JP2012170346A - Suspension for parallel reaction, method for parallel reaction, and screening method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a suspension for parallel reaction, to provide a parallel reaction using the suspension, and to provide a screening method using the parallel reaction.SOLUTION: Gel particles each composed of a low-melting agarose and dispersed in an organic solvent have each a network structure made up through hydrogen bonds among polysaccharide chains. The surface of each of the gel particles is covered with a semipermeable membrane. Inside each of these gel particles, DNA and an enzyme for conducting a cell-free synthesis of protein or peptide are included. Because of having high molecular weight, the DNA and the enzyme do not elute via the semipermeable membrane to the outside. Besides, because of also having high molecular weight, the synthesized protein or peptide is retained inside the gel particles.

Description

  The present invention relates to a suspension for simultaneously performing a plurality of chemical reactions and biochemical reactions in a large number of gel particles, a parallel reaction method using the suspension, and a screening method using the parallel reaction. .

To search for enzymes and bioactive peptides with the desired activity from among many candidate proteins and peptides with random mutations, or to search for a catalyst with the desired activity from many candidate substances, It is often used in drug discovery and the search for new chemical substances.
For example, in Non-Patent Document 1, when cloning a cell that expresses the target protein, the cell is cultured from a concentration that allows one cell to enter one container by the limiting dilution method, and a clone that expresses the target protein is obtained. A method has been proposed.

  Non-Patent Document 2 and Non-Patent Document 3 disclose that a library is prepared by randomly substituting some amino acids of a certain enzyme, and that an enzyme having industrially useful activity is searched from this library. Has been.

In the above-described method, a reactor having a volume of about several hundred microliters to several milliliters such as a 96-well plate is generally used.
For example, a cell suspension containing target cells is dispensed into a 96-well plate, and analytical reagents including antibodies are sequentially added to determine the amount of substance expressed on the cell surface or secreted from the cells. When selecting a clone that expresses a highly active enzyme from a library that quantifies or expresses an enzyme containing various amino acid substitutions, the enzyme is expressed in a reactor such as a 96-well plate. The enzyme activity is measured by adding a reaction substrate or the like.
In other words, when a 96-well plate or the like is used, it is necessary to sequentially dispense a plurality of reagents into a large number of reactors (holes), and the reactors required as the number of enzymes and cells to be analyzed increases. The number increases, and the dispensing operation of the solution / reagent associated therewith becomes complicated.

  Non-Patent Document 4 proposes making a minute reaction vessel using semiconductor processing technology, but the problem of dispensing operation has not been solved. Therefore, proposals that further reduce the reaction vessel and do not require a dispensing operation are proposed in Patent Documents 1, 2, and 3 and Non-Patent Documents 5 and 6.

  Patent Document 1 includes a content that induces a specific reaction by separately containing hydrophilic molecules and functional molecules such as enzymes in water droplets constituting a W / O emulsion and fusing these water droplets together. It is disclosed. Further, Patent Document 1 discloses that DNA is contained in addition to molecules.

  In Patent Document 2, a reactive substance is included in one charged vesicle, a substance that reacts with the reactive substance is included in the other charged vesicle, and the two vesicles are fused and fused. It is disclosed that a reaction is caused to occur in one vesicle.

  Patent Document 3 includes a solid phase carrier to which a primer for PCR (polymerase chain reaction) is bound in an aqueous phase (water droplet) of an emulsion, and a nucleic acid complex is formed on the solid phase carrier by causing PCR in the water droplet. It is disclosed that, after ligation, the emulsion is broken to recover the conjugate of the solid phase carrier-nucleic acid complex, and cell-free protein synthesis is performed using this solid phase carrier-nucleic acid complex.

  Non-Patent Document 5 discloses a technique in which a target molecule is contained in a phospholipid bilayer (liposome), and these phospholipid bilayers are moved and fused by a laser.

  Non-Patent Document 6 discloses the contents of supplying a substrate into the gel particle through a polymer film on the surface of the gel particle in order to cause a protein synthesis reaction in the gel particle.

JP 2000-271475 A JP 2008-29952 A JP 2009-178067 A

Bio Experiment Illustrated, 6, 128-130 AISTToday 5 (4): 18-19,2005 Biochemistry, vol.72 (12) pp.1430-1433,2000 Analytical Chemistry, vol.77 (24) pp.8050-8056,2005 Nature Biotechnol., 16, 652-656 (1998) Journal of Biotechnology, 143, 183-189 (2000)

  By using vesicles, emulsions, or gel particles as a reaction vessel as disclosed in Patent Literatures 1 to 3 or Non-Patent Literature 5, the reaction vessel can be made smaller and a large number of parallel reactions can be performed. However, even if the target protein or peptide is obtained by causing a protein or peptide synthesis reaction in a micro container such as gel particles, there is a problem that these substances are easily eluted from the micro reaction container such as gel particles. is there.

  Further, as disclosed in Non-Patent Document 6, the surface of the gel particle is covered with a polymer film, so that the protein synthesized in the gel particle can be retained inside. However, in Non-Patent Document 6, there is only one kind of DNA included in the gel particles, and a plurality of different synthesis reactions are not performed simultaneously.

In order to solve the above problems, the present invention is a suspension for parallel reaction in which gel particles are dispersed. The gel particles contain protein synthase and DNA (deoxyribonucleic acid). Covered with a semipermeable membrane that allows the introduction of low-molecular substances such as substrates and energetic substances necessary for the synthesis of proteins and peptides into the gel particles and prevents the elution of proteins and peptides synthesized in the gel particles .
The size of the gel particles is suitably 1 to 500 μm, and the number of gel particles can be 10 ¹⁶ to 10 ¹² / ml.

  As a method of producing gel particles covered with a semipermeable membrane, for example, a gel solution and a solution that reacts with this to form a semipermeable membrane are separated through a partition wall, and then from a fine hole formed in the partition wall. A method of extruding the gel solution as fine particles into a solution that forms a semipermeable membrane, or a method of sending the gel solution to a solution that forms a semipermeable membrane by reacting with the gel solution through a narrow channel, etc. Applied.

  A suspension is obtained by dispersing the gel particles thus prepared in a solution containing a low molecule such as a substrate or an energy substance. As the substrate, amino acids and nucleic acids can be considered, and as the energy substance, ATP (adenosine triphosphate) can be considered.

  Ideally, one molecule of DNA is contained in one gel particle in order to simultaneously perform different parallel reactions within individual gel particles. For this purpose, at the stage of preparing gel particles, a solution containing a plurality of types of DNA is subjected to limiting dilution to a concentration at which one molecule of DNA is contained in one gel particle.

  In practice, there may be a case where a plurality of DNA molecules are included in one gel particle even when limiting dilution is performed, or there are no DNA molecules included in one gel particle. The meaning of limiting dilution in the present invention is a concentration at which one DNA is included in one gel particle on average.

  By doing so, only a specific protein or peptide is synthesized with one gel particle. Examples of the target substance encoded by DNA include enzyme proteins such as protease, lipase, amylase, and cellulase, fluorescent proteins, and physiologically active peptides.

Examples of the gel include agarose and sodium alginate, and examples of the semipermeable membrane include a polyion complex membrane composed of a polyvalent anion polymer and a polyvalent cation polymer, such as Alg-PEI (alginate-polyethyleneimine). It is done.

  In the parallel reaction using the above suspension, a substrate and an energy substance necessary for protein and peptide synthesis are added to the suspension, and the substrate and the energy substance are passed through a semipermeable membrane on the surface of the gel particle. It introduce | transduces in a gel particle and performs a cell-free protein synthesis reaction simultaneously in parallel in each gel particle.

  In the screening method according to the present invention, following the parallel reaction described above, the characteristics of the protein or peptide contained in each gel particle are examined, and DNA encoding the target substance is selected. For example, DNA selection is performed by separating gel particles that emit fluorescence. Specific means for sorting include flow cytometry and micromanipulators.

By using the parallel reaction suspension according to the present invention, for example, 10 ¹² to 10 ¹⁶ reactions can be carried out simultaneously in parallel.
In addition, elution of DNA, protein, and peptide from the gel particle is prevented, the reaction in the gel particle is efficiently performed, and the reaction product, protein and peptide, is also efficiently recovered.

Schematic diagram of gel particles in suspension according to the present invention Schematic diagram of the state of introduction of substances necessary for cell-free synthesis of proteins and peptides into gel particles Schematic diagram of cell-free synthesis of proteins and peptides in gel particles Cell-free protein synthesis in gel particles covered with semipermeable membrane. (A) is a micrograph of gel particles before adding a substrate. (B) is a fluorescence microscopic image of gel particles before adding a substrate. (C) is a photomicrograph of gel particles after protein synthesis. (D) is a fluorescence microscopic image of gel particles after protein synthesis. Synthesis of green fluorescent protein GFP is detected as fluorescence. Cell-free protein synthesis from single-molecule DNA in gel particles. (A) is a micrograph after protein synthesis of gel particles encapsulating one molecule of DNA. (B) is a confocal laser micrograph after protein synthesis of gel particles encapsulating one molecule of DNA. Arrows indicate gel particles in which DNA is encapsulated and GFP is synthesized by a protein synthesis reaction. The synthesis of GFP has been detected as fluorescence. Gel particles in which DNA is not encapsulated do not have fluorescence.

  As shown in FIG. 1, gel particles made of low melting point agarose dispersed in an organic solvent have a network structure due to hydrogen bonding between polysaccharide chains, and the surface thereof is covered with a semipermeable membrane. The gel particles covered with the semipermeable membrane contain DNA and an enzyme for cell-free protein synthesis. Since these DNAs and enzymes have large molecular weights, they do not permeate through the semipermeable membrane.

  In order to perform cell-free protein synthesis or cell-free peptide synthesis using the above suspension, substances necessary for protein or peptide synthesis, that is, amino acids, nucleic acids, Add ATP or the like. Since these amino acids, nucleic acids, and ATP are small molecules, they penetrate through the semipermeable membrane on the surface of the gel particle and enter the gel particle as shown in FIG.

  In the gel particle, protein or peptide is synthesized as shown in FIG. Specifically, RNA polymerase unwraps a part of the double helix of DNA to strip the base, and mRNA is transcribed using the stripped base as a template. Based on this mRNA information, an amino acid that requires tRNA To synthesize proteins or peptides with ribosomes. And the synthesized protein and peptide cannot permeate the semipermeable membrane and are retained in the gel particles.

  The above is the reaction in one gel particle in the suspension according to the present invention. In the parallel reaction using this suspension, the DNA library is first subjected to limiting dilution to produce gel particles. The dilution concentration and the size of the gel particles are such that only one molecule of DNA is contained in each gel particle. In reality, the number of gel particles is so large that not all gel particles contain exactly one molecule of DNA, but some do not contain DNA or some contain gel particles. There is no problem in performing parallel reactions.

  Next, a substance necessary for the synthesis of the protein or peptide is supplied into the gel particle, and cell-free synthesis is performed in the gel particle. In order to divide, it sorts using flow cytometry (flow cytometory), such as FACS (trademark). Specifically, gel particles in which proteins or peptides are synthesized are sent one by one to the sheath flow, and laser light is irradiated in the middle of the flow path. Gel particles in which the target protein or peptide is synthesized are stained with a fluorescent antibody and distinguished from other gels. Therefore, the gel particles are charged positively and negatively charged on the downstream side by a deflecting plate. Separated and recovered from other gels.

  By performing parallel reaction and screening in this way, DNA that synthesizes the target protein or peptide can be identified.

Example 1
W / O / W droplets are prepared using an aqueous solution containing template DNA (30 DNA molecules / gel particles) encoding 1 wt% sodium alginate, protein synthase and GFP (green fluorescent protein) gene as the inner aqueous phase, By mixing this with an aqueous solution containing 0.1 mol / L calcium chloride and 0.1 wt% PEI (polyethyleneimine), gel particles of micrometer size covered with a semipermeable membrane composed of a polyion complex of alginic acid and PEI. Obtained (FIG. 4A).
Cell-free protein synthesis was performed in the gel particles by mixing 25 μL of the centrifuged sample and 25 μL of the NTP / amino acid solution and incubating at 30 ° C. for 6 hours.
Then, after leaving still at 5 degreeC for 24 hours, as a result of observing with a microscope, the fluorescence derived from GFP synthesized from the inside of a gel particle was able to be detected (FIG. 4B, 4C).

(Example 2)
In Example 1, the same operation as in Example 1 was performed except that the template DNA was limit-diluted to a concentration of 1 DNA molecule / gel particle, and a cell-free protein of GFP was obtained from a single molecule of DNA in each gel particle. Synthesis was performed. As a result of observation with a microscope, GFP-derived fluorescence synthesized from within the gel particles was detected (FIG. 5).

  The suspension according to the present invention searches for a cell with a large amount of secreted target substance from a large number of cells, or has a target activity from among many candidate proteins or peptides randomly mutated. It can be used for parallel reactions when searching for substances.

Claims (9)

A suspension for parallel reaction in which gel particles are dispersed. The gel particles contain protein synthase and DNA (deoxyribonucleic acid), and the surface of the gel particles is necessary for protein synthesis into the gel particles. Suspension for parallel reaction, characterized by being covered with a semipermeable membrane that allows the introduction of small molecules such as substrates and energetic substances and prevents the elution of target substances such as proteins and peptides synthesized in gel particles Muddy liquid.   2. The parallel reaction suspension according to claim 1, wherein each gel particle dispersed in the suspension contains an average of 1 molecule or less of DNA. . The suspension for parallel reaction according to claim 1, wherein the semipermeable membrane is a polyion complex membrane composed of a polyvalent anion polymer and a polyvalent cation polymer. The suspension for parallel reaction according to claim 1, wherein the gel is composed of agarose or sodium alginate. The suspension for parallel reaction according to claim 1, wherein the gel particles have a size of 1 to 500 μm.   A substrate and an energy substance necessary for protein or peptide synthesis are added to the suspension according to any one of claims 1 to 5, and the substrate and the energy substance are introduced into the gel particle through a semipermeable membrane on the surface of the gel particle. A parallel reaction method comprising introducing and performing individual cell-free synthesis reactions in parallel in individual gel particles.   The parallel reaction method according to claim 6, wherein the substrate is an amino acid and a nucleic acid, and the energy substance is ATP (adenosine triphosphate).   The parallel reaction according to claim 6 or claim 7, characterized by examining the characteristics of the protein or peptide contained in each gel particle and selecting DNA encoding the target protein. Screening method.   9. The screening method according to claim 8, wherein the selection of the DNA involves sorting gel particles that emit fluorescence.