# Patent

## Title
Artificial bioparticle and method of manufacturing the same

## Abstract

According to an artificial bioparticle characterized in that a leucine zipper is integrated in each N terminal of an MVP constituting a waist of a vault and a method of manufacturing an artificial bioparticle in which a leucine zipper gene is integrated and expressed in a side to be an N terminal of an MVP gene, a novel artificial bioparticle including a vault of which large internal space can effectively be made use of, which can be used as a nanocapsule applicable to a drug delivery system (DDS), and a method of manufacturing the same are provided.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This patent application is the U.S. national phase of International Patent Application No. PCT/JP2013/080219, filed Nov. 8, 2013, which claims the benefit of Japanese Patent Application No. 2012-253031, filed on Nov. 19, 2012, which are incorporated by reference in their entireties herein.
INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: 42,877 bytes ASCII (Text) file named “720653ReplacementSequenceListing.txt,” created Aug. 25, 2015.
TECHNICAL FIELD
The present invention relates to a novel artificial bioparticle including a vault, which can be used as a nanocapsule applicable to a drug delivery system (DDS), and a method of manufacturing the same.
BACKGROUND ART
A vault 2 is a huge ovoid bioparticle having a particle size of 40 nm×40 nm×67 nm, and it is a nucleic acid-protein complex having a largest molecular weight within a cell (see FIG. 2). Vault 2 present in an organism is constituted of three types of proteins (major vault protein (MVP), vault poly(ADP-ribose)polymerase (VPARP), and telomerase-associated protein-1 (TEP1)) and one type of RNA. Vault 2 is such that 39 MVPs 3, which are main components and have a molecular weight of approximately 100 kDa, gather to form a half vault in a shape of a bowl (each site being referred to as a cap 5, a shoulder 6, a body 7, and a waist 8) and two halves are associated at waist 8 as if edges of the bowls were coupled, to thereby form an outer shell of the ovoid particle. Components other than the MVP are present in an internal space formed by the outer shell.
MVP 3 forming the outer shell of vault 2 is constituted of 12 domains in total including 9 repeating structures (3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i) formed from antiparallel β sheets and shoulder 6, a cap helix 9, and a cap ring 10 (FIG. 3), and intermolecular hydrophobic bond between domains of cap helix 9 is important for formation of a half vault in a bowl shape. Two half vaults form the ovoid vault particle by associating N terminals of MVPs 3, and such association is formed only based on very weak interaction of ionic bond and a short intermolecular β sheet. Such structural information of the vault and a mechanism of formation of a particle have been clarified as the present inventor succeeded in overall structure determination of a rat liver derived vault in 2009 (see, for example, Hideaki Tanaka et al., “The Structure of Rat Liver Vault at 3.5 Angstrom Resolution,” Science, Vol. 323, pp. 384-388 (2009) (NPD 1)).
It has previously been known that as an MVP which is a main component of a vault is expressed in an insect cell, an ovoid particle the same as in an organism is formed (see, for example, Andrew G. Stephen et al., “Assembly of Vault-like Particles in Insect Cells Expressing Only the Major Vault Protein,” The Journal of Biological Chemistry, Vol. 276, No. 26, pp. 23217-23220 (2001) (NPD 2)). Owing to a characteristic shape of a vault, development of a drug delivery system (DDS) by using the vault as a nanocapsule has progressed (see, for example, Valerie A. Kickhoefer et al., “Engineering of vault nanocapsules with enzymatic and fluorescent properties,” PNAS, Vol. 102, No. 12, pp. 4348-4352 (2005) (NPD 3) and Valerie A. Kickhoefer et al., “Targeting Vault Nanoparticles to Specific Cell Surface Receptors,” ACS nano, 3 (1): 27-36.doi: 10.1021/nn800638x(2009) (NPD 4)).
For example, Japanese National Patent Publication No. 2013-509202 (PTD 1) has disclosed use of a vault particle which is a recombinant particle having an MVP as well as a fusion protein and mINT and a protein of interest (cytokine) for delivery of the protein of interest to a cell or a tumor, or a target. In addition, for example, Japanese National Patent Publication No. 2007-508846 (PTD 2) has disclosed a technique directed to a composition for delivering a polynucleotide packaged by a polypeptide, having a leucine zipper as a polynucleotide-bound domain.
In a conventional method, for taking a drug into a particle, a C terminal 160 residue (an INT domain: bonding to an MVP) of a VPARP which is a constituent component of a vault and present in a particle is used as a tag. Such use is also for having the particle retain the drug therein. This method, however, has not yet successfully made full use of a large internal space in the vault.
CITATION LIST
Patent Document


    PTD 1: Japanese National Patent Publication No. 2013-509202
    PTD 2: Japanese National Patent Publication No. 2007-508846


Non Patent Document


    NPD 1: Hideaki Tanaka et al., “The Structure of Rat Liver Vault at 3.5 Angstrom Resolution,” Science, Vol. 323, pp. 384-388 (2009)
    NPD 2: Andrew G. Stephen et al., “Assembly of Vault-like Particles in Insect Cells Expressing Only the Major Vault Protein,” The Journal of Biological Chemistry, Vol. 276, No. 26, pp. 23217-23220 (2001)
    NPD 3: Valerie A. Kickhoefer et al., “Engineering of vault nanocapsules with enzymatic and fluorescent properties,” PNAS, Vol. 102, No. 12, pp. 4348-4352 (2005)
    NPD 4: Valerie A. Kickhoefer et al., “Targeting Vault Nanoparticles to Specific Cell Surface Receptors,” ACS nano, 3 (1): 27-36.doi: 10.1021/nn800638x(2009)


SUMMARY OF INVENTION
Technical Problem
The present invention was made to solve the problems above, and an object thereof is to provide a novel artificial bioparticle including a vault of which large internal space can effectively be made use of, which can be used as a nanocapsule applicable to a drug delivery system (DDS), and a method of manufacturing the same.
Solution to Problem
An artificial bioparticle according to the present invention is characterized in that a leucine zipper is integrated in each N terminal of an MVP constituting a waist of a vault.
In the artificial bioparticle according to the present invention, preferably, a linker is interposed between the N terminal of the MVP and the leucine zipper. In this case, preferably, the linker includes 3 to 6 glycines.
In the artificial bioparticle according to the present invention, preferably, the leucine zipper is derived from GCN4 which is a transcription activator factor of yeast.
The present invention also provides a method of manufacturing the artificial bioparticle according to the present invention described above, which includes integrating and expressing a leucine zipper gene in a side of an MVP gene, which is to be an N terminal.
In the method of manufacturing the artificial bioparticle according to the present invention, the MVP gene and the leucine zipper gene are coupled by a gene encoding a linker, without a restriction enzyme site being interposed.
Advantageous Effects of Invention
According to the method of manufacturing an artificial bioparticle in the present invention, an artificial bioparticle can be obtained at yields remarkably higher than in a conventional example. The artificial bioparticle according to the present invention is expected as a nanocapsule which can be used for a DDS and of which internal space can effectively be made use of, and according to the present invention, yields are higher by an order of magnitude, which leads to significant reduction in cost. Based on the artificial bioparticle according to the present invention, progress in development of a particle of which opening and closing can reversibly be controlled depending on pH can be expected.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a schematic diagram conceptually showing bond between an N terminal 3a of an MVP 3 constituting a vault 2 and a leucine zipper 4 in an artificial bioparticle 1 according to the present invention.
FIG. 2 is a diagram schematically showing vault 2 used for artificial bioparticle 1 according to the present invention.
FIG. 3 is a diagram schematically showing MVP 3 constituting vault 2.
FIG. 4 is a diagram schematically showing application of artificial bioparticle 1 according to the present invention as a nanocapsule.
FIG. 5 is a diagram schematically showing one example of a drug delivery system in which artificial bioparticle 1 according to the present invention is applied as a nanocapsule.
FIG. 6 shows on the left, a photograph showing results of SDS-PAGE after disruption and showings a supernatant (a lane A) and a precipitate (a lane B) of LZMVP_Gly3 and a supernatant (a lane C) and a precipitate (a lane D) of LZMVP_Gly6, and shows on the right, a photograph showing results of SDS-PAGE after sucrose density gradient centrifugation, in which a group a on the left shows results of LZMVP_Gly3 and a group b on the right shows results of LZMVP_Gly6.
FIG. 7 shows an electron micrograph showing a final purified preparation of LZMVP_Gly3.



DESCRIPTION OF EMBODIMENTS
FIG. 1 is a schematic diagram conceptually showing bond between an N terminal 3a of MVP 3 constituting vault 2 and leucine zipper 4 in an artificial bioparticle 1 according to the present invention. FIG. 2 is a diagram schematically showing vault 2 used for artificial bioparticle 1 according to the present invention and FIG. 3 is a diagram schematically showing MVP 3 constituting vault 2. As described above, vault 2 is such that 39 MVPs 3 representing main components gather to form a half vault in a shape of a bowl (each site being referred to as cap 5, shoulder 6, body 7, and waist 8) and two halves are associated at waist 8 as if edges of the bowls were coupled, to thereby form an outer shell of the ovoid particle (FIG. 2). MVP 3 forming the outer shell of vault 2 is constituted of 12 domains in total including 9 repeating structures (3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i) formed from antiparallel β sheets and shoulder 6, cap helix 9, and cap ring 10 (FIG. 3). Artificial bioparticle 1 according to the present invention is characterized in that leucine zipper 4 is integrated in each N terminal of MVP 3 constituting waist 8 of vault 2.
In artificial bioparticle 1 according to the present invention, a linker is preferably interposed between the N terminal of MVP 3 and leucine zipper 4. As such a linker is interposed, a degree of freedom of movement of leucine zipper 4 in artificial bioparticle 1 is ensured.
The linker between the N terminal of MVP 3 and leucine zipper 4 in artificial bioparticle 1 according to the present invention is preferably formed from 1 to 6 small amino acid(s) aligned in a straight chain. Though artificial bioparticle 1 having leucine zipper 4 is expressed in any case of 1 to 6 amino acids forming the linker, from a point of view of uniformity in obtained artificial bioparticles 1 and a greater amount of expression and from a point of view of a degree of freedom of movement of leucine zipper 4, the linker is preferably formed from 3 to 6 small amino acids as aligned in a straight chain. The amino acid constituting the linker is exemplified by an amino acid having a small side chain such as glycine or alanine. In a present experimental example which will be described later, an example in which a linker is formed from 3 or 6 straight-chain glycines is shown.
Leucine zipper 4 is a motif like a zipper formed in such a manner that two α-helices each composed of approximately 30 amino acid residues establish hydrophobic bond at leucine residues thereof. For leucine zipper 4 in the present invention, for example, a leucine zipper derived from GCN4 which is a transcription activator factor of yeast or other leucine zippers derived from other proteins can be used without particularly being restricted. Among those, an X-ray crystal structure of a leucine zipper derived from GCN4 which is a transcription activator factor of yeast was determined in 1991 at 1.8 Å resolution, and it was clarified on an atomic level that peptides each constituted of 33 amino acid residues establish hydrophobic bond at leucine residues thereof to thereby form a strong coiled coil. Therefore, a leucine zipper derived from GCN4 which is a transcription activator factor of yeast can suitably be employed.
Such an artificial bioparticle according to the present invention is expected as a nanocapsule of which internal space can effectively be made use of and which can be made use of for a DDS, and according to the present invention, yields are higher by an order of magnitude, which leads to significant reduction in cost. Based on the artificial bioparticle according to the present invention, progress of development of a particle of which opening and closing can reversibly be controlled depending on pH can be expected.
Here, FIG. 4 is a diagram schematically showing application of artificial bioparticle 1 according to the present invention as a nanocapsule and FIG. 5 is a diagram schematically showing one example of a DDS in which artificial bioparticle 1 according to the present invention is applied as a nanocapsule. According to artificial bioparticle 1 in the present invention, for example, by introducing a cysteine residue in a leucine zipper attached to an N terminal of an MVP, as shown in FIG. 4, a nanocapsule of which opening and closing can reversibly be controlled depending on pH in such a manner that a stable ovoid particle is formed based on disulfide bond under a neutral condition and the particle opens as if an egg were just split into two as a result of cleavage of disulfide bond under an acid condition can be developed. By using such a nanocapsule, for example, as shown in FIG. 5, such a DDS is also possible that a drug 11 is placed in an internal space of vault 2 under the acid condition followed by administration so that the nanocapsule is taken into a target cell 13 as a result of an antigen-antibody reaction between a specific antigen 12 on a surface of target cell 13 and an antibody (Fab) attached in advance to the surface of vault 2, the nanocapsule opens as an acid condition is established in endosome 14, and drug 11 is released. Such an artificial bioparticle according to the present invention has a very large space therein, and hence it is considered to be viable as a carrier in a gene therapy.
In addition, use of artificial bioparticle 1 according to the present invention as a nanocapsule in which cosmetic components are confined for permeation deep into skin is also possible.
Recently, such a technique has also been established that an internal space of a protein complex is used as a template, a metal is polymerized, and the polymerized product is regularly sequenced so that a substrate serving as a base for an extremely small semiconductor. Though a spherical protein such as ferritin is currently used, by using an oval particle like vault 2 by making use of artificial bioparticle 1 according to the present invention, a novel, unprecedented substrate may be made.
The present invention also provides a method of manufacturing artificial bioparticle 1 characterized in that a leucine zipper gene is integrated and expressed in a side of an MVP gene, which is to be an N terminal. Thus, as will be described later in the experimental example, artificial bioparticle 1 according to the present invention can be obtained at yields significantly higher than, or at least 10 times as high as, in a conventional expression system in which an insect cell having a W-vault formed of wild type MVPs (W-MVP) is employed. Base sequences of the leucine zipper gene and the MVP gene have already been known, and by combining as appropriate conventionally known genetic engineering techniques, a leucine zipper gene can be integrated in a side of an MVP gene which is to be an N terminal. In the experimental example which will be described later, an example in which a fragment of a leucine zipper gene cut from GCN4 which is a transcription activator factor of yeast and purified and a fragment of a rat derived MVP gene similarly cut and purified are ligated (through a linker which will be described later) is shown.
Though a cell, in which a product obtained by integrating a leucine zipper gene in a side of an MVP gene which is to be an N terminal is expressed, is not particularly restricted, an insect cell or a cell of higher forms of life than an insect cell is exemplified. It has been known that a bioparticle is not successfully formed in some cases, for example, when Escherichia coli lower than an insect cell is used, and expression by using an insect cell normally used in the field of the art is preferred. SD is exemplified as a specific example of an insect cell. An expressed artificial bioparticle can be purified with a conventionally known appropriate method.
In the method of manufacturing artificial bioparticle 1 according to the present invention, the MVP gene and the leucine zipper gene are coupled by a gene encoding a linker, without a restriction enzyme site being interposed. When a fragment of the leucine zipper gene derived from GCN4 which is a transcription activator factor of yeast and the rat derived MVP gene described above are used, in an attempt for coupling, a site of Ecoki (GAATTC) remains therebetween, which may inhibit formation of a vault particle. In the experimental example which will be described later, an Ecoki site was substituted with a Gly linker which was a series of 3 residues or 6 residues of glycines which were amino acids having a smallest side chain, and thereafter expression in an insect cell was carried out. Specifically, a primer is designed in accordance with a linker to be introduced, and after PCR, the purified fragment is desirably ligated such that it is interposed between an N terminal of an MVP gene and a leucine zipper gene.
In the method of manufacturing artificial bioparticle 1 according to the present invention as well, what is preferred as a leucine zipper or a linker is as described above with regard to artificial bioparticle 1.
Though the present invention will be described in further detail with reference to an experimental example, the present invention is not limited thereto.
EXPERIMENTAL EXAMPLE
[1] Construction of Abundant Expression System of Wild-Type MVP (W-MVP) Using Insect Cell Sf9
[A] Preparation of W-MVP Cloned Recombinant Baculovirus Genome (Bacmid DNA)
An operation was performed in a procedure below.
(1) A DNA of a rat liver derived MVP (W-MVP) was introduced into a pFastBac vector by using a restriction enzyme site of EcoRI and SphI.
(2) Obtained pFastBac was transformed to Escherichia coli (DH5α), and the resultant product was placed on an LB plate containing Ampicillin (100 μg/mL) and subjected to standing culture at 37° C. for 24 hours.
(3) Several colonies were picked up with a platinum loop and whether or not a target gene was amplified was observed with colony PCR.
(4) The colony in which amplification of the gene was observed in (3) above was inoculated in 5 mL of an LB liquid culture medium containing Ampicillin (100 μg/mL), and subjected to shake culture at 37° C. overnight.
(5) W-MVP cloned pFastBac was purified from Escherichia coli (DH5α) cultured overnight, with QIAprep Spin Miniprep Kit of QIAGEN.
(6) To twenty microliters of DH10Bac, 0.1 μg of W-MVP cloned pFastBac was added and lightly mixed, and thereafter the mixture was rested on ice for 20 minutes.
(7) Heat shock at 42° C. for 1 minute was provided, followed by resting on ice for 2 minutes. Thereafter, 200 μL of an SOC culture medium was added, and the resultant product was subjected to shake culture at 37° C. for 4 hours.
(8) Twenty microliters of the culture solution in (7) above were poured over an LB plate containing kanamycin (50 μg/mL), gentamicin (7 μg/mL), tetracycline (10 μg/mL), X-gal (100 μg/mL), and IPTG (50 μg/mL), and subjected to standing culture at 37° C. for 24 hours (until presence or absence of coloring of the colony (either blue or white) was determined).
(9) A white colony was picked up with a platinum loop and inoculated on a new LB plate (similar to the above) and subjected to standing culture at 37° C. overnight. Thereafter, coloring was again checked.
(10) Escherichia coli (DH5α) derived from the white colony checked again in (9) above was inoculated on a 5 mL of an LB liquid culture medium containing kanamycin (50 μg/mL), gentamicin (7 μg/mL), and tetracycline (10 μg/mL), and subjected to shake culture at 37° C. overnight.
(11) W-MVP cloned Bacmid was purified from Escherichia coli (DH10Bac) cultured overnight, with QIAprep Spin Miniprep Kit of QIAGEN. Since Bacmid has a very large size, in purification, an elution buffer heated to 70° C. was used.
[B] Multiplication of W-MVP Cloned Recombinant Baculovirus (Production of Virus Solution)
A procedure below was performed in a safety cabinet.
(1) A concentration of 519 cells (1.5-2.0×106 cells/mL) being cultured in an Sf900-II culture medium (containing 10% serum) of Invitrogen was prepared to 1.2×105 cells/mL by being diluted with an Sf900-II culture medium (containing 10% serum) in a 15-mL tube.
(2) In a 12-well culture plate, 1 mL of a cell culture solution prepared to a final concentration of 0.4×105 cells/mL was introduced. Three hundred microliters of the culture solution at 1.2×105 cells/mL described above and 700 μL of the Sf900-II culture medium (containing 10% serum) were added to obtain a volume of 1 mL in total.
(3) The cells were attached to a bottom surface of the cell culture plate by resting them at 27° C. for 20 minutes.
(4) In a 1.5-mL tube, 214 μL of Grace medium unsupplement, 8 μL of Cellfectin, and 1 μg of Bacmid were mixed and left at a room temperature for 30 minutes in the safety cabinet (Cellfectin was used after being stirred for approximately 10 seconds with a vortex mixer).
(5) The 12-well culture plate in (3) above was observed with an inverted microscope and attachment of the cells onto the bottom surface of a container was confirmed. Then, the culture medium was removed (suctioned with a pipetman while the plate was inclined toward the back and the lid was placed as standing in the front).
(6) After the culture medium was removed, the cells were washed with 1 mL of Grace medium unsupplement. A cleaning solution was discarded.
(7) Thereafter, a solution mixture of Bacmid and Cellfectin described above was poured over the cells.
(8) The 12-well plate was placed in a sealed Tupperware® and was rested at 27° C. for 4 hours. In order to maintain a humidity in the Tupperware®, several sheets of KimwipeTM wetted with pure water and 1 mL of 0.5M EDTA were placed in a corner of the Tupperware®.
(9) After 4 hours, 400 μL of Grace medium unsupplement (containing 10% serum) was layered and cultured at 27° C. for two days.
(10) After culturing for two days, the culture solution was transferred to a 1.5-mL tube, and the cells were precipitated by high-speed centrifugation (4,000×g, 3 min.). Since a supernatant was a virus solution (P0), the supernatant was transferred to a new 1.5-mL tube. The virus solution (P0) was stored in a chromatography chamber at 4° C. having a light-shielding film put thereon.
(11) A culture solution was added to a culture flask having an area of base of 25 cm2 such that the number of cells was 3×106 cells (the volume of the solution was 5 mL in total). For example, in a case that the number of Sf9 cells being cultured in the Sf900-II culture medium (containing 10% serum) was 1×106 cells/mL, a solution of 5 mL which was obtained by adding 2 mL of the 51900-II culture medium (containing 10% serum) to 3 mL of the culture solution was introduced in the culture flask.
(12) The cells were attached to the bottom surface of the cell culture flask by resting them at 27° C. for 15 minutes.
(13) Two hundred microliters of the P0 virus solution were added and cultured for three days.
(14) After culturing for three days and before collection of a virus solution (P1), a culture solution was added to a culture flask having an area of base of 75 cm2 such that the number of cells was 9×106 cells (the volume of the solution was 15 mL in total). By preparing 15 mL of the culture solution at 6×105 cells/mL, the total number of cells was 9×106 cells.
(15) The cells were attached to the bottom surface of the cell culture flask by resting them at 27° C. for 15 minutes.
(16) Before collection of the virus solution (P1), 0.5 mL of a supernatant was taken out of the culture flask having an area of base of 25 cm2 described above with the use of a pipet, and added to the culture solution in (14) above (for prevention of contamination). Thereafter, the culture solution was cultured at 27° C. for three days.
(17) While the remaining culture solution was suctioned and discharged by the pipet, the cells attached to the bottom surface of the flask were removed and transferred to a 15-mL tube and precipitated by high-speed centrifugation (4,000×g, 3 min.). Since the supernatant was the virus solution (P1), the supernatant was transferred to a new 15-mL tube. The Sf9 cells which fell as the precipitate were subjected to freeze preservation at −80° C. for checking expression. The virus solution (P1) was stored in a chromatography chamber at 4° C. having a light-shielding film put thereon.
(18) After culturing for three days in the culture flask having an area of base of 75 cm2, while the culture solution was suctioned and discharged by a pipet, the cells attached to the bottom surface of the flask were removed and transferred to a 50-mL tube and precipitated by high-speed centrifugation (4,000×g, 3 min.). Since a supernatant was a virus solution (P2), the supernatant was transferred to a new 50-mL tube. The Sf9 cells which fell as the precipitate were subjected to freeze preservation at −80° C. for checking expression. The virus solution (P2) was stored in a chromatography chamber at 4° C. having a light-shielding film put thereon.
(19) Thirty milliliters of a culture solution at 1×106 cells/mL were prepared in a 1-L spinner flask, 3 mL (a quantity equivalent to 1% of a culture medium) of the virus solution (P2) was added, and the resultant product was cultured at 27° C. for three days.
(20) After culturing for three days, the culture solution was transferred to a centrifugal tube and the cells were precipitated by high-speed centrifugation (4,000×g, 30 min.). Since a supernatant was a virus solution (P3), the supernatant was transferred to a new 500-mL medium bottle. The centrifugal tube, a lid of the centrifugal tube, and the medium bottle used here had been sterilized by autoclaving as being wrapped in an aluminum foil. The 519 cells which fell as the precipitate were subjected to freeze preservation at −80° C. for purification. The virus solution (P3) was stored in a chromatography chamber at 4° C. having a light-shielding film put thereon.
(21) Five hundred milliliters of the culture solution at 1×106 cells/mL were prepared in a 3-L spinner flask, 5 mL (a quantity equivalent to 1% of a culture medium) of the virus solution (P3) was added, and the resultant product was cultured at 27° C. for three days.
(22) After culturing for three days, the culture solution was transferred to a centrifugal tube and the cells were precipitated by high-speed centrifugation (4,000×g, 30 min.). Since a supernatant was a virus solution (P4), the supernatant was transferred to a new 500-mL medium bottle. The centrifugal tube, a lid of the centrifugal tube, and the medium bottle used here had been sterilized by autoclaving as being wrapped in an aluminum foil. The Sf9 cells which fell as the precipitate were subjected to freeze preservation at −80° C. for purification. The virus solution (P4) was stored in a chromatography chamber at 4° C. having a light-shielding film put thereon.
[2] Construction Using Insect Cells, of Abundant Expression System of MVP (LZ-MVP) Having Leucine Zipper Added to N Terminal
An operation was performed in a procedure below.
(1) A DNA (a leucine zipper gene) (containing restriction enzyme sites of BamHI and EcoRI) (SEQ ID NO: 2) (an amino acid sequence of the expressed leucine zipper shown in SEQ ID NO: 3) encoding 249th arginine to 281th arginine in an amino acid sequence (SEQ ID NO: 1) of yeast-derived GCN4 (281 amino acid) was introduced in a pGEM-T vector of Promega KK, and leucine zipper-cloned plasmid was purified with the use of QIAprep Spin Miniprep Kit of QIAGEN. A base sequence of the leucine zipper gene introduced in pFastBac (containing a restriction enzyme site (BamHI, EcoRI) and starting Met) is shown in SEQ ID NO: 4 and an amino acid sequence of the expressed leucine zipper is shown in SEQ ID NO: 5.
(2) The leucine zipper gene was cut from the pGEM-T vector by using restriction enzymes BamHI and EcoRI.
(3) Similarly, pFastBac cloned with a rat derived W-MVP was also treated with restriction enzymes BamHI and EcoRI.
(4) A fragment of the leucine zipper gene and a fragment of the MVP gene obtained in (2) and (3) above were subjected to agarose gel electrophoresis, a target band was cut, and a target product was purified from the gel with the use of QIAquick Gel Extraction Kit of Qiagen.
(5) The both fragments purified in (4) above were ligated with the use of DNA Ligation Kit (Mighty Mix) of Takara Bio Inc. (a base sequence after ligation is shown in SEQ ID NO: 6 and amino acid sequences of an expressed leucine zipper and an MVP are shown in SEQ ID NOs: 7 and 8).
(6) An operation the same as in preparation of a W-MVP cloned recombinant Baculovirus genome (the procedures (1) to (11)) and multiplication of the W-MVP cloned recombinant baculovirus (the procedures (1) to (20)) described above was performed to multiply LZ-MVP cloned recombinant baculoviruses, to prepare a virus solution (P4).
[3] Insertion of Glycine Linker Between Leucine Zipper of LZ-MVP and MVP
In the constructed expression system above, a site of EcoRI (GAATTC) remained between a leucine zipper gene and an MVP gene, and may inhibit formation of a vault particle. Therefore, the present inventor substituted these with a Gly linker which was a series of 3 residues of an amino acid (glycine (Gly)) having a smallest side chain (of which amino acid sequence is shown in SEQ ID NO: 9) or 6 residues (Gly6) (of which amino acid sequence is shown in SEQ ID NO: 10).
(1) The following three types of primers were made (a base sequence encoding Gly is gcc).









forward primer (lzmvp_0g_f)



(SEQ ID NO: 11)



atggcaactgaagaggccat



 



forward primer (lzmvp_3g_f)



(SEQ ID NO: 12)



ggcggcggcatggcaactgaagaggccatcatccgcatc



 



reverse primer (lzmvp_3g_r)



(SEQ ID NO: 13)



GCCAGATTAAAGAAATTAGTTGGCGAACGCggcggcggc






A complementary sequence of a reverse primer is as follows.









(SEQ ID NO: 14)



gccgccgccgcgttcgccaactaatttctttaatctggc






(2) When a linker formed of the Gly3 residues was inserted, lzmvp_0g_f and lzmvp_3_g_r were used as primers, and when a linker formed of the Gly6 residues was inserted, lzmvp_3g_f and lzmvp_3g_r were used as primers. With pFastBac of an earlier made LZ-MVP being used as a template, PCR (2 minutes at 94° C.→10 seconds at 98° C.→8 minutes at 68° C. (an underlined part being performed 10 cycles)) was carried out with the use of KOD-plus Mutagenesis kit manufactured by Toyobo Co., Ltd.
(3) After PCR, DpnI included in KOD-plus Mutagenesis kit manufactured by Toyobo Col, Ltd. was used to digest a template plasmid. In a 1.5-mL tube, 50 μL of a PCR product and 2 μL of DpnI were mixed, tapped and spun down, and thereafter incubated at 37° C. for 1 hour.
(4) In a 1.5-mL tube, 2 μL of a reaction solution in (3) above, 7 μL of sterilized water, 5 μL of Ligation high, and 1 μL of T4 polynucleotide kinase were added in this order, tapped and spun down, and thereafter incubated at 16° C. for 1 hour.
(5) Five microliters of the reaction solution in (4) above were added to 50 μL of Escherichia coli DH5α and rested on ice for 30 minutes.
(6) Heat shock was provided at 42° C. for 45 seconds.
(7) The solution was rested on ice for 2 minutes.
(8) Four hundred and fifty microliters of an SOC culture medium were added and subjected to shake culture at 37° C. for 1 hour.
(9) Fifty microliters of the culture solution in (8) above were poured over an LB plate containing Ampicillin (100 μg/mL).
(10) The plate was subjected to standing culture at 37° C. overnight.
Henceforth, an operation the same as in the procedures (2) to (20) for preparation of a W-MVP cloned recombinant baculovirus genome described above was performed to make a virus solution (P4) of LZMVP in which a Gly linker had been introduced between a leucine zipper and an MVP (LZMVP_Gly3, LZMVP_Gly6).
[4] Purification of Vault (W-Vault, LZ-Vault)
(1) In a 3-L spinner flask, 500 mL of Sf9 cells was cultured, and P4 or P3 virus solution was added for infection at the time point when the number of cells attained to 1×106 cells/mL, and cultured for three days.
(2) After culturing for three days, the culture solution was transferred to a centrifugal tube and the cells were precipitated by high-speed centrifugation (4,000×g, 30 min.).
(3) The precipitated cells were suspended in a PBS Buffer, and the suspension was transferred to a centrifugal tube and subjected to high-speed centrifugation (4,000×g, 30 min.). Thus, the cells were washed to remove medium components.
(4) The cells obtained as the precipitate were suspended in 100 mL of Buffer A for cell disruption (50 mM Tris-HCl (pH 7.5), 75 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, 1 mM PMSF, and two protease inhibitors for animal cells (manufactured by Nacalai Tesque, Inc.)), and subjected to ultrasonic disruption (TOMY UD-201, OUTPUT 2, DUTY 60, 2 min.×2).
(5) A homogenate was subjected to high-speed centrifugation at 14,300 rpm for 30 minutes at 4° C. with the use of a high-speed centrifuge (Beckman HP-26XP, JA25.50 rotor), and deposits resulting from cell disruption were removed as the precipitate.
(6) A supernatant was subjected to ultra-high-speed centrifugation at 40,000 rpm for 2 hours at 4° C. with the use of an ultra-high-speed centrifuge (Hitachi CP80WX, P45AT rotor), to thereby shed a vault fraction as a precipitate.
(7) A small amount of Buffer A for purification (50 mM Tris-HCl (pH 7.5), 75 mM NaCl, 1.5 mM MgCl2, 1 mM DTT, and 1 mM PMSF) was added to the vault fraction obtained as the precipitate, and the vault fraction was suspended with a Dounce homogenizer.
(8) To the solution in (7) above, equal parts of Ficoll/Sucrose Buffer (90 mM MES-NaOH (pH 6.5), 10 mM Sodium phosphate, 1 mM MgCl2, 0.5 mM EGTA, 0.02% NaN3, 14% Ficoll-PM70, and 14% Sucrose) were added and mixed well.
(9) The solution in (8) above was subjected to ultra-high-speed centrifugation at 25,200 rpm for 10 minutes at 4° C. with the use of an ultra-high-speed centrifuge (Hitachi CP80WX, P45AT rotor), to thereby shed an unwanted substance as a precipitate.
(10) A vault fraction of the supernatant was four-fold diluted with Buffer A for purification, and subjected to ultra-high-speed centrifugation at 40,000 rpm for 2 hours at 4° C. with the use of an ultra-high-speed centrifuge (Hitachi CP80WX, P45AT rotor), to thereby shed a vault fraction as a precipitate.
(11) A small amount of Buffer A for purification was added to the vault fraction obtained as the precipitate and the vault fraction was suspended with a Dounce homogenizer.
(12) A density gradient of sucrose was created in a centrifugal tube of the ultra-high-speed centrifuge (Hitachi CP80WX, P28S rotor). From the bottom of the centrifugal tube, 4 mL of 60% Sucrose, 5 mL of 50% Sucrose, 5 mL of 45% Sucrose, 5 mL of 40% Sucrose, 5 mL of 30% Sucrose, and 5 mL of 20% Sucrose were layered. Four such tubes were created.
(13) Five milliliters of the solution in (11) above were layered on a 20% layer of the density gradient of sucrose in (12) above.
(14) Ultra-high centrifugation was carried out at 25,000 rpm for 16 hours at 4° C. with the use of an ultra-high-speed centrifuge (Hitachi CP80WX, P28S rotor).
(15) Since vaults were contained in a part of 40-45% fractions and a 50% fraction, they were collected with a pipet. The 40 and 45% fractions were collected totally (5 mL each) and half of the 50% fraction (2.5 mL) was collected.
(16) The solution collected in (15) above was four-fold diluted with Buffer A for purification and subjected to ultra-high-speed centrifugation at 40,000 rpm for 2 hours at 4° C. with the use of an ultra-high-speed centrifuge (Hitachi CP80WX, P45AT rotor), to thereby shed a vault fraction as a precipitate.
(17) A small amount of Buffer A was added to the vault fraction obtained as the precipitate and the vault fraction was suspended with a Dounce homogenizer.
(18) A vault sample in (17) above was filtered through a 0.22-μm filter to thereby remove debris.
(19) Two milliliters of the sample in (18) above were applied to a gel filtration column (manufactured by GE Healthcare Japan, Sephacryl S-500, 26/60) equilibrated by 2 bed volumes of Buffer A for gel filtration (50 mM Tris-HCl (pH 7.5), 75 mM NaCl, 1.5 mM MgCl2, and 1 mM DTT), and the sample was fractionated by 4 mL at a flow rate of 0.5 mL/min.
(20) Since a target vault fraction comes out around 140 to 190 mL (fraction Nos. 39-49) after application of the sample, this vault fraction was collected and subjected to ultra-high-speed centrifugation at 40,000 rpm for 2 hours at 4° C. with the use of an ultra-high-speed centrifuge (Hitachi CP80WX, P45AT rotor), to thereby shed a vault fraction as a precipitate. (Around 90 to 110 mL (fraction Nos. 27-30), an aggregate of vaults comes out. Therefore, uniform vault particles can be obtained by removing the aggregate).
(21) A small amount of Buffer A was added to the vault fraction obtained as the precipitate and the vault fraction was suspended with a Dounce homogenizer, which was adopted as a final purified preparation.
FIG. 6 shows on the left, a photograph showing results of SDS-PAGE after disruption (the procedure (9) above) and showing a supernatant (a lane A) and a precipitate (a lane B) of LZMVP_Gly3 and a supernatant (a lane C) and a precipitate (a lane D) of LZMVP_Gly6. FIG. 6 shows on the right, a photograph showing results of SDS-PAGE after sucrose density gradient centrifugation (the procedure (14) above), in which a group a on the left shows results of LZMVP_Gly3 and a group b on the right shows results of LZMVP_Gly6. FIG. 7 shows an electron micrograph showing a final purified preparation of LZMVP_Gly3.
[5] Quantification of Protein of Purified Vault
(1) Quantification of proteins of W-MVP, LZMVP_Gly3, and LZMVP_Gly6 obtained as described above was carried out with the use of BCA Protein Assay Reagent Kit of Pierce.
(2) In a 15-mL tube, 5 mL of a reagent A of the kit and 100 μL of a reagent B were introduced and mixed well.
(3) Seven tubes each obtained by introducing 500 μL of the solution mixture in (2) above in a 1.5 mL Eppendorf tube were prepared.
(4) Twenty five microliters of each of 5 BSA standard solutions (1000, 500, 250, 125, and 62.5 μg/ml) prepared in advance were taken, introduced in the 1.5-mL tube in (3) above, and mixed well in a vortex mixer.
(5) Two types of solutions each obtained by diluting a vault solution to be quantified (containing W-MVP, LZMVP_Gly3, or LZMVP_Gly6) with ultrapure water were prepared (for example, 5-fold dilution and 10-fold dilution, or 25-fold dilution and 50-fold dilution).
(6) The two types of the diluted vault solutions fabricated in (5) above were each introduced in the 1.5 mL tube in (3) above and mixed well in a vortex mixer.
(7) The 1.5-mL tubes in (4) above and (6) above were incubated at 37° C. for 30 minutes.
(8) An absorbance of (7) above at 562 nm was measured with a spectrophotometer (colorimetric analysis based on coordination of reduced Cu (+) and bicinchoninic acid (BCA) resulting from Biuret test).
(9) Initially, measurement results of BCA were plotted with the ordinate representing an absorbance and the abscissa representing a BSA concentration, and an approximate curve (a standard curve) was found with the method of least squares.
(10) A concentration of the vault diluted solution was found from the standard curve in (9) above and then a concentration of a vault undiluted solution was found from a dilution factor.
(11) The concentration of the vault undiluted solution was multiplied with a total fluid volume, to thereby calculate total yields of vaults.
(12) Consequently, a W-vault (Comparative Example 1) weighed 3 to 5 mg per 1 L culture, whereas yields from 70 to 80 mg exceeding approximately 10 times were achieved with an LZ-vault (LZMVP_Gly3 (Example 1) and LZMVP_Gly6 (Example 2)).
It should be understood that the embodiments and the experimental examples disclosed herein are illustrative and non-restrictive in every respect. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.
REFERENCE SIGNS LIST


    
    
        1 artificial bioparticle; 2 vault; 3 MVP; 4 leucine zipper; 5 cap; 6 shoulder; 7 body; 8 waist; 9 cap helix; 10 cap ring; 11 drug; 12 antigen; 13 target cell; and 14 endosome.

## Claims

1. An artificial bioparticle comprising multiple, self-assembling copies of Major Vault Protein (MVP), wherein a leucine zipper is fused to the N-terminus of each MVP via a linker comprising 3 to 6 glycines.

2. The artificial bioparticle according to claim 1, wherein the leucine zipper is derived from GCN4 which is a transcription activator factor of yeast.

3. A method of manufacturing the artificial bioparticle according to claim 1, comprising expressing a protein comprising a leucine zipper linked to the N-terminus of an MVP via a linker comprising 3 to 6 glycines.

4. The artificial bioparticle according to claim 1, wherein the linker consists of 3 to 6 glycines.

5. The artificial bioparticle according to claim 1, wherein the linker consists of 6 glycines.