JP4264745B2 - Enzyme and biotin modified anisotropic polymer microparticle, microtubule and microparticle complex using this, and ATP self-supply nanobiomachine - Google Patents

Description

  The present invention relates to the transport of functional polymer microparticles by motor proteins, or the movement of motor proteins in the presence of non-ATP, more specifically, by the reaction between the ATP-producing enzyme immobilized on the microparticles and the substrate, ATP self-supplied, microtubules move on a kinesin-immobilized substrate, biotin-modified anisotropic polymer microparticles, microtubule and microparticle complexes using this, and ATP self-supply motor protein nanobiomachine and ATP It relates to a self-supplied transfer method.

(Background of the Invention)
The intracellular transport mechanism utilizes microtubules, which are fibrous polymers that act as pathways that induce molecular motion. Molecular transport is performed by the microtubule motor proteins kinesin and dynein. These proteins use the energy from ATP hydrolysis to move in one direction along microtubules and transport molecules to specific destinations.
Microtubules, 24 nm diameter cytoskeletal fibers, play multiple roles within the cell. For example, microtubules pass through nerve cell axons, allowing bidirectional transport of substances and membrane vesicles between the cell body and nerve endings. Microtubules are macromolecules composed of GTP-bound tubulin protein subunits. Each subunit is a heterodimer of α-tubulin and β-tubulin, each having multiple isoforms. Tubulin binds to two GTP molecules at two different sites, and all three tubulins share an invariant glycine-rich region that is involved in controlling access to one of the nucleotide binding sites. It is thought that.

  Research on transporting target substances using the above kinesin-microtubule system has been actively conducted. In recent years, there is a method of fixing the microtubule on the substrate and observing the motion of kinesin, but it is difficult to fix the microtubule. For this reason, in vitro motility assay (Non-patent Document 1) is generally used in which kinesin is physically immobilized on a glass substrate and microtubules are moved.

  Hess et al. Fabricated a 1 μm-high uneven substrate and moved the microtubules on the substrate. The microtubule moved around the convex part without moving. Thereby, surface unevenness imaging by a microtubule-kinesin system is possible (Non-Patent Document 2).

  Bachand et al. Successfully immobilized and transported avidinized quantum dots on microtubules using biotinylated microtubules. (Non Patent Literature 3)

It has been reported that a microtubule is moved in a single direction using a microfabrication technique, and it is possible to transport a target substance to another place artificially separated. (Non-Patent Document 4)
In addition, there is an example (Non-Patent Document 5) in which particles such as quantum dots have been transported so far, but it is not possible to place further objects on the quantum dots. There has been no report of a case where a functional substance (enzyme) is placed on a fine particle and transported.
On the other hand, the present inventors used anisotropic latex fine particles each having an epoxy ring and a hydroxyl group in different domains of the fine particles, reacted the hydroxyl side with an active ester biotin derivative, and biotinylated only one side of the fine particles. Development of anisotropic polymer microparticles carrying enzyme and biotin by preparing anisotropic bithionized latex microparticles and further immobilizing pyruvate kinase on biotinylated microparticles through epoxy on the microparticles A patent application has already been filed (see Patent Document 1).
In addition, hydroxyethyl methacrylate monomer, methyl methacrylate monomer and ethylene dimethacrylate monomer are soap-free emulsion polymerized in the presence of a polymerization initiator to synthesize seed polymer particles, then styrene monomer, glycidyl methacrylate monomer, divinylbenzene monomer and solvent In addition, anisotropic anisotropic latex fine particles in which soap-free seed emulsion polymerization is carried out in water and the functional groups have hydroxyl groups and epoxy rings in domains of different sizes. Anisotropic bitionated latex fine particles in which epoxy group is converted to amino group and biotin is bound. Furthermore, anisotropic bithionized latex fine particles in which hydroxyl groups of anisotropic bithionized latex fine particles are reacted with epichlorohydrin or the like to convert them into epoxy groups and to fix enzymes. In addition, anisotropic bithionized latex microparticles whose enzyme is pyruvate kinase have been developed, and a patent application has already been filed (see Patent Document 2).
However, since such anisotropic bithionized latex fine particles have too short a linker, they cannot be efficiently bound to biotinylated microtubules via streptavidin.

JP 2005-58101 A Japanese Patent Laid-Open No. 2005-113034 Vale, R. D .; Reese, T. S .; Sheetz, M. P. Cell. 1985, 42: 39-50. Surface Imaging by Self-Propelled Nanoscale Probes Hess, H .; Clemmens, J .; Howard, J .; Vogel, V .; Nano Lett .; (Communication); 2002; 2 (2); 113-116.) Assembly and Transport of Nanocrystal CdSe Quantum Dot Nanocomposites Using Microtubules and Kinesin Motor Proteins Bachand, GD; Rivera, SB; Boal, AK; Gaudioso, J .; Liu, J .; Bunker, BC; Nano Lett .; (Communication); 2004 ; 4 (5); 817-821. Hiratsuka, Y .; Tada, T .; Oiwa, K .; Kanayama, T .; Uyeda, T. Q. Controlling the direction of kinesin-driven microtubule movement along microlithographic tracks. Biophys. J. 81, 1555-1561 (2001). Bachand, G. D. et al. Assembly and transport of nanocrystal CdSe quantum dot nanocomposites using microtubules and kinesin motor proteins.Nano Lett. 4, 817-821 (2004).

The present inventor continued research to provide enzyme-modified anisotropic bithionized latex microparticles that can be bound to microtubules, a microtubule-microparticle complex using this, and an ATP self-feeding nanobiomachine.
The usefulness of motor protein transport ability has been suggested so far, but it is difficult to modify a substance to be transported directly to a microtubule, particularly a high molecular weight substance, and the modification efficiency is low.
Thus, there is no example in which functional polymer fine particles are immobilized on a microtubule and transported. Moreover, various chemical and biological substances can be immobilized on the fine particles through the epoxy functional groups on the fine particles, and transport by molecular motor can be realized.
The presence of ATP is essential for kinesin to drive as a molecular motor. Conventionally, when moving microtubules using kinesin, it was necessary to add ATP to the buffer used. This ATP is not a problem because it exists in a large amount in the living body, but the artificially synthesized one has a problem that it is expensive. Therefore, if a function for synthesizing ATP is added to the microtubule itself, kinesin can be driven and moved in the presence of non-ATP. This time, by immobilizing microparticles modified with pyruvate kinase (PK), an ATP-generating enzyme, in the microtubule, the motor protein nanobiomachine itself synthesizes ATP without requiring expensive ATP, That movement can be realized.

In order to achieve the above-mentioned object, the present invention is directed to immobilizing pyruvate kinase (PK) covalently on anisotropic polymer fine particles, with an enzyme only on one side, and a linker represented by chemical structural formula 1 on the other side. Succeeded in regiospecifically immobilizing biotin containing polyethylene glycol (where n = 3 to 100).
That is, the present invention is anisotropic latex fine particles in which an epoxy group in a wide domain A and a hydroxyl group in a narrow domain B are provided in fine particles, respectively, and the epoxy side is reacted with an enzyme so that the hydroxyl side The chemical structural formula 1
(Where n = 3 to 100)
It is the enzyme modification anisotropic bitionated latex fine particle couple | bonded with the biotin derivative shown by these.
In the present invention, the enzyme is preferably pyruvate kinase.
Furthermore, the present invention is an anisotropic latex fine particle in which an epoxy group in a wide area domain A and a hydroxyl group in a narrow area domain B are provided in fine particles, respectively, and the epoxy side is reacted with an enzyme, and the hydroxyl side Is further bound to biotinylated microtubules by further binding streptavidin to enzyme-modified anisotropic biotinylated latex microparticles bound to biotin containing polyethylene glycol represented by the chemical structural formula 1 (where n = 3 to 100). It is a complex of bound microtubules and fine particles.
Further, the present invention is preferably an ATP self-feeding motor protein nanobiomachine capable of moving a complex of microtubules and microparticles on a kinesin-immobilized substrate by using pyruvate kinase as an enzyme. can do.
Furthermore, the present invention is an anisotropic latex fine particle in which an epoxy group in a wide area domain A and a hydroxyl group in a narrow area domain B are provided in fine particles, respectively, and the epoxy side is reacted with an enzyme, and the hydroxyl side Is transferred to a kinesin-immobilized substrate while self-supplied with ATP using a complex of microtubules and microparticles bound to a biotin derivative represented by the chemical structural formula 1, and further bound to streptavidin and biotinylated microtubules It is a nano bio machine.

The enzyme-modified anisotropic polymer fine particles of the present invention can be easily bound to biotinylated microtubules via biotin-avidin interaction. As a result of developing enzyme-modified anisotropic polymer microparticle-immobilized microtubules, the microtubules moved on the kinesin-immobilized substrate in the presence of ATP after loading the microparticles. (2) Even in the presence of non-ATP, kinesin is produced by self-supply of ATP by pyruvate kinase (enzyme) on the microparticles using phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) as substrates in solution. The microtubules can be moved by driving.

In the present invention, examples of the enzyme include PGA kinase, adenylate kinase, ATP synthase, and pyruvate kinase is particularly preferably used.
As avidin, streptavidin with high purity is preferably used.
The enzyme and biotin-modified anisotropic polymer microparticle-coupled microtubule of the present invention is in a form in which an enzyme, which is a biomolecule, is introduced onto the microtubule through the microparticle (FIG. 1). This microtubule does not deteriorate its motility even after the microparticles are immobilized, so that a nanobiomachine capable of transporting substances can be constructed. In addition, the type of immobilized enzyme is not particularly limited because it is a product of a reaction between an amino group on the protein surface and an epoxy group on the fine particle. Since it is an ATP-producing enzyme this time, ATP can be self-supplied in the presence of a substrate. It is very economical and useful to achieve microtubule migration in the presence of non-ATP while adding expensive ATP in solution.
When producing the enzyme-modified anisotropic polymer fine particle-bound microtubule of the present invention, the microtubule used may be one prepared using rhodamine or biotinylated tubulin, which is a fluorescent substance.
As the fluorescent substance that can be used in the present invention, anything may be used, but examples of familiar substances include rhodamine, FITC, BODIPY FL-X [6-((4,4-difluoro-5,7-dimethyl-4- bora-3a, 4a-diaza-s-indacene-3-propionyl) amino) hexanoic acid succinimidyl ester], OregonGreen514, Cye dye series. In particular, rhodamine is preferably used.
In the present invention, the length of the linker of the biotinylation reagent is not particularly limited when biotinylating the tubulin monomer constituting the microtubule, but as a familiar one, Biotin- (Pierce, Biotin- ( Long Arm) -NHS and EZ-link NHS-LC-LC-Biotin. In particular, Biotin- (Long Arm) -NHS is preferably used.

(Preparation of enzyme and biotin-modified anisotropic polymer particles)
<Preparation of anisotropic polymer fine particles>
By the same method as Patent Document 1, anisotropic latex fine particles were prepared.
First, hydrophilic poly (glycidyl methacrylate-divinylbenzene) seed particles [P (GMA-DVB)] were prepared and prepared by soap-free emulsion polymerization of glycidyl methacrylate (GMA) and divinylbenzene.
Polymerization was carried out at a rotation speed of 200 rpm at 70 ° C. for 15 hours using 14 g of GMA / DVB 1 g / polymerization initiator V-50 0.45 g / about 285 g of water. The monomer change rate and particle size are 100 wt% and about 180 nm, respectively. The use of initiator V-50 is effective for keeping the pH of the reaction solution neutral during polymerization and preventing ring-opening reaction of the epoxide group of GMA. On the other hand, when a persulfuric acid-based initiator is used, the reaction solution becomes acidic with the polymerization reaction, and the epoxide is ring-opened at 70 ° C.
Thereafter, the hydrophilic poly (glycidyl methacrylate-divinylbenzene) [P (GMA-DVB)] prepared above was used as seed particles, and styrene monomer, 2-mercaptoethanol, polymerization initiator V-50 and water. And anisotropic poly (glycidyl methacrylate-divinylbenzene) and poly (styrene) composite fine particles [P (GMA-DVB) / P (St)] were prepared by soap-free seed emulsion polymerization. Before use, P (GMA-DVB) seeds were dialyzed for 24 hours with tap water and pure water using a cellulose membrane to remove unreacted monomers, initiators, oligomers, and the like.
To P (GMA-DVB) seed particles, add 2 g styrene monomer 0.02 g / 0.02 g polymerization initiator (V-50) 0.04 g / toluene 2 g / pure water about 130 g, Polymerization was performed at 70 ° C. and 200 rpm for 24 hours to obtain the desired fine particles. The morphology of the obtained composite fine particles was observed with a transmission electron microscope.

<Biotinylation on the hydroxyl side of anisotropic polymer particles>
Due to the low reactivity of the hydroxyl group, active ester biotin, biotin-PEG-CO ₂ -NHS (Shearwater Polymers, Inc .; weight average molecular weight of about 3400 was used in the presence of dimethylaminopyridine (DMAP) (about 69 ethylene glycol repeating units), and the structure is shown in chemical structural formula 1. 10 g of latex was washed three times with dehydrated acetonitrile and then dispersed in 20 g of acetonitrile. Thereafter, an acetonitrile solution (10 g) containing biotin-PEG-CO ₂ —NHS (10 mg) and DMAP (0.05 g) was mixed. The reaction was carried out at 65 ° C. under argon gas for 24 hours.

<Immobilization of anisotropic polymer particles of pyruvate kinase>
Pyruvate kinase was directly immobilized on the microparticles via an epoxy group on the surface of the microparticles. 1 g of latex was washed twice with a pH 7.4 phosphate buffer and then dispersed in 2 ml of a pH 7.4 phosphate buffer. Then about 790 mg of pyruvate kinase was added. The enzyme immobilization reaction was performed for 16 hours while rotating at room temperature (about 25 ° C.). After immobilization, the microparticles were centrifuged, and the physically adsorbed enzyme was washed four times with a 1 M potassium chloride (KCl) solution. The supernatant after centrifugation and the potassium chloride solution separated after washing were collected, the protein in the solution was measured by UV absorption at 280 nm, and the amount of enzyme immobilized on the microparticles was calculated. The amount immobilized on fine particles was about 75 mg per 1 g of latex fine particles.

(Preparation of enzyme, biotin-modified anisotropic polymer particles and microtubule complex (MTs-Particle))
<Preparation of microtubules>
The microtubules used in the present invention consist of rhodamine-modified / biotinylated (hereinafter referred to as RB) tubulin (feeding ratio 1: 1) with BRP80 (80 mM PIPES, pH 6.8, 1 mM MgCl ₂ , 1 mM EGTA, 10% glycerol, 5 RB-microtubules were prepared by suspending in mM GTP) buffer and incubating at 37 ° C for 40 minutes, and finally stabilizing the microtubules with taxol.
Avidin modified with Alexa Fluor 488 was used for the purpose of examining the distribution of biotinylation and rhodamineation in the obtained RB-microtubules. Avidin can specifically bind to biotin distributed in microtubules. As a result of fluorescence observation, it was confirmed that biotinylated tubulin and rhodamine labeled tubulin were randomly polymerized to form microtubules (FIG. 2).
<Modification of enzyme and biotin-modified anisotropic polymer particles>
In order to bind the enzyme shown in Example 1 above and biotin-modified anisotropic polymer fine particles to the microtubule, further modification was performed. First, in order to visualize the fluorescence of the microparticles, the immobilized enzyme (pyruvate kinase) was labeled with BODIPY FL-X as a fluorescent agent. 2 ml of BODIPY FL-X solution (1 mg / ml) was added to 0.1 g of enzyme-immobilized fine particle pH 7.4 phosphate buffer and reacted for 2 hours. After washing by centrifugation, a reaction was performed by adding 2 ml of a streptavidin solution (1 mg / ml) to the fine particles. After the reaction, unreacted streptavidin and fine particles were separated by centrifugation.
<Binding of enzyme, biotin-modified anisotropic polymer fine particle and microtubule>
The 0.5 ml microtubule prepared above was diluted with 200 ml assay buffer (10 mM Tris-acetate (pH 7.5), 50 mM potassium acetate, 2.5 mM EGTA, 4 mM magnesium sulfate, 20 mM taxol), 50 ml of the fine particle solution modified as described above was added and reacted for 2 to 10 minutes to prepare a complex of fine particles and microtubules. Figure 3 shows the binding between microparticles and microtubules in the solution.

(Production of kinesin motor nanobiomachine that self-supplied ATP)
A kinesin motor nanobiomachine that self-supplied ATP was prepared using a flow chamber as shown in Fig. 4 that was prepared using a slide glass and a cover glass.
A kinesin solution dissolved in assay buffer (10 mM Tris-acetate (pH 7.5), 50 mM potassium acetate, 2.5 mM EGTA, 4 mM magnesium sulfate) is inserted into the flow chamber, and the kinesin is physically fixed to the substrate for 2 minutes. Turned into. Kinesin (K 560) used was expressed from Escherichia coli, which is Escherichia coli. After washing with assay buffer (10 mM Tris-acetate (pH 7.5), 50 mM potassium acetate, 2.5 mM EGTA, 4 mM magnesium sulfate), microtubule assay buffer solution (10 mM Tris-acetate (pH 7.5) prepared above) , 50 mM potassium acetate, 2.5 mM EGTA, 4 mM magnesium sulfate) were similarly inserted into the flow chamber, and the microtubules were immobilized on kinesin on the substrate for 2 minutes. After washing with assay buffer (10 mM Tris-acetate (pH 7.5), 50 mM potassium acetate, 2.5 mM EGTA, 4 mM magnesium sulfate), enzyme-modified anisotropy with high fluorescence labeling and streptavidin prepared above was bound. 20 ml of the solution of molecular fine particles was further added to the flow chamber, and the enzyme-modified anisotropic polymer fine particles were bound to the microtubules. After washing with assay buffer (10 mM Tris-acetate (pH 7.5), 50 mM potassium acetate, 2.5 mM EGTA, 4 mM magnesium sulfate), the microtubule loading capacity and movement were evaluated with a fluorescence microscope.
First, in order to clarify the motion characteristics of MTs-Particles, a 1 mM ATP buffer solution was added to the flow chamber. MTs-Particles moved smoothly without cross-linking even when two MTs-Particles overlapped. It was also confirmed that MTs-Particles were loaded with free enzyme-immobilized fine particles on the substrate and fine particles in the solution while moving.
Next, ADP and PEP buffer solution were added into the flow chamber instead of 1 mM ATP buffer solution. It was confirmed that MTs-Particle moved on the kinesin-immobilized substrate when the ADP and PEP concentrations were 1 mM. As can be seen from FIGS. 5A-5C, MTs-Particles move only in one direction. In other words, this movement is not random walking due to heat. Conversely, there is a difference in each observed MTs-Particle velocity. It can be inferred that this is due to the MT length, fine particle density, and kinesin immobilization density. In the MTs-Particle shown in FIG. 5B, the points represented by green fluorescence were fine particles, and 90% of the fine particles were observed on the MTs.
The change of speed with time was examined (Fig. 6A). MTs-Particles started to move 15 minutes after inserting the substrates (ADP, PEP). After that, the speed began to increase rapidly, reaching a maximum of 3-4.5 nm / sec, and slowly decreased. Since the PEP concentration (1 mM) is in great excess, this decrease in rate is not due to a decrease in PEP concentration (PEP Michelis constant; Km = 256 μM). Fig. 6B shows the existence frequency of MTs-Particle length. The average MTs-Particle length was 11 μm, and 50% or more was 10 μm or less.
MTs added with 30μM ATP solution moved smoothly onto kinesin, and the rate decreased exponentially with time (Fig. 6C). From this figure, the relationship between the microtubule movement speed and the ATP concentration was determined, and it was found that the ATP concentration at the steady state of the MTs-Particle movement speed shown in Fig. 6A was about 4.4 mM.
Until now, there are examples where streptavidin-coated microparticles or quantum dots (Qdot) (Non-patent Document 5) are mounted on MTs through biotin-avidin bonds, but functional biomolecules (such as the present inventors) There is no application example to nanobiomachines in which (enzyme) is immobilized on anisotropic fine particles and mounted on MTs. In other words, although there have been reports on the construction of nanobiomachines so far, microparticles and MTs aggregate at multiple locations, hindering the transport of MTs by kinesin. With the anisotropic fine particles we used, the enzyme (target substance immobilization site) and biotin (modification site for MTs) can be easily separated and immobilized respectively. Aggregation with MTs does not occur by controlling the biotin site area on the microparticles. In addition, the application of biotin containing polyethylene glycol (wherein n = 3 to 100) represented by chemical structural formula 1 significantly improved the binding efficiency of microparticles to microtubules.

The enzyme and biotin-modified anisotropic polymer microparticles of the present invention, the microtubule-microparticle complex using this, and the nanobiomachine that self-supplies ATP are epoch-making, and biotin on the microparticles is mediated by avidin. Because it can be combined with various substances, "immobilized enzyme cassette" or "immobilized enzyme device", etc. Also, microtubule and microparticle complex and ATP self-supplying nanobiomachine are mass transport and sensing in micro space Various applications in a wide range of fields are conceivable and the industrial applicability is extremely high.

Conceptual diagram of enzyme-modified anisotropic polymer fine particle-bound microtubule of the present invention Fluorescence image of streptavidin-modified microtubules (a) Rhodamine: Microtubule fluorescence, (b) BODIPI: Streptavidin fluorescence Fluorescence picture of a complex of enzyme, biotin-modified anisotropic polymer particles and microtubules Conceptual diagram of flow chamber Microtubule movement after ATP self-supply (a) 31, (b) 45, (c) Image after 61 minutes Enzyme-modified anisotropic polymer microparticles and fluorescent images of microtubules with them Time course of enzyme-modified anisotropic polymer microparticle-coupled microtubules Length distribution of enzyme-modified anisotropic polymer microparticle-bound microtubules Time course of microtubule movement speed at 30μM ATP concentration

Claims (5)

An anisotropic latex fine particle in which the epoxy group in the wide domain A and the hydroxyl group in the domain B in the narrow region are respectively provided in the fine particle, the epoxy group side is reacted with an enzyme, and the hydroxyl side is represented by a chemical structural formula 1
(Where n = 3 to 100)
Enzyme-modified anisotropic bithionized latex fine particles bound to a biotin derivative represented by
The enzyme-modified anisotropic bithionized latex microparticle according to claim 1, wherein the enzyme is pyruvate kinase. An anisotropic latex fine particle in which a fine particle has an epoxy group in a wide domain A and a hydroxyl group in a narrow domain B, the epoxy group side is reacted with an enzyme, and the hydroxyl side is represented by the chemical structural formula 1
(Where n = 3 to 100)
A complex of enzyme-modified anisotropic bithiolated latex microparticles bound to biotinylated microtubules further bound to biotinylated microtubules via avidin with enzyme-modified anisotropic bithionized latex microparticles bound to the biotin derivative shown in FIG. The ATP self-supplying motor protein nanobiomachine capable of moving the microtubule and microparticle complex according to claim 3 onto a kinesin-immobilized substrate, wherein the enzyme is pyruvate kinase and avidin is streptavidin. An anisotropic latex fine particle in which the epoxy group in the wide domain A and the hydroxyl group in the narrow domain B are respectively attached to the fine particles, the epoxy side is reacted with pyruvate kinase, and the hydroxyl side is represented by the chemical structural formula 1
(Where n = 3 to 100)
Using a motor protein nanobiomachine that is bound to the biotin derivative shown in Fig. 1 and then bound to biotinylated microtubules via avidin, the complex of microtubules and microparticles on the kinesin-immobilized substrate while self-supplying ATP ATP self-supply moving method to move to.