JP6425327B2 - Protein-magnetic particle complex and method for producing the same - Google Patents

Description

  The present invention relates to a protein-magnetic particle complex and a method for producing the same.

  Magnetic particles can be easily separated and guided by magnets, and their operation can be relatively easily automated. Therefore, applications in fields such as medical care and the environment are expected in recent years. For example, as an application in the medical field, a complex (hereinafter referred to as "protein-magnetic particle") in which a functional protein or peptide such as an antigen or an antibody (hereinafter referred to as "protein") is immobilized on the surface of a magnetic particle Techniques for detecting an antibody or an antigen by an antigen-antibody reaction using a “complex” are proposed (Patent Documents 1 to 3). Complexes reacted with the antibody or antigen can be separated magnetically.

  Such a protein-magnetic particle complex has been prepared by producing a protein of interest and a magnetic particle in separate systems, and immobilizing the protein on the magnetic particle. For example, the protein of interest is produced using bacteria such as E. coli and yeast, while the magnetic particles are magnetic bacteria that produce magnetic bacterial particles (hereinafter referred to as "BacMPs") in the cells. A protein-magnetic particle complex is formed by producing the protein and immobilizing the protein on the magnetic particle by chemical treatment using a polyfunctional compound, a crosslinking agent or the like. Specifically, it has been reported that proteins can be immobilized on lipid bilayer membranes on the surface of magnetic particles by mixing magnetic particles whose surface is modified with amino groups or carboxyl groups with proteins in the presence of a crosslinking agent. (Patent Documents 2 and 3). However, such an approach causes the amino group or carboxyl group of the functional protein to be immobilized on the carrier at random, resulting in denaturation or inactivation of the protein. Furthermore, in addition to the need for many proteins for immobilization, the operation is also complicated.

  In addition, using genetic engineering techniques, the gene encoding an anchor protein localized to the lipid bilayer membrane of magnetic bacterial particle (BacMPs) surface has binding affinity with the target functional protein (here, antibody). A method has also been proposed that utilizes a plasmid produced by fusing a gene encoding a protein (here, antibody binding domain) that it has (Patent Document 4). The transformant obtained by introducing the plasmid into magnetic bacteria is cultured to obtain magnetic bacterial particles displaying an anchor protein and the antibody binding domain. Then, the magnetic bacterial particles are isolated from the cells, and a solution containing the antibody is added to the particles to immobilize the antibody on the magnetic particles. However, this method also requires complicated operations to obtain the complex.

Patent No. 2948935 gazette Unexamined-Japanese-Patent No. 5-209884 Japanese Patent Application Laid-Open No. 5-099926 Patent No. 5119398 gazette

In recent years, with the expansion of applications of recombinant protein-magnetic particle complexes, it has been required to make the complexes multifunctional and highly functional by modifying magnetic particles with various functional molecules. There is also a need for the development of methods for producing protein-magnetic particle complexes without the need for complicated procedures.
Therefore, the problem to be solved by the present invention is to provide a novel and useful protein-magnetic particle complex, and to provide a method for producing the complex without requiring complicated operations. is there.

A Gram-negative magnetic bacterium, Magnetospirillum magneticum AMB-1, produces cytosolic nanosized BacMPs arranged in a chain. For example, various applications of genetically modified BacMPs such as immunological assay, ligand-receptor interaction or cell separation have been proved by expressing the target protein on BacMPs, but the gram negative which is a reducing environment Since no disulfide bond is formed in the cytoplasm of magnetic bacteria, a protein requiring formation of an intramolecular disulfide bond for proper folding (hereinafter referred to as "disulfide bond protein") is retained on BacMPs while retaining its function. It was difficult to express it.
As a result of intensive studies, the present inventors have developed a dual expression system in which disulfide bond proteins are expressed in the periplasm, which is an oxidative environment, and proteins that are docking partners of disulfide bond proteins are expressed in the cytoplasm. did. After bacterial culture, cells can be disrupted, and magnetic bacterial particles and disulfide bond protein can be docked via the docking partner to produce a complex in which disulfide bond protein is functionally expressed on BacMPs. According to this method, a complex can be formed by in vitro docking of a disulfide-linked protein and a protein that is a docking partner thereof, and hence this method is referred to as “in vitro docking method”.

That is, the first aspect of the present invention is
(1) A method for producing a complex of a disulfide bond protein and a magnetic particle in a gram negative magnetic bacterium,
(I) in the bacteria,
(A) a first fusion protein comprising a disulfide linked protein fused to a binding protein, and (b) a second fusion protein comprising a binding partner protein fused to a protein capable of being bound or integrated into a magnetic bacterial particle A co-expression step of co-expression, wherein expression of said first fusion protein is induced to the periplasm of said bacteria and expression of said second fusion protein is induced to the cytoplasm of said bacteria;
(Ii) culturing the bacteria of step (i) under conditions sufficient to produce said first and second fusion proteins;
(Iii) a step of destroying the bacterial cell membrane of the step (ii), wherein the complex of the disulfide bond protein and the magnetic bacterial particle is formed;
Manufacturing method.

  Here, the term "protein" includes not only proteins but also peptides. "Disulfide linked protein" refers to a protein comprising at least one disulfide bond in the molecule. Also, "binding protein" and "binding partner protein" refer to proteins that have binding affinity to each other and can specifically form a bond. Furthermore, "magnetic bacterial particles" refers to magnetic particles produced by magnetic bacteria, also referred to herein as "BacMPs". The "protein that can be bound or integrated into magnetic bacterial particles" refers to a protein that can be bound or integrated into magnetic bacterial particles, and even proteins intrinsic to organelles of magnetic bacteria can be genetically engineered. It may be a protein which is bound or integrated to magnetic bacterial particles by a technique.

In one embodiment of the present invention,
(2) The production method according to (1) above, which further comprises an isolation step (iv) of isolating the complex of the step (iii) from the disrupted cells.

Furthermore, a preferred embodiment of the present invention is
(3) The method according to the above (1) or (2), further comprising a recovery step of recovering the disulfide bond protein from the complex;
(4) The method according to any one of the above (1) to (3), wherein the binding protein is an immunoglobulin Fc domain;
(5) The method according to any one of the above (1) to (4), wherein the binding partner protein is a ZZ domain of protein A derived from Staphylococcus aureus ;
(6) The method according to any one of (1) to (5) above, wherein the disulfide-linked protein is a single chain variable fragment (scFv); and (7) bound or integrated to the magnetic bacterial particle It is a manufacturing method in any one of said (1)-(7) whose protein to be obtained is Mms13 derived from Magnetospillum magneticum .

  As described above, with the expansion of applications of protein-magnetic particle complexes, multifunctionalization and functionalization are required, and attempts are made to modify magnetic particles with various functional molecules. For example, in immobilizing a functional protein to organelle in vivo, there is a method of immobilizing a plurality of proteins by fusing a plurality of proteins via a peptide linker and expressing it as a single fusion protein. However, fusion of proteins with complex structures and fusion of multiple proteins may induce aggregate formation, misfolding and degradation by proteases. Therefore, there is a need for a technique for efficiently immobilizing a plurality of functional proteins on organelles without the problems as described above.

As a result of intensive studies, the present inventors have established a technique for immobilizing a plurality of target proteins on BacMPs in vivo, using the magnetic bacterium Magnetospirillum magneticum AMB-1 as a protein expression host. According to this method, while the scaffold protein is localized on BacMPs, a plurality of target proteins are co-expressed as a fusion protein fused with a tag protein (binding protein) capable of binding to the scaffold protein (binding partner protein). By expressing, a plurality of target proteins can be immobilized on BacMPs. This method is referred to herein as the “in vivo docking method” because it forms a complex by docking a plurality of target proteins with a scaffold protein in vivo.

That is, the second aspect of the present invention is
(8) with magnetic particles;
A scaffold protein fused to the protein that can be bound or integrated to the magnetic particle and comprising a first binding partner protein and a second binding partner protein;
A first fusion protein comprising a first binding protein and a first protein of interest fused to the first binding protein;
A second fusion protein comprising a second binding protein and a second protein of interest fused to said second binding protein;
A complex of protein and magnetic particles,
The first target protein is immobilized on the magnetic particle via binding of the first binding protein to the first binding partner protein.
The complex, wherein the second target protein is immobilized on the magnetic particle through the binding of the second binding protein and the second binding partner protein.

  Here, the “first binding protein” and the “first binding partner protein” can form a bond with each other by affinity, and the “second binding protein” and the “second binding partner protein” are also the same. Have a relationship of

Also, a preferred embodiment of the present invention is
(9) The complex according to (8), wherein the function of one of the first target protein and the second target protein is expressed by the presence of the other protein.

Also, another aspect of the present invention is
(10) A method for producing a complex of a plurality of proteins and magnetic particles in a gram negative magnetic bacterium,
(I) in the bacteria,
(A) a first fusion protein comprising a first binding protein and a first target protein fused to the first binding protein,
(B) a second fusion protein comprising a second binding protein and a second protein of interest fused to the second binding protein, and (c) a fusion to a protein that can be bound or integrated to a magnetic bacterial particle And co-expressing a scaffold protein comprising a first binding partner protein and a second binding partner protein;
(Ii) a culture step of culturing the bacteria of the step (i) under conditions sufficient to produce the first and second target proteins and the scaffold protein, the culture step comprising the first and second steps Forming a complex of the target protein of the present invention and the magnetic bacterial particle;
Manufacturing method.
The preferred embodiment of the present invention is
(11) (iii) a cell membrane disrupting step of disrupting the bacterial cell membrane of the step (ii);
(Iv) isolating the complex formed in the step (ii) from the cells disrupted in the step (iii);
The method according to (10), further comprising

  According to the present invention, novel and useful protein-magnetic particle complexes are provided. There is also provided a method of producing the complex without the need for complicated operations.

2 shows an in vitro docking method according to an aspect of the present invention. (A) Disulfide bond proteins of interest migrate to the periplasm, while the binding proteins are expressed on cytoplasmic BacMPs. Disulfide linked proteins dock to bound proteins on BacMPs while magnetically separating BacMPs from cell lysates. (B) Expression vector. All fusion genes represented here, M. It is expressed under the mms 16 promoter from Magneticum AMB-1. IgG binding assay is shown. Alkaline phosphatase prior to cell lysis (AP) coupled donkey IgG and Mms13ZZ transformant or M. Added to Magneticum AMB-1 wild type. BacMPs were extracted and binding of IgG-AP to Mms13-ZZ was detected by chemiluminescence. The time course of scFv-Fc binding on ZZ-BacMPs is shown. BacMPs were magnetically recovered from cell lysates at 0, 10, 30 and 60 minutes after cell disruption. Subsequently, membrane proteins extracted from BacMPs were subjected to Western blot using mouse anti-FLAG antibody and donkey anti-mouse IgG-AP. FIG. 7 shows Western blot analysis of Mms13-ZZ, Mms13-scFv and scFv-Fc prepared under reducing conditions ((A)) and non-reducing conditions ((B) and (C)). (A) and (B): scFv fusion proteins were detected by anti-FLAG IgG-AP and Mms13-ZZ by donkey anti-mouse IgG-AP. (C): Mms 13-ZZ and scFv fusion proteins were detected by rabbit anti-human λ light chain polyclonal antibody and AP conjugated anti-rabbit IgG. “Mms13-ZZ” is a BacMPs membrane protein derived from a transformant contained in pUM13ZZ; “Mms13-ZZ + scFv-Fc” is a BacMPs membrane protein derived from a transformant contained in pUM13ZZ / scFvF; “Mms13-scFv” is The BacMPs membrane protein derived from the transformant contained in pUM13 scFv is shown. Figure 3 shows [beta] -galactosidase binding assay on BacMPs. “ZZ” is BacMPs extracted from transformants contained in pUM13ZZ; “scFv” is BacMPs extracted from transformants contained in pUM13 scFv; “ZZ + scFv-Fc” is a trait contained in pUM13ZZ / scFvc BacMPs extracted from transformants are shown. M. Figure 2 shows in silico analysis of magneticum AMB-1 DsbA protein. C. using ClustalW 2.1 . DsbA derived from magneticum AMB-1 was compared with that derived from E. coli. Shades indicate confirmed sequence motifs. M. Figure 2 shows in silico analysis of magneticum AMB-1 DsbB protein. C. using ClustalW 2.1 . DsbB derived from magneticum AMB-1 was compared with that derived from E. coli. Dark shading indicates a confirmed sequence motif and light shading indicates a predicted sequence motif. M. FIG . 16 shows in silico analysis of magneticum AMB-1 DsbC protein. C. using ClustalW 2.1 . DsbC derived from magneticum AMB-1 was compared with that derived from E. coli. Dark shading indicates a confirmed sequence motif and light shading indicates a predicted sequence motif. M. FIG . 7 shows in silico analysis of magneticum AMB-1 DsbD protein. C. using ClustalW 2.1 . DsbD derived from magneticum AMB-1 was compared with that derived from E. coli. Dark shading indicates a confirmed sequence motif and light shading indicates a predicted sequence motif. M. FIG . 7 shows in silico analysis of magneticum AMB-1 DsbG protein. C. using ClustalW 2.1 . DsbG derived from magneticum AMB-1 was compared with that derived from E. coli. Dark shading indicates a confirmed sequence motif and light shading indicates a predicted sequence motif. 2 shows an in vivo docking method according to an aspect of the present invention. (A) shows the schematic of the expression vector used for in vivo docking, (B) shows Mms13-miniscaffoldin, GFP-DocC and mCherry-DocR. FIG. 7 shows expression and localization analysis of Mms13-miniscaffollidin. (A) shows the western blotting of the membrane fraction of BacMPs, (B) shows the antibody binding assay. Lane 1, lane 2 and lane 3 show a transformant carrying pUMtetDoc-M13miniscaf, a transformant carrying pUMtetDoc and a wild type, respectively. Figure 5 shows expression analysis of whole cell extract by western blotting. (A) was detected by anti-GFP antibody and (B) by anti-mCherry antibody. Lane 1, lane 2 and lane 3 show a transformant carrying pUMtetDoc-M13miniscaf, a transformant carrying pUMtetDoc and a wild type, respectively. Figure 12 shows antibody binding assay to BacMPs surface. (A) was detected by anti-GFP antibody and (B) by anti-mCherry antibody. Lane 1, lane 2 and lane 3 show BacMPs derived from a transformant carrying pUMtetDoc-M13miniscaf, BacMPs derived from a transformant carrying pUMtetDoc and BacMPs derived from a wild type, respectively. The western blotting of the membrane fraction of BacMPs is shown. (A) was detected by anti-GFP antibody and (B) by anti-mCherry antibody. Lanes 1 and 2 show the membrane fraction of BacMPs derived from a transformant carrying pUMtetDoc-M13miniscaf and the membrane fraction of BacMPs derived from wild type, respectively. In order from the top, (A) to (C) show fluorescence images of a wild type, a transformant carrying pUMtetDoc, and a transformant carrying pUMtetDoc-M13 miniscaf, respectively. 2 shows an in vivo docking method according to an aspect of the present invention. (A) shows the schematic of the expression vector used for in vivo docking, (B) shows Mms13-miniscaffoldin, eMHC-DocC and DM-DocR.

Hereinafter, the present invention will be described in more detail.
<1> Creation of Protein-Magnetic Particle Complex by In Vitro Docking A first aspect of the present invention relates to a method for producing a complex of a disulfide bond protein and a magnetic particle in a gram negative magnetic bacterium. That is, in the production method, gram-negative magnetic bacteria are used to produce a recombinant protein-magnetic particle complex, which comprises magnetic particles and a disulfide-linked protein immobilized on the magnetic particles.

In general, the cytoplasm of Gram-negative bacteria is a reducing environment. Therefore, when a foreign protein whose disulfide bond (hereinafter, also referred to as "S-S bond") influences structure formation, such as an antibody molecule, is expressed on magnetic bacterial particles present in the cytoplasm, the S-S bond is Do not form. On the other hand, since the periplasm of gram negative bacteria is an oxidative environment, it is suitable for expression of a protein containing S-S bond. Therefore, in the production method, the expression of a disulfide bond protein is induced to the periplasm and the production of BacMPs is performed in the cytoplasm.
As Gram-negative magnetic bacteria, Magnetospirillum magneticum (M. magneticum) AMB-1 can be used. M. magneticum AMB-1 cytoplasmically synthesizes nanosized BacMPs arranged in a chain. BacMPs contain a lipid bilayer membrane and pure magnetite (50 to 100 nm in diameter) surrounded by the lipid bilayer membrane and show strong ferrimagnetism. Other gram negative strains that produce similar magnetite can also be used in the present invention.

The production method includes the steps (i) to (iii) described below.
(I) Co-expression step In this step, in Gram-negative bacteria, (a) a first fusion protein comprising a disulfide-linked protein fused to a binding protein, and (b) bound or integrated to a magnetic bacterial particle It is coexpressed with a second fusion protein comprising the binding partner protein fused to the protein.

Examples of the disulfide bond protein include scFv (single chain variable fragment) shown in FIG. 1 (A). The scFv is generally readily available, and the Fc-fusion scFv forms an antibody-like dimerization structure by the interaction between the disulfide bond between the hinge region and the CH3 region, and has an affinity of bivalent binding Provide an effect. It also improves the solubility and in vivo folding in the cytoplasm of E. coli.
An alternative approach has been reported to functionally express single domain antibody fragments (referred to as sdAbs, VHHs or Nanobodies) on BacMPs in the cytoplasm. Although sdAbs are suitable for internal application due to their tight folding and stability, they have limited utility because they are prepared by camelid or shark immunity. In contrast, scFv are commonly available and are expected to expand the development possibilities of antibody and magnetic particle complexes as they form antibody-like dimerization structures.
The disulfide bond protein is not limited to the scFv shown in FIG. 1, and immunoglobulins (antibodies) such as IgG and IgM, surface antigens such as bacteria and viruses among antigens which they specifically recognize, interleukins and chemokines And the like, various growth factors such as EGF and TG, their receptors, their fragments, and their variants can be used.

The disulfide bond protein is fused to the binding protein.
In the example shown in FIG. 1, an immunoglobulin Fc domain (Fc) is used as a binding protein, but the binding protein is not limited thereto, and Strep-tag, p53, p16, E2F, fragments thereof and mutations thereof You can use the body.
Binding proteins have binding affinity with binding partner proteins. The binding partner protein is ZZ (ZZ domain of protein A from Staphylococcus aureus ) in FIG.
The binding partner protein is not limited to the ZZ domain, and Streptavidin, MDM2, Cyclin D, DP, their fragments and variants thereof can be used.

By selecting an appropriate combination of the binding protein and the binding partner protein, these interactions allow the disulfide bond protein of interest to be efficiently immobilized on BacMPs.
For example, combinations having K _D values in the range of 10 ⁻¹² to 10 ⁻⁶ can be used. Specifically, a combination of Strep-tag and Streptavidin, a combination of p53 and MDM2, a combination of p16 and Cyclin D, and a combination of E2F and DP can be mentioned as a combination of a binding protein and a binding partner protein. One skilled in the art will readily appreciate that, if desired, the binding protein and the binding partner protein can be interchanged with one another.

Examples of proteins that can be bound or integrated to magnetic bacterial particles include Mms13 derived from Magnetospillum magneticum shown in FIG. 1 (A). Mms13 protein is integrated with lipid bilayers on the surface of magnetite and can bind tightly to magnetite of BacMPs.
As proteins that can be bound or integrated to magnetic bacterial particles, in addition to Mms13, anchor proteins Mms16 and Mms24 like Mms13, Mms5, Mms6 and Mms7 involved in magnetite formation, and iron ion transport protein MagA It can be mentioned.

  In the step (i), the expression of the first fusion protein (in FIG. 1, scFv-Fc) is induced to the periplasm of the bacteria, and the expression of the second fusion protein (in FIG. 1, ZZ-Mms13) is It is induced to the cytoplasm of the bacteria. Therefore, the disulfide bond protein contained in the first fusion protein forms a disulfide bond in the oxidative environment of periplasm and is folded. As a result, in the complex formed in step (iii) described below, the disulfide bond protein is immobilized on the magnetic particles while maintaining its function.

Induction of the expression of the first fusion protein into the periplasm is performed by a signal peptide added to the N-terminus of the protein. As the signal peptide, for example, those from E. coli DsbC shown in FIG. 1 can be used. Endogenous DsbC is a protein that should be efficiently transferred to the periplasm for establishment of in vitro docking method, and there is an advantage that fusion with future full-length DsbC can also be envisioned. Of course, other known signal peptides capable of inducing protein expression into the periplasm can also be used in the present invention. Such signal peptides include those derived from DsbA, OmpA, PhoA, MdoD and TorA of E. coli, those derived from PelB of Erwinia carotovora , those derived from pIII of M13 phage, and the like.

(Ii) Culture Step In this step, the bacteria of the step (i) are cultured under conditions sufficient to produce the first and second fusion proteins and the scaffold protein. Here, conditions sufficient for producing a fusion protein are, for example, temperature 25 to 29 ° C., pH 6.0 to 7.5, microaerobic conditions, using Magnetic spirillum growth medium (MSGM) medium 3 It is culture conditions which culture | cultivate for-7 days.

(Iii) Cell membrane destruction step In this step, the cell membrane of the bacteria of the step (ii) is destroyed. And this step forms a complex of the disulfide bond protein and the magnetic bacterial particle.
That is, disruption of the cell membrane enables interaction between the binding protein and the binding partner protein, and the disulfide bond protein is trapped on the magnetic bacterial particle, and a complex of the disulfide bond protein and BacMPs is formed.
The disruption of the cell membrane of said bacteria may be performed alone or in combination with methods known in the art. For example, when the cell wall is broken with lysozyme etc., nonionic surfactants such as Triton X-100, Tween 20, Briji 35 etc., zwitterionic surfactants such as CHAPS, Zwittergent 3-12 etc, or SDS or N It is preferable to add an anionic surfactant such as lauroyl sarcosine. In addition, in this step, in order to prevent decomposition of a disulfide bond protein or the like, it is also desirable to have a protease inhibitor such as PMSF.

(Iv) Isolation step The production method of the present invention further comprises the isolation step of isolating the complex of disulfide bond protein and BacMPs formed in the step (iii) from the disrupted cells (iv ) May be included.

  The method of isolating the complex is not particularly limited, and may be a method known in the art. The isolation of disulfide bond proteins is performed, for example, by recovering BacMPs using magnetism. For recovery using magnetism, magnets such as columnar neodymium-boron (Nd-B) magnets are used.

  The isolation of the complex may be performed immediately after complete solubilization of the cells, but may be performed preferably after 10 minutes or more. At this timing, since the desired disulfide bond protein is completely captured on BacMPs by the interaction between the first fusion protein and the second fusion protein, the complex is isolated in a good yield. It can be released.

Another aspect of the invention relates to a method of producing a disulfide linked protein.
The production method further includes a recovery step of recovering the disulfide-linked protein from the complex in addition to the steps (i) to (iii).
For example, the disulfide bond from the complex using an acidic buffer of pH 2 to 4, an alkaline buffer of pH 10 to 12, a high ionic strength buffer of 3 M or more, a specific protein for binding protein or binding partner protein, etc. Protein can be recovered.

Next, the vector used in the above-mentioned production method and the transformant containing the vector will be described.
In the production method, in the step (ii), a transformant into which a vector has been introduced is usually cultured. Therefore, the scope of the present invention also includes a vector used for producing a protein-magnetic particle complex and a transformant containing the vector.

A vector according to one aspect of the present invention comprises a gene encoding a first fusion protein; a gene encoding a second fusion protein; and a signal sequence linked to a gene encoding the first fusion protein. Including.
Here, the gene encoding the first fusion protein is a fusion of the gene encoding the binding protein and the gene encoding the disulfide bond protein, and the gene encoding the second fusion protein is linked to BacMPs Alternatively, it is a fusion of a gene encoding a protein that can be integrated with a gene encoding a binding partner protein.

  A signal sequence forms a disulfide bond in the molecule in order to guide the first fusion protein containing the disulfide bond protein to the oxidative environment, periplasm. Thus, a protein-magnetic particle complex can be obtained while the disulfide bond protein retains its function.

  An example of the vector of the present invention is shown in (c) of FIG. 1 (B). In the example shown in (c) of FIG. 1 (B), the gene encoding the first fusion protein is “scFv-Fc”, the gene encoding the second fusion protein is “mms13-ZZ”, and the signal sequence is It is "dsbC". However, the gene encoding the first fusion protein linked to the signal sequence and the gene encoding the second fusion protein may be incorporated into separate vectors to transform the host cell.

In addition, the vector may contain a gene encoding a tag peptide that is advantageous for separation and purification of a target protein. In the example shown in FIG. 1 (B), the vector contains a gene (FLAG) encoding FLAG tag linked to the end of the scFv-Fc fusion gene.
The vector may further contain a gene to improve protein synthesis efficiency.

In addition, the vector should contain a gene encoding the first fusion protein and a promoter controlling the expression of the gene encoding the second fusion protein.
In the example shown in FIG. 1 (B), the mms16 promoter which is constitutively expressed is used as the promoter, and the scFv-Fc fusion gene and the mms13-ZZ fusion gene are controlled by the mms16 promoter.
The vector of the present invention may be any vector capable of transforming gram negative magnetic bacteria, and examples include plasmid vectors and cosmid vectors. Among them, preferably, a plasmid vector can be used.

According to the method for producing protein-magnetic particles according to the first aspect of the present invention, and the vector and transformant used in the method, the protein is correctly folded in one step without complicated operations. A protein-magnetic particle complex can be obtained in which the immobilized disulfide bond protein is immobilized on BacMPs. Therefore, the disulfide bond protein maintains its function in the obtained complex.
More specifically, the cytoplasm of the magnetic bacteria in which BacMPs are produced is a reductive environment. Thus, conventional methods of cytoplasmic disulfide bond protein expression do not form protein disulfide bonds on BacMPs. In such conventional methods, disulfide bond formation is dependent on air oxidation during the extraction and purification process of BacMPs, and the rate and yield of disulfide bond formation by air oxidation is very slow compared to in vivo And, there has been a concern that protein aggregation or degradation may occur due to misfielding or unfolding. In the production method according to the first aspect of the present invention, while the expression of the second fusion protein containing a protein that can be bound or integrated into the magnetic bacterial particle is induced to the cytoplasm of the bacteria, Since the expression of the first fusion protein containing the fused disulfide bond protein is induced to the periplasm in the oxidative environment, a complex in which the disulfide bond protein is immobilized on the magnetic particle while maintaining the function I was able to get
In addition, for preparation of the complex, purification of the target protein and a pretreatment step for forming an S-S bond were not required. The prepared complex was directly available after extraction from cells.
From the above facts, according to the first aspect of the present invention, a completely new method of preparing an S-S binding protein (in vivo docking method) is provided. The method is expected to expand the possibilities of developing complexes of antibodies and BacMPs.
In addition, it is possible to express not only antibodies but also various disulfide bond proteins on magnetic bacterial particles, and provide an innovative method to realize high throughput of targeting ligand or lead compound screening in drug discovery research. be able to. For example, the disulfide bond protein-BacMPs complex obtained by the production method is useful as a magnetic carrier for ligand-receptor interaction or affinity generation, and provides efficient screening of potential ligands or drugs. .

Hereinafter, the second aspect of the present invention will be described.
<2> Creation of Multiple Protein-Magnetic Particle Complex by In Vivo Docking Another aspect of the present invention relates to a complex of multiple proteins and magnetic particles.
The complex is described with reference to FIG.

The protein-magnetic particle complex according to the present aspect includes a plurality of proteins that are target proteins (that is, first and second target proteins), magnetic particles, and a scaffold protein. In the complex, a scaffold protein is bound or integrated to a magnetic particle, and a plurality of proteins are immobilized on the magnetic particle via the scaffold protein.
In the example shown in FIG. 11, the first and second target proteins are shown as a fluorescent green protein (Green Fluorescent Protein (GFP)) and mCherry, respectively. Also, magnetic particles are shown as magnetic bacterial particles (BacMPs), and scaffold proteins are shown as Mms13-miniscaffoldin. In this example, the magnetic particles are shown as magnetic bacterial particles (BacMPs) produced by gram negative magnetic bacteria, but in the protein-magnetic particle complex according to this aspect, the magnetic particles are not otherwise known as magnetic bacteria. It may be a magnetic particle produced by the method of

As mentioned above, the scaffold protein comprises a protein that can be bound or integrated to the magnetic particle, as well as a first binding partner protein and a second binding partner protein.
In the example of FIG. 11, the proteins that can be bound or integrated to the magnetic particle are shown as Mms13, and the first binding partner protein and the second binding partner protein are shown as CohC and CohR, respectively. Here, the first binding partner protein and the second binding partner protein play a role of a scaffold for binding the first binding protein and the second binding protein to the scaffold protein. Typically, the first and second binding proteins are different proteins.

The first binding partner protein and the second binding partner protein are usually linked by a peptide linker. As a peptide linker, a peptide linker generally used in the art can be used, and it is a peptide consisting of 5 to 100, preferably 5 to 20 amino acids.
As the peptide linker, for example, a peptide represented by (N ₄ S) _n (wherein n is an integer) can be used. The peptide linker used in the example shown in FIG. 11 is an (N ₄ S) ₂ peptide consisting of 10 amino acids.
Also, any one of the first binding partner protein and the second binding partner protein and the protein that can be bound or integrated to the magnetic particle are usually linked by a peptide linker. As a peptide linker, a peptide linker generally used in the art can be used, and it is a peptide consisting of 5 to 200, preferably 50 to 100, amino acids. In the example shown in FIG. 11, the first binding partner protein and the protein which can be bound or integrated to the magnetic particle are linked by a peptide linker, and the peptide linker consists of 100 amino acids (N ₄ S ) ₂₀ peptides.

  The first binding protein and the second binding protein bind to the first binding partner protein and the second binding partner protein, respectively, and the first and second target proteins are formed on the magnetic particle through these bindings. It is fixed to That is, the first and second binding proteins play a role as a tag for binding the first and second target proteins to the scaffold, respectively.

As a combination of the binding protein and the binding partner protein, one having a K _D value in the range of 10 ⁻¹⁰ to 10 ⁻⁹ can be used.
Specific examples of the combination of binding protein and binding partner protein include, for example, combination of cohesin and dockerin domain, combination of protein G and antibody Fc region domain, combination of protein A and antibody Fc region domain, basic leucine zipper A combination of a motif and an acidic leucine zipper motif is included.

In the example shown in FIG. 11, the cohesin and dockerin domains of the protein complex cellulosome produced by the cellulolytic bacteria were used as the binding protein and the binding partner protein, respectively. The combination of Cohesin and dockerin has a strong binding with a K _D value of 10 ^-10 to 10 ^-9, and further, there is species specificity in Cohesin / dockerin binding. It was found that multiple proteins can be functionally immobilized on BacMPs by co-expressing multiple cohesin domains as scaffolds on BacMPs and co-expressing multiple fusion proteins in which the dockerin domain is fused to the protein .
In the example shown in FIG. 11, the first binding protein and the second binding proteins DocC and DocR respectively bind to the first binding partner protein and the second binding partner proteins CohC and CohR. The first and second target proteins (GFP and mCherry) are immobilized on BacMPs.

  Here, a system in which the function of one of the first target protein and the second target protein is expressed by the presence of the other protein is an object of particular interest in the present invention. Here, "the function of a protein is expressed" means that the protein is correctly folded in addition to the enhancement of the activity of the protein, and the protein has an ability to bind to its target molecule. It is also intended to provide, the structure of the protein is stabilized.

As a specific example of the combination of the first target protein and the second target protein, for example,
-A combination of GFP (Green Fluorescent Protein) and mCherry,
-A combination of MHC II (class II major histocompatibility complex (MHC II)) and HLA-DM (Human leukemia antigen DM),
-Combination of Tropomyosin receptor kinase A and p75 neurotrophin receptor,
-Combination of Endoglycanase and beta-glucosidase,
-Combination of Endoglucanase and Cellobiohydrolase,
-Combination of beta-glucosidase and Cellobiohydrolase,
-A combination of Horseradish peroxidase and glucose oxidase-A combination of Acyl-ACP reductase and Aldehyde deforming oxygenase is mentioned.

As a preferable combination of the first target protein and the second target protein, a combination of MHC II and HLA-DM is intriguing.
eMHC II (empty MHC II) is an important protein responsible for human immune mechanism, and is associated with autoimmune diseases such as rheumatoid arthritis and rejection in organ transplantation. And, MHC II is a heterodimeric, single-pass transmembrane protein composed of an α chain and a β chain, and it is expressed on the cell membrane and degradation products of antigenic proteins from various diseases and pathogens (antigen peptides) And form an antigenic peptide-MHC II complex (pMHC II). The recognition of the antigen peptide bound to MHC II by helper T cells through T cell receptors specifically activates the cells. Therefore, in the development of peptide vaccine therapy using an antigenic peptide as a vaccine, analysis of the binding ability of MHC II to the antigenic peptide and identification of the antigenic peptide are important.
On the other hand, HLA-DM is a protein whose structure is similar to that of MHC II, has a chaperone-like function that stabilizes the structure by interacting with eMHC II, and is involved in the selection of an antigen peptide that binds to MHC II. It is believed that

  Therefore, in a protein-magnetic particle complex in which MHC II and HLA-DM having chaperone-like function are colocalized on BacMPs, MHC II can retain antigen binding ability by the presence of HLA-DM. The advantages of will be apparent to those skilled in the art. Such protein-magnetic particles are useful, for example, in peptide binding tests for the purpose of screening antibody peptides. In addition, since it contains magnetic particles, it is easy to separate from the mixture. However, prior to the present invention, there was no evidence that two proteins could be functionally localized through the scaffold on magnetic particles.

In a preferred embodiment of the present invention, the gram-negative magnetic bacteria M. aeruginosa also used in the first aspect of the present invention . magneticum AMB-1 can be used. Therefore, one embodiment of the second aspect of the present invention is a method for producing a magnetic particle-multiprotein complex, which comprises a magnetic particle and a plurality of proteins immobilized on the magnetic particle in gram negative magnetic bacteria. .
More specifically, the following steps (i) and (ii) are included.

(I) Co-expression step In this step, the first and second fusion proteins and the scaffold protein are co-expressed in gram negative magnetic bacteria.
Specifically, (a) a first fusion protein comprising a first binding protein and a first target protein fused to the first binding protein, (b) a second binding protein and the second A second fusion protein comprising a second protein of interest fused to a binding protein of (1), and (c) a protein fused to or capable of being bound to or integrated with a magnetic bacterial particle, and a first binding partner protein and a first A scaffold protein containing the two binding partner proteins is coexpressed.

  As the first and second target proteins, the first and second binding proteins, and the first and second binding partner proteins that can be used in the step, the above-mentioned proteins can be used .

(Ii) Culture Step In this step, the bacteria of the step (i) are cultured under conditions sufficient to produce the first and second target proteins and the scaffold protein. Here, "a condition sufficient for producing a fusion protein" is a condition similar to that described in the first aspect of the present invention.
In this step, a complex of the first and second target proteins and BacMPs is formed. That is, both the first and second fusion proteins and the scaffold protein are expressed in the cytoplasm of magnetic bacteria. By culturing the bacteria under the conditions described above, these proteins are produced in the cytoplasm, and the first and second target proteins are immobilized on the BacMPs via the scaffold protein by the binding affinity between the binding protein and the binding partner protein. And multiple protein-magnetic particle complexes are formed. Of course, the first aspect of the invention may be used for those in which one or both of the proteins of interest are disulfide linked proteins. That is, the expression of one or both may be induced into the periplasm.

(Iii) Cell membrane destruction step In this step, the cell membrane of the bacteria of the step (ii) is destroyed.
The disruption of the bacterial cell membrane may be carried out by the same method as described in the cell membrane disruption step (iii) of the first aspect of the present invention.
(Iv) Isolation step The production method further includes an isolation step (iv) of isolating the complex formed in the step (ii) from the cells disrupted in the step (iii). It may be. The isolation of the complex may be performed by the same method as described in the isolation step (iv) of the first aspect of the present invention.
In addition, the production method may further include a recovery step of recovering the first and second target proteins from the complex. This process is the same process as the recovery process described in the first aspect.

  In the production method, in the step (ii), a transformant into which a vector having the following constitution is introduced is cultured. Therefore, the scope of the present invention also includes a vector used for producing a protein-magnetic particle complex and a transformant containing the vector.

  FIG. 11A shows an example of a vector used in the above-mentioned production method. The vector contains a gene encoding a scaffold protein (Mms13-miniscaffolidin in FIG. 1) and a gene encoding a first fusion protein and a second fusion protein (Target-Dockerin in FIG. 1).

The gene encoding the scaffold protein is a gene encoding a protein (Mms13 in FIG. 11) which can be bound to or integrated with the magnetic particle; and a gene encoding a peptide linker (FIG. 11 a hydrophilic polypeptide linker NS liker (N ₄₎ S) ₂₀ ) and; genes encoding the first and second binding partner proteins (CohC and CohR in the cohehin domain in FIG. 11) and genes encoding the first binding partner protein and the second binding partner protein And a gene encoding a peptide linker which links with the gene encoding; and a gene for expression confirmation (c-myc tag in FIG. 11).

The genes encoding the first and second fusion proteins (Target-Dokerin) respectively correspond to the genes encoding the first and second target proteins (GFP and mCherry in FIG. 11); and the first and second binding proteins It is composed of a coding gene (in FIG. 11, DocC and DocR). The genes encoding the first and second binding proteins are linked to the genes encoding the first and second target proteins, respectively. That is, in FIG. 11, DocC is fused at the end of GFP (GFP-DocC), and DocR is fused at the end of Cherry (mCherry-DocR).
And, as also shown in FIG. 11 (B), it is preferable that a ribosome binding site (RBS) is included between the gene encoding the first fusion protein and the gene encoding the first fusion protein. Alternatively, the genes may be contained on separate vectors. The vectors that can be used in the second aspect of the invention are also as described for the first aspect of the invention.

Expression of a gene encoding a scaffold protein, and expression of a gene encoding a first fusion protein and a gene encoding a second fusion protein are controlled by a promoter, respectively.
In the example shown in FIG. 11, the expression of Mms13-miniscaffofdin is controlled by the constitutively expressing mms16 promoter, and the expression of GFP-DocC and mCherry-DocR constructs an artificial operon and Pmsp1 (tetO, a tetracycline expression induction system. Controlled by).

In step (ii) of the above-mentioned production method, the vector having the above-described configuration is introduced into magnetic bacteria, and the transformants thus produced are cultured.
In the example shown in FIG. 11 (B), the expressed scaffold protein Mms13-miniscaffoldin is localized on BacMPs, and then GFP-DocC and mCherry-DocR whose expression is induced by the addition of tetracycline are in Mms13-miniscaffoldin Bind to CohC and CohR. As a result, the target proteins GFP and mCherry are immobilized on BacMPs via the Mms13-miniscaffoldin.

According to a method of producing a plurality of protein-magnetic particles according to the second aspect of the present invention, and a vector and a transformant used in the method, production of functional protein and immobilization on a magnetic particle can be achieved. It can be done in one step and does not require complicated purification steps of functional proteins. Also, by combining the binding protein (scaffold molecule) and the binding partner protein (tag molecule), a plurality of proteins can be immobilized in any number and at any position. Furthermore, since the binding protein that plays the role of a tag and the functional protein of interest are gene-fused, they can be immobilized with orientation maintained.
So far, in the immobilization of functional proteins to organelles in vivo, a method of immobilizing a plurality of proteins by fusing a plurality of proteins via a peptide linker and expressing it as a single fusion protein However, there has been a problem that fusion of complex proteins or fusion of multiple proteins induces formation of aggregates, misfolding and degradation by protease. According to the production method of the present invention, and the vector and the transformant, since a plurality of proteins are expressed via a scaffold protein, the plurality of proteins can be immobilized on magnetic particles without such problems. It is possible.
In addition, according to the multiple protein-magnetic particles according to the second aspect of the present invention, for example, it is possible to improve the reaction efficiency by immobilizing a group of enzymes performing a series of metabolic reactions and to stabilize the target protein. It is expected to do. In addition, a complex in which a target protein that requires a specific protein to be a chaperone or cofactor for activation and the chaperone or cofactor are colocalized is expected to be applied in the medical field and the like.

Examples of the present invention will be described below, but the present invention is not limited thereto.
<Example 1> Functional Expression of scFv on Magnetic Bacterial Particles by In Vitro Docking Method (1-1) Sample Phusion DNA Polymerase (NEB) is used for gene amplification of DNA fragment for expression vector construction. In-Fusion HD Cloning Kit (Clontech) and Quick Ligation Kit (NEB) were used as reagents for AP (alkaline phosphatase) labeled donkey anti-mouse IgG (Novus Biologicals, Littleton) for IgG binding test, biotin β-Galactosidase (AVIVA SYSTEMS) and Alkaline Phosphatase Streptavidin (Vector) for evaluation of the binding activity of Anti-β-Galactosidase scFv Laboratories were used.
As a host for gene cloning, E. coli strain TOP10 (Life technologies, Carlsbad, Calif., USA) is used, and E. coli transformants are prepared in Luria-Bertani (LB) medium containing ampicillin (100 μg / ml). Incubated at ° C.
The plasmid vector is T. Yoshino, and T. Matsunaga. , Efficient and Stable Display of Functional Proteins on Bacterial Magnetic Particles Using Mms13 as a Novel Anchor Molecule, Society. It was constructed based on pUM13 ZZ described in (2006).

(1-2) M.I. In silico analysis of magneticum AMB-1 derived Dsb protein family
M .. The sequence of each Dsb protein of magneticum AMB-1 was searched from the protein database (http://www.ncbi.nlm.nih.gov/protein) of NCBI. As for the signal peptide of Dsb protein, whether it is experimentally confirmed or predicted from the sequence, or not on the database, using the database first on the Signal Peptide Website (http://www.signalpeptide.de/) I checked. For those not in the database, prediction of signal sequence was performed using PrediSi (http://www.predisi.de/). The transmembrane domain was predicted using SOSUI (http://bp.nuap.nagoya-u.ac.jp/sosui) except for E. coli DsbB, for which a predicted region has been reported in the literature.
Moreover, ClustalW (http://clustalw.ddbj.nig.ac.jp/) was used for the homology analysis of Dsb protein.

M. from the NCBI Protein database . As a result of searching the Dsb protein family derived from magneticum AMB-1, DsbB, DsbC, DsbD and DsbG were found as in E. coli. On the other hand, no protein corresponding to DsbA having an oxidase function was found. For each Dsb protein, the motifs and S-S binding sites analyzed in E. coli are summarized in FIG.
M. D. excluding DsbA . Although each Dsb protein derived from magneticum AMB-1 has little homology with E. coli, it has a CXXC motif at the catalytic site. Also, DsbB was predicted to have another pair of cysteine pairs in addition to the CXXC motif involved in the oxidation of DsbA, like E. coli. For the membrane proteins DsbB and DsbD, SOSUI was predicted to have four and eight transmembrane (TM) regions, respectively, as in E. coli.

The analyzed M. Although DsbA was not found from magneticum AMB-1, it is functionally a partner molecule of DsbB . In the magneticum AMB-1 strain, there is a possibility that part of the genome containing the dsbA gene has been deleted. As another possibility, in E. coli, overexpression of the dsbC gene which is an S-S-linked isomerase complements the function of the dsbA-deficient strain . Also in magneticum AMB-1, it is conceivable that DsbC plays a part of the function of DsbA.
From the above analysis results, M. Similar to E. coli, magneticum AMB-1 was also suggested to have oxidation of the S-S binding protein in the periplasm and the accompanying folding mechanism. This is an important finding as a premise for the in vitro docking method to function.

(1-3) Construction of Plasmid and Cultivation of Magnetic Bacteria Plasmids were prepared by standard cloning techniques (J. Sambrook, D. W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 2001) or In-Fusion. Constructed by cloning technology (Clontech, Mountain View, CA, USA).
A vector (pUM13ZZ / scFvFc) ((c) in FIG. 1 (B)) expressing an anti-β-galactosidase single chain antibody (scFv) and a fusion gene of a constant region (Fc) of human IgG1 has NsiI sites at both ends A scFv-Fc fusion gene artificially synthesized to have a signal sequence (DsbCss) of E. coli periplasmic protein DsbC and a sequence encoding a FLAG tag at the 5 'end, mms13 and Protein A encoding anchor protein Mms13 The gene was constructed by inserting it into the NsiI site (corresponding to the start codon of the Mms13-ZZ gene) of pUM13ZZ ((a) of FIG. 1 (B)) expressing a fusion gene of the IgG binding domain (ZZ) of In order to express as a single operon from the mms16 promoter to the scFv13R4-Fc fusion gene and the Mms13-ZZ fusion gene, the Shine-Dalgarno (SD) sequence lost due to the insertion of the scFv-Fc gene is the Mms13-ZZ gene. The artificially synthesized gene was designed to be added upstream.
On the other hand, in order to construct an Mms13-scFv expression vector expressed as a conventional Mms13 fusion protein as a control vector, a scFv gene PCR-amplified with SspI site at both ends was inserted into SspI digested pUM13ZZ (pUM13 scFv) ((B) in FIG. 1 (B)).

Each expression plasmid (pUM13ZZ, pUM13scFv and pUM13ZZ / scFvFc) was purified by Y.3. Okamura, H .; Takeyama, T .; Sekine, T. Sakaguchi, A .; T. Wahyudi, R .; Sato, et al. Design and Application of Cryptic-Plasmid-Based Shuttle Vector for Magnetospirillum magneticum Design and Application of a New Cryptic-Plasmid-Based Shuttle Vector for Magnetospirillum magneticum, Society. By electroporation according to the method described in (2003) . It transformed to magneticum AMB-1.

M .. magneticum AMB-1 (ATCC 700264), as described in T.M. Matsunaga, T .; Sakaguchi, F .; Tadakoro, Magnetite formation by a magnetic bacterium capable of growing aerobically, Appl Microbiol. Biotechnol. 35 (1991) 651-655, according to the method described in U.S. Pat . magneticum AMB-1 transformants were cultured under the same conditions, using 5 μg / ml of ampicillin.

(1-4) Preparation of magnetic bacterial particles Collected AMB-1 wild strain or each transformant (equivalent to 250 ml culture) to 2.5 ml of Extraction buffer A (50 mM Tris-HCl, 5 mM EDTA, pH 8.0) After suspension, lysozyme and PMSF were added to final concentrations of 0.4 mg / ml and 2 mM, respectively, and allowed to stand at room temperature for 5 minutes. Subsequently, 0.5 ml of Extraction buffer B (1.5 M NaCl, 0.1 M CaCl ₂ , 0.1 M MgCl ₂ , 0.02 mg / ml DNase I) was added, and allowed to stand at room temperature for 5 minutes. Next, Triton X-100 was added to a final concentration of 0.2%, and after gently stirring for 0 to 120 minutes at room temperature, the magnetic bacterial particle fraction was magnetically recovered using an Nd-B magnet. Thereafter, the plate was washed five times with PBS-T to obtain purified magnetic bacterial particles.

(1-5) IgG Capture Test In order to prove the concept of the present invention, an IgG binding test was performed using an AMB-1 transformant of pUM13ZZ encoding a Mms13-ZZ fusion protein.
0 to 2 μg / ml of AP-labeled donkey anti-mouse IgG was added to cell pellets (corresponding to 45 ml culture) of AMB-1 wild strain or pUM13 ZZ transformant to extract magnetic bacterial particles. After suspending 50 μg of each of the magnetic bacterial particles in 50 μl of PBS, 50 μl of Lumi-Phos 530 was added, and substrate reaction was performed at room temperature for 20 minutes, and then the luminescence intensity was measured using a luminometer.
As a result, a dose-dependent binding of IgG-AP was observed, and it was shown that even a low concentration of at least 16 ng / ml can be detected (FIG. 2). Although the binding between Donkey IgG and Protein A is weaker compared to human IgG1, this concentration corresponds to only 0.18 μg / l culture, and the system functions even when the expression amount of functional molecules is very low. It was shown to do.

(1-6) Western Blotting 1% containing 10 mM DTT and 5 mM EDTA in the case of reduction against 0.1 to 0.2 mg of magnetic bacterial particles obtained from AMB-1 wild strain or each AMB-1 transformant The LDS solution was added with 10 to 20 μl of a 1% SDS solution containing 5 mM EDTA if not reduced, heat-treated at 95 ° C. for 15 minutes, and the supernatant collected by magnetic separation was used as a particle membrane fraction. The obtained supernatant is mixed with a sample buffer, electrophoresed on an SDS-PAGE gel (10 to 20% gradient), and then applied to a PVDF membrane by a semi-dry method using Transfer Buffer (48 mM Tris, 39 mM Glycine, 20% Methanol) Blotted. The antigen-antibody reaction was performed using 1 μg / ml of primary antibody or AP-labeled secondary antibody dissolved in PBS-T (2% BSA / PBS-T) containing 2% BSA. After the secondary reaction, the PVDF membrane was washed three times with PBS-T, and then detection of AP activity was performed using BCIP / NBT-Blue.

(1-6-1) Analysis of binding time course of scFv-Fc and Mms13-ZZ Next, a co-expression vector of a mutant human anti-β-Galactosidase scFv-Fc fusion protein and Mms13-ZZ, which are model proteins (pUM13) The time required for the binding of Fc to ZZ was examined using AMB-1 transformants of The mutant scFv (scFv13R4) used here is one that has been modified so as to express at a high level in the cytoplasm of E. coli.
Triton X-100 was added to the cells partially disrupted with lysozyme and DNase I for 10 minutes to completely disrupt and solubilize the cells, and the magnetic bacterial particles were separated after 0, 10, 30 and 60 minutes . As a result, contrary to expectation, most of the binding between Fc and ZZ occurred at time 0, and reached the maximum level 10 minutes after the addition of Triton X-100 (FIG. 3). From these results, it was shown that recovery after 10 minutes after completely disrupting the cells was sufficient to obtain magnetic bacterial particles to which Fc fusion proteins were bound from AMB-1 transformants.

(1-6-2) Expression Analysis of scFv Fusion Protein In order to confirm the expression level of Mms13-scFv and scFv-Fc fusion gene expression vectors in AMB-1 transformants, antibodies of linear epitope or structural epitope were used respectively Western blotting was performed.
As a result, Mms13-scFv was detected as a single 41 kDa band predicted by anti-FLAG antibody of linear epitope recognition under reducing and non-reducing conditions, but anti-human λ light chain of structural epitope recognition (VL -Λ) not detected with antibody (Figure 4). On the other hand, the scFv-Fc monomer (52 kDa) was detected by the anti-FLAG antibody under reducing conditions, but the dimer (105 kDa) and trimeric amounts of both anti-FLAG antibody and anti-human VL-λ were detected under non-reducing conditions It was detected as two bands corresponding to the body (157 kDa) (FIG. 4).
From these results, Mms13-scFv was not correctly folded on the magnetic bacterial particle in the cytoplasm, while the scFv-Fc expressed in the periplasm was a dimer that seems to be mediated through the hinge region of Fc, or As a trimer in which a non-covalent bond is added to the dimer, it is considered to be correctly folded with an S-S bond. In addition, multimer formation of scFv-Fc fusion protein is also reported by other groups.

(1-7) β-Galactosidase Binding Test In order to confirm whether the scFv-Fc captured on the magnetic particle has antigen binding ability, β-Galactosidase binding test was performed.
Each 50 μg of magnetic bacterial particles extracted from AMB-1 into which pUM13ZZ, pUM13scFv or pUM13ZZ / scFvFc has been introduced is added with biotin-β-Galactosidase (1 μg / ml) suspended in 2% BSA / PBS-T at room temperature Let stand for 15 minutes. After washing once with 200 μl of PBS-T, Alkaline Phosphatase Streptavidin (1 μg / ml) was added and allowed to stand at room temperature for 15 minutes. After washing three times with 200 μl of PBS-T, the cells were suspended in 50 μl of PBS. To this, 50 μl of Lumi-Phos 530 was added and reacted for 30 minutes, and the luminescence intensity was measured by a luminometer.

The binding of biotin-labeled β-Galactosidase was detected in magnetic bacterial particles extracted from co-expressing transformants of scFv-Fc and Mms13-ZZ (FIG. 5, ZZ + scFv-Fc), whereas Mms13-ZZ alone or Mms13 -It was not recognized in the magnetic bacterial particle extracted from the transformant of scFv (FIG. 5, ZZ and scFv). From this, it was shown that the cytoplasmically expressed Mms13-scFv was not functionally expressed on the magnetic bacterial particle, whereas the periplasmic expressed scFv-Fc was functionally expressed on the magnetic bacterial particle.
As used herein, scFv (scFv13R4) is a variant of anti-β-Galactosidase scFv (scFv13) that has been functionally modified to have high solubility and folding ability in the cytoplasm of E. coli. However, even the highly soluble scFv13R4 did not express its function on magnetic bacterial particles by the conventional direct expression method.

Examples 2 and 3 described below correspond to the second aspect of the present invention.
Example 2 Co-localization of GFP and mCherry on Magnetic Bacteria Particles by In Vivo Docking Method In this example, GFP and mCherry were used as a plurality of target proteins to be immobilized on BacMPs.
(2-1) Samples All reagents used were commercially available special grade products for research or those based thereon, and the preparation of reagents etc. used distilled water and pure water obtained by treating distilled water with MilliQ Lab (Nippon Millipore). Alkaline phosphatase (ALP) -labeled mouse-derived anti-c-myc monoclonal antibody was purchased from ACRIS. Mouse-derived anti-GFP monoclonal antibody and mouse-derived anti-mCherry monoclonal antibody were purchased from Clontech Laboratories. Goat-derived ALP-labeled anti-mouse IgG antibody was purchased from Santa Cruz Biotechnology. Recombinant GFP and mCherry were purchased from CELL BIOLABS. SH-9000 (Corona Electric) was used as a plate reader. Lumiphos 530 manufactured by Wako Pure Chemical Industries, Ltd. was used as a light emission substrate of ALP.
Also, in this example, as the expression host of the fusion protein, the magnetic bacteria M. coli were used. The mms13 gene deficient strain (Δmms13 strain) of magneticum AMB-1 strain was used. E. coli TOP10 (Invitrogen) was used for plasmid construction.

The plasmid vector is T. Yoshino, A. et al. Shimojo, Y. Maeda, T .; Matsunaga. , Inducible Expression of Transmembrane Protein on Bacterial Magnetic Particles in Mamnetospirillum magneticum AMB-1, Appl Environ Microbiol. It was constructed based on pUMtOR13GFP described in (2010).

(2-2) Construction of plasmid and culture of magnetic bacteria FIG. 11 (A) shows the plasmid construct pUMtetDoc-M13miniscaf constructed in this example. The plasmid is a fusion protein (GFP-DocC) of GFP and Clostridium thermocellum 's CelS derived Dockerin (DocC) downstream of the Pmsp1 (tetO) promoter, and a fusion protein (mCherry-DocR) of mCherry and Ruminococcus flavefaciens ScaA derived Docker (DocR) ), And downstream of the Pmms16 promoter Mms13, NS linker, C.I. thermocellum from Cohesin (CohC), R. It contains the gene of flavesfaciens-derived Cohesin (CohR), a fusion protein of c-myc tag (Mms13-miniscaffoldin).
Furthermore, pUMtetDoc (not shown) containing GFP-DocC and mCherry-DocR genes downstream of Pmms16 promoter was constructed.

  The Δmms13 strain was transformed by electroporation using each of the prepared plasmids. For the culture of magnetic bacteria, magnetic spirillum growth medium (MSGM) was used as a culture medium, and preculture was inoculated 1/1000 in 5 L of culture medium, and culture was carried out by standing at room temperature of 26 to 29 ° C. At the time of inoculation, microaerobicity was made by bubbling with argon gas (15 minutes). In addition, when culturing the transformant, a final concentration of 5.0 μg / ml ampicillin was added, and a final concentration of 500 ng / ml ATc was added in the middle of the logarithmic growth phase, followed by overnight culture. Recovery of BacMPs was performed by the method described below.

(2-3) Preparation of Magnetic Bacteria Particles After bacterial cell culture, centrifugation (9000 g, 4 ° C., 10 minutes) was performed to recover magnetic bacteria from the culture solution. The collected magnetic bacteria were suspended in HEPES buffer solution (10 mM, pH 7.4) containing 1 mM CaCl ₂ and disrupted at 1800 kg / cm ² using a French press (Otake Corporation, 5501 M) (3 times). BacMPs were recovered by transferring the disrupted solution to a glass container and attaching a Nd-Fe-B (neodymium-iron-boron) magnet. The supernatant was removed, HEPES buffer containing 1 mM CaCl ₂ was further added, and magnetic separation was performed to wash BacMPs (10 times). BacMPs was dispersed in an appropriate amount of PBS, the absorbance (660 nm) was measured, and the dry weight was converted from the calibration curve.

(2-4) Western Blotting and Antibody Binding Test on BacMPs A cell culture solution of wild type (WT) and each transformant was centrifuged (8000 g, 10 minutes) to recover the cells. To 2 × 10 ⁹ cells, 20 μl of a 1% SDS solution was added and boiled at 99 ° C. for 30 minutes. Furthermore, 3 × SDS sample buffer was added and heated at 99 ° C. for 5 minutes to prepare a cell sample. Further, 30 μl of a 1% SDS solution was added to 25 to 500 μg of BacMPs obtained from WT and each transformant, and the mixture was boiled while being diffused by ultrasonic waves (100 ° C., 30 minutes). The supernatant was collected by centrifugation (18000 g, 5 minutes), 15 μl of 3 × SDS sample buffer was added to the sample, and the mixture was heated at 99 ° C. for 5 minutes to obtain a particle membrane fraction sample. As controls, recombinant GFP and recombinant mCherry were used.

  Each sample was subjected to SDS-PAGE using a gel with acrylamide concentration 15%. The gel after electrophoresis was subjected to semi-dry blotting on a PVDF membrane. ALP-labeled anti-c-myc antibody (1 μg / ml, PBST) or mouse-derived anti-GFP monoclonal antibody (0.05 μg / ml, PBST), or mouse-derived anti-mCherry monoclonal antibody to the PVDF membrane after blotting (2 μg / ml, PBST), the secondary antibody was reacted with Goat-derived ALP-labeled mouse IgG antibody (0.5 μg / ml, PBST) (room temperature, 1 hour). After washing for 10 minutes three times with PBS-T, NBT / BCIP-Blue Liquid Substrate was added as a substrate to cause color development. In addition, pixel intensity was calculated using image analysis software Image J for analysis.

Next, antibody binding tests on BacMPs were performed.
ALP-labeled anti-c-myc antibody (1 μg / ml, PBST), mouse-derived anti-GFP monoclonal antibody (1 μg / ml, PBST) or mouse-derived anti-mCherry monoclonal antibody (1 μg / ml, PBST) in WT and transformant-derived BacMPs 50 μg 100 μl was reacted for 30 minutes at room temperature. After washing, BacMPs reacted with mouse-derived anti-GFP monoclonal antibody or mouse-derived anti-mCherry monoclonal antibody were further reacted with goat-derived ALP-labeled anti-mouse IgG antibody (1 μg / ml) as a secondary antibody. After washing, BacMPs were suspended in 50 μl of TBS. After transferring to a 96-well plate, 50 μl of lumiphos 530 was added, and the luminescence intensity after 5 minutes was measured.

(2-4-1) Confirmation of localization of Mms13-miniscaffoldin in transformants Expression of Mms13-miniscaffoldin (57 kDa) in transformants using an antibody against c-myc tag fused to the C-terminus of Mms13-miniscaffoldin Did. As a result of western blotting, the expression of Mms13-miniscaffoldin could be confirmed in the transformant carrying pUMtetDoc-M13 miniscaf (FIG. 12 (A), lane 1).
Moreover, the localization of Mms13-miniscaffoldin on purified BacMPs was confirmed by the antibody binding test to BacMPs described above. As a result, binding of anti-c-myc antibody was confirmed in BacMPs purified from a transformant carrying pUMtetDoc-M13 miniscaf (FIG. 12 (B), lane 1).
From the above results, it was shown that Mms13-miniscaffoldin expressed in transformants was localized and immobilized on BacMPs membrane.

(2-4-2) Expression of GFP-DocC and mCherry-DocR in Transformant and Confirmation of Binding on BacMPs In order to confirm expression of GFP-DocC and mCherry-DocR in transformant, GFP by Western blotting -Detection of DocC (36 kDa) and mCherry-DocR (38 kDa) was performed. As a result, in the transformant, the expression of GFP-DocC and mCherry-DocR could be confirmed (Fig. 13 (A) and (B), lane 1).
Moreover, the band of GFP-DocC and mCherry-DocR of each transformant was analyzed using image analysis software Image J, and the comparison of pixel intensity was performed. When the pixel intensity of GFP-DocC and mCherry-DocR of the transformant carrying pUMtetDoc-M13 miniscaf is 100%, in the transformant carrying pUMtetDoc, GFP-DocC is 18% and mCherry-DocR is 69% there were. Thus, it was shown that in the transformant carrying pUMtetDoc, the expression levels of GFP-DocC and mCherry-DocR were reduced as compared to the transformant carrying pUMtetDoc-M13 miniscaf. Dockerin is reported to be structurally unstable in the absence of binding to Cohesin and to be degraded by proteases in cells. Therefore, in the transformant carrying pUMtetDoc not expressing Mms13-miniscaffoldin, it is considered that GFP-DocC and mCherry-DocR were degraded and the expression amount was reduced.

In addition, as a result of antibody binding test on BacMPs using GFP antibody and anti-mCherry antibody, binding of anti-GFP antibody and anti-mCherry antibody was confirmed in BacMPs purified from a transformant carrying pUMtetDoc-M13miniscaf ( Fig. 14 (A) and (B), lane 1). Therefore, it was shown that GFP-DocC and mCherry-DocR which were expressed in the transformant were bound on BacMPs in which Mms13-miniscaffoldin was localized.
From these results, it was shown that two types of proteins can be immobilized on BacMPs in one step by co-expressing Mms13-miniscaffoldin as a scaffold, GFP-DocC, and mCherry-DocR.

(2-4-3) Confirmation of Co-localization of GFP-DocC and mCherry-DocR on BacMPs The scaffold Mms13-miniscaffoldin contains CohC and CohR each binding to DocC and DocR, respectively. Therefore, when GFP-DocC and mCherry-DocR co-localize on Mms13-miniscaffoldin, the binding amount ratio of GFP-DocC and mCherry-DocR is 1: 1.
In order to calculate the immobilized amount of GFP-DocC and mCherry-DocR bound on BacMPs, the amount of GFP-DocC and mCherry-DocR contained in the membrane fraction of purified BacMPs was quantified by Western blotting. Standard curves were prepared using known concentrations of GFP and mCherry, and analysis with Image J was performed (FIG. 15).
As a result, the binding amount of GFP-DocC and mCherry-DocR was 380 ng and 570 ng per 1 mg BacMPs, respectively. Therefore, it was shown that comparable GFP-DocC and mCherry-DocR were immobilized on BacMPs. The reason why the amount of binding of GFP-DocC is slightly lower than the amount of binding of mCherry-DocR is considered to be because GFP-DocC is susceptible to degradation by protease.

(2-5) Fluorescence microscope observation of transformant The fluorescence microscope observation of GFP and mCherry of a transformant was performed, and the localization analysis in a cell was performed. Cultures of WT and transformants were dropped onto a slide glass coated with 2% agarose gel for observation. For detection of GFP fluorescence, a filter for GFP detection (excitation / detection: 466 nm: 525 nm) was used. A CY3 filter (excitation / detection: 513-556 nm / 570-613 nm) was used for detection of mCherry. For observation of FRET of GFP / mCherry, a FRET detection filter combined with an excitation filter (460-495 nm) and a detection filter (580 nm) was used.
As a result of the fluorescence observation, the fluorescence of mCherry was detected in the transformant carrying pUMtetDoc, but the fluorescence of GFP was not detected (FIG. 16 (B)). This is considered to be due to the low expression level of GFP-DocC. In the transformant carrying pUMtetDoc-M13 miniscaf, fluorescence of GFP and mCherry was detected (FIG. 16 (C)). In addition, their fluorescence showed that GFP and mCherry were localized at the same position in the cell.

Furthermore, by irradiating the excitation wavelength of GEP and detecting the fluorescence wavelength of mCherry, FRET (Fluorescence resonance energy transfer) between GFP and mCherry was observed. Here, FRET is a phenomenon in which, when two fluorescent molecules are positioned in close proximity (10 nm or less), the energy absorbed by one of the molecules is transferred to the other molecule to emit fluorescence.
As a result of observation, in the transformant carrying pUMtetDoc-M13miniscaf, fluorescence of mCherry by FRET was detected at the same position as the localization position of GFP and mCherry (lane of “FRET” in FIG. 16 (C)) . Therefore, it was shown that GFP-DocC and mCherry-DocR are located in proximity.
From these results, in the transformant carrying pUMtetDoc-M13 miniscaf, GFP-DocC and mCherry-DocR expressed in the cell were bound and co-localized to Mms13-miniscaffoldin localized on BacMPs. Indicated.

  In this Example, it was shown that co-localization on BacMPs can be achieved by co-expressing a fluorescent protein in which miniscafollldin having a cohesin domain and dockerin are fused in the magnetic bacterium AMB-1. In this method, there is no need to perform complicated operations for purification and immobilization of proteins, and a protein-magnetic particle complex can be produced very simply.

<Example 3> Functional expression of MHC by colocalization of MHC class II and chaperone-like protein HLA-DR on magnetic particles by in vitro docking method In this example, as a plurality of target proteins to be immobilized on BacMPs, eMHC II and HLA-DM were used.

(3-1) Samples The reagents used were all commercially available special grade products for research or those based thereon, and the preparation of reagents etc. used distilled water and pure water obtained by treating distilled water with MilliQ Lab (Nippon Millipore). Alkaline phosphatase (ALP) -labeled anti-FLAG monoclonal antibody was purchased from Sigma-Aldrich Japan. Alkaline phosphatase (ALP) -labeled anti-HA tag monoclonal antibody was purchased from abcam. N-terminal biotin-labeled HA _306-318 (PKYVKQNTLKLAT) was purchased from BEX Corporation. ALP labeled streptavidin was purchased from Roche. SH-9000 (Corona Electric) was used as a plate reader. Lumiphos 530 manufactured by Wako Pure Chemical Industries, Ltd. was used as a light emission substrate of ALP.
Also, in this example, as the expression host of the fusion protein, the magnetic bacteria M. coli were used. The mms13 gene deficient strain (Δmms13 strain) of magneticum AMB-1 strain was used. E. coli TOP10 (manufactured by Invitrogen) was used for plasmid construction.
The plasmid vector is T. Yoshino, A. et al. Shimojo, Y. Maeda, T .; Matsunaga. , Inducible Expression of Transmembrane Protein on Bacterial Magnetic Particles in Mamnetospirillum magneticum AMB-1, Appl Environ Microbiol. It was constructed based on pUMtOR13GFP described in (2010).

(3-2) Construction of plasmid and culture of magnetic bacteria FIG. 17 (A) shows the vector construct pUMMHC II-DMDoc-M13 miniscaf constructed in this example.
The vector is composed of an extracellular domain of MHC II downstream of the Pmsp1 (tetO) promoter, a FLAG tag, a fusion protein of CelS-derived Dockerin (DocC) from Clostridium thermocellum (MHC II-DocC), HLA-DM, HA-tag, Ruminococcus flavefaciens of ScaA from Dockerin (DocR) of the fusion protein (DM-DocR), and downstream of the Pmms16 promoter Mms13, NS linker, C. thermocellum from Cohesin (CohC), R. It contains the gene of flavesfaciens-derived Cohesin (CohR), a fusion protein of c-myc tag (Mms13-miniscaffoldin).
Furthermore, Mms 13, NS linker, C.I. A vector construct pUMMHC II-DMDoc-M13cohC (not shown) was constructed which contains the gene of thermocellum- derived Cohesin (CohC) and a fusion protein of c-myc tag (Mms13-cohC).

The Δmms13 strain was transformed by electroporation using each of the prepared plasmids.
In addition, for culture of magnetic bacteria, magnetic spirillum growth medium (MSGM) is used as a culture medium, and preculture is inoculated 1/1000 in 5 L of culture medium and left to stand at room temperature (26-29 ° C.) Did. At the time of inoculation, microaerobicity was made by bubbling with argon gas (15 minutes). In addition, when culturing the transformant, a final concentration of 5.0 μg / ml ampicillin was added, and a final concentration of 500 ng / ml ATc was added in the middle of the logarithmic growth phase, followed by overnight culture.

(3-3) Preparation of Magnetic Bacterial Particles Preparation of magnetic bacterial particles was performed by the same method as described in (2-3) of Example 2 above.

(3-4) Western Blotting and Antibody Binding Test on BacMPs A cell culture solution of WT and each transformant was centrifuged (8000 g, 10 minutes) to recover the cells. To 2 × 10 ⁹ cells, 20 μl of a 1% SDS solution was added and boiled at 99 ° C. for 30 minutes. Furthermore, 3 × SDS sample buffer was added and heated at 99 ° C. for 5 minutes to obtain a cell sample. After adding 30 μl of a 1% SDS solution to 200 μg of BacMPs obtained from WT and each transformant, the mixture was boiled while being diffused by ultrasonic waves (100 ° C., 30 minutes). The supernatant was recovered by centrifugation (18000 g, 5 minutes), and 15 μl of 3 × SDS sample buffer was added to the sample and heated at 99 ° C. for 5 minutes to obtain a particle membrane fraction sample.
Each sample was subjected to SDS-PAGE using a gel with an acrylamide concentration of 12.5%. The gel after electrophoresis was subjected to semi-dry blotting on a PVDF membrane. The blotted PVDF membrane was reacted with ALP-labeled anti-FLAG monoclonal antibody (1 μg / ml, PBST) or ALP-labeled anti-HA tag monoclonal antibody (1 μg / ml, PBST) (room temperature, 1 hour). After repeating washing for 10 minutes three times with PBS-T, NBT / BCIP-Blue Liquid Substate was added as a substrate to develop color.

Next, an antibody binding test was performed to confirm that the target proteins eMHC II-DocC and DM-DocR were bound on the transformant-derived BacMPs.
50 μg of WT and transformant-derived BacMPs were reacted with 100 μL of ALP-labeled anti-FLAG monoclonal antibody (1 μg / mL, PBST) and ALP-labeled anti-HA tag monoclonal antibody (1 μg / ml, PBST) at room temperature for 30 minutes. After washing, BacMPs were suspended in 50 μl of TBS. After transferring to a 96-well plate, 50 μl of lumiphos 530 was added, and the luminescence intensity after 5 minutes was measured.

(3-4-1) Confirmation of Expression of eMHC II-DocC and DM-DocR As shown in FIG. 17 (A), in the plasmid pUMMHC II-DMDoc-M13 miniscaf, the eMHC-DocC and DM-DocR are ribosome binding sites (RBS The operon is configured via). Moreover, pUMMHC II-DMDoc-M13cohC (not shown) also has the same configuration.
From the results of western blotting, the expression of eMHC-DocC and DM-DocR was confirmed in the transformant carrying pUMMHC II-DMDoc-M13 miniscaf and in the transformant carrying pUMMHC II-DMDoc-M13cohC. Here, detection was performed using an FLAG tag introduced into eMHC-DocC and an antibody against HA tag introduced into DM-DocR.

(3-4-2) Confirmation of Binding of eMHC II-DocC and DM-DocR on BacMPs An antibody is used to confirm that eMHC II-DocC and DM-DocR are bound on transformant-derived BacMPs. The binding test was done. In transformant-derived BacMPs carrying pUMMHC II-DMDoc-M13 miniscaf, it was possible to confirm the binding of eMHC II-DocC and DM-DocR. On the other hand, in transformant-derived BacMPs carrying pUMMHC I-DMDoc-M13cohC, binding of eMHC II-DocC could be confirmed.

(3-5) Peptide binding assay and evaluation of peptide binding ability of eMHC II-DocC A peptide binding assay was performed to evaluate peptide binding ability of eMHC II-DocC. Specifically, influenza hemagglutinin-derived antigenic peptide (HA _306-318 ) is used for eMHC II-DocC and DM-DocR binding BacMPs (eMHC II / DM-BacMPs) and eMHC II-DocC binding BacMPs (eMHC II-BacMPs). ) Was evaluated.
The peptide was adjusted to 5 mg / ml with DMSO and stored at -20 <0> C until use. Biotin-labeled HA _{306-318 (} 100 μl) adjusted to 0.1 to 10 μM (PBST) was added to 50 μg of BacMPs obtained from WT and each transformant, and reacted at 37 ° C. for 3 hours. Thereafter, the plate was washed three times with 100 μl PBS, 100 μL of ALP-labeled streptavidin (ALP-SA: 0.5 U / ml, PBST) was added, and allowed to react at room temperature for 30 minutes. After that, washing was performed twice using 100 μl of Tris-HCl buffer (pH 7.4), and suspended in 50 μl of TBS was used as a sample. After transferring to a 96-well microtiter plate, adding lumiphos 530 (50 μl) and reacting at room temperature for 5 minutes, luminescence intensity was measured using a luminescence plate reader.

As a result, binding of the antigen peptide was confirmed in eMHC II / DM-BacMPs, while the antigen peptide did not bind to eMHC II-BacMPs in which DM-DocR is not colocalized.
Furthermore, the _kinetics of HA _306-318 concentration and reaction time were analyzed.

In this example, co-localization of eMHC II and the chaperone HLA-DM in magnetic bacteria succeeded in preparation of eMHC II, which was conventionally difficult to produce.
As mentioned above, MHC II molecules are important proteins responsible for human immune system, and bind to degradation products (antigen peptides) of antigenic proteins derived from various diseases and pathogens, and thus they can be used as antigenic peptide-MHC II complex ( Form pMHC II). Therefore, analysis of peptide binding ability of MHC II and identification of antigenic peptide are important.
MHC II (eMHC II) not bound to antigenic peptides is structurally unstable and forms aggregates. Therefore, recombinant MHC II is produced, for example, by separately producing an α chain and a β chain as inclusion bodies and then mixing with an antigen peptide and refolding, and this method requires complicated operations. . In addition, it has been reported that addition of HLA-DM to pMHC II into which a UV-degradable antigenic peptide has been introduced, and irradiation of UV produces eMHC II with peptide binding ability, but in this system High concentrations of HLA-DM are required to generate binding-capable eMHC II. That is, conventionally, in order to evaluate the peptide-binding ability of MHC II with a target antigenic peptide candidate, a complicated experimental step was required or a very high concentration of sample was required. In addition, sufficient stability could not be obtained unless pMHC II was produced using a high affinity antigen peptide. Such complexity and difficulty of producing recombinant MHC II has been a bottleneck for MHC II research.
However, in this example, co-localization of eMHC II and the chaperone HLA-DM in magnetic bacteria enabled simple and efficient maintenance of the function of eMHC II which was conventionally difficult to produce. It was possible to prepare in the state. In this example, eMHC II and HLA-DM are coexpressed in the limited space of the bacterial cell of magnetic bacteria, and co-localized via a scaffold protein to eMHC II and HLA-DM. Can be placed in close proximity, which makes it possible to efficiently produce an eMHC II that retains peptide binding ability. Furthermore, since eMHC II and HLA-DM are immobilized on BacMps, they can be easily recovered using an external magnetic field, so that simplification and application of antigen peptide screening techniques can be expected.

Claims (7)

A method for producing a complex of a disulfide bond protein and a magnetic particle in a gram negative magnetic bacterium,
(I) in the bacteria,
(A) a first fusion protein comprising a disulfide linked protein fused to a binding protein, and (b) a second fusion protein comprising a binding partner protein fused to a protein capable of being bound or integrated into a magnetic bacterial particle A co-expression step of co-expression, wherein expression of said first fusion protein is induced to the periplasm of said bacteria and expression of said second fusion protein is induced to the cytoplasm of said bacteria;
(Ii) culturing the bacteria of step (i) under conditions sufficient to produce said first and second fusion proteins;
(Iii) a cell membrane disruption step of disrupting the bacterial cell membrane of the step (ii), wherein a complex of the disulfide bond protein and the magnetic bacterial particle is formed;
Including the manufacturing method.   The production method according to claim 1, further comprising: (iv) isolating the complex of step (iii) from the disrupted cells.   The production method according to claim 1, further comprising a recovery step of recovering the disulfide bond protein from the complex.   The method according to any one of claims 1 to 3, wherein the binding protein is an immunoglobulin Fc domain. The method according to any one of claims 1 to 4, wherein the binding partner protein is a ZZ domain of protein A derived from Staphylococcus aureus .   The method according to any one of claims 1 to 5, wherein the disulfide-linked protein is a single chain variable fragment (scFv). The method according to any one of claims 1 to 6, wherein the protein capable of being bound or integrated to the magnetic bacterial particle is Mms13 derived from Magnetospillum magneticum .

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