfide bond, original Mouse Hinge (M. Hinge WT: CKPCIC: SEQ ID NO: 15) was used. As a negative control that does not form a disulfide bond, HBs85 WT (that contains hinge sequence and tag polypeptide and does not form a dimer) was used.
As shown in
Next, using the Fab′ portion of the refolded HBs85 monoclonal antibody, binding to the antigen under non-reducing conditions was confirmed by ELISA.
Biotin was bound to the Fab′ portion of the refolded HBs85 monoclonal antibody using a maleimide reaction, and a HBs antigen was detected by ELISA. The reactivity of each Fab′ portion in ELISA is shown in
Next, using MBP as the target polypeptide as in Example 1, whether there was an effect of maintaining the monomer ratio of the fusion polypeptide with respect to sequences of other tag polypeptides was investigated. In addition to the tag polypeptides represented by SEQ ID NOs: 1, 2, and 5, the sequences shown in Table 5 were investigated. In SEQ ID NOs: 9 to 13, a neutral amino acid alanine was inserted into the tag polypeptides. MBP was used as a control not forming a disulfide bond, and M. Hinge2 (CKPSIS: SEQ ID NO: 16) was used as a control forming a disulfide bond.
In
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-223373 | Nov 2018 | JP | national |
