JP2004065238A - Method for introducing peptide, dna protein complex or metallic fine particle into prokaryotic cell by using transfection reagent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To simply, safely, and efficiently introduce a peptide, a DNA protein complex or a metallic fine particle into a prokaryotic cell having a cell wall consisting mainly of a peptideglycan. <P>SOLUTION: A method comprises introducing the peptide, the DNA protein complex or the metallic fine particle into the prokaryotic cell having the cell wall consisting mainly of the peptideglycan, wherein the method is conducted in coexistence of a transfection reagent. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for introducing a peptide, a DNA-protein complex, or metal microparticles into a prokaryotic cell having a cell wall mainly composed of peptidoglycan.
[0002]
[Prior art]
The introduction of peptide and DNA protein complexes into prokaryotic cells to express proteins and genes has not been performed so far, but is a technology that is expected to develop in the future.
[0003]
As a method for introducing a peptide and DNA protein complex into prokaryotic cells, mainly a physical method and a chemical method are known (for example, see Non-Patent Document 1).
[0004]
Physical methods include an electroporation method, a particle gun method, and the like.For example, in the case of the electroporation method, in E. coli, cells in the logarithmic growth phase are washed with a solution containing almost no salt such as pure water, and placed on ice. After coexisting with the peptide or DNA-protein complex, a high voltage is applied for a short time to temporarily create small pores in the cells, and the peptide or DNA-protein complex is introduced. For expression of a peptide or DNA protein complex.
[0005]
For example, in the case of Escherichia coli, for example, in the case of Escherichia coli, the cells are treated with a calcium chloride solution or the like, coexist with a peptide or DNA protein complex on ice, warmed briefly at 42 ° C., and cooled again on ice for about 2 minutes. After that, the cells are cultured at 37 ° C. for about 1 hour in an activation medium to express a peptide or DNA protein complex.
[0006]
In the case of eukaryotic cells, especially cultured cells, a lipofection method is known as a chemical method in addition to the above method. The lipofection method mainly comprises forming a complex of a positively charged liposome (cationic liposome) and a polynucleotide, adsorbing the cationic liposome on the surface of the negatively charged cell, and fusing it with the cell membrane. A method of introducing a peptide or DNA protein complex into cells.
[0007]
These methods are conventionally used methods and have many problems as described below.
[0008]
For example, in the chemical method, the operation of treating cells is complicated, and requires a skilled technique. In addition, heat shock treatment or the like is also required, which places a burden on cells. The transfection efficiency of the peptide or DNA protein complex into cells is also very low.
[0009]
The electroporation method shows high transfection efficiency for most cells such as bacteria, fungi, yeast, plant cells, and animal cells, but causes many cells to die and causes damage or polarization to the transfected cells. In addition, there may be differences in introduction efficiency. It also requires special equipment.
[0010]
It has been considered that the lipofection method is limited to cells having no cell wall in cells having a cell wall such as a peptidoglycan layer or a cellulose layer because a cell membrane fused with a lipofection reagent is not exposed. Recently, the inventor has found that it is also possible in bacteria (see, for example, unpublished patent applications 1 and 2).
[0011]
[Unpublished patent application 1]
Japanese Patent Application No. 2002-132197
[0012]
[Unpublished patent application 2]
Japanese Patent Application No. 2002-337529
[0013]
[Non-patent document 1]
Sambrook, J. and Dee. Wu. DW Russell, "Molecular Cloning: A Laboratory Manual", (USA), 3rd edition, Cold Spring Harbor Laboratory Press, 2001. [0014]
[Problems to be solved by the invention]
A main object of the present invention is to provide a method for simply, safely and efficiently introducing a peptide or DNA protein complex or metal fine particles into a prokaryotic cell having a cell wall mainly composed of peptidoglycan.
[0015]
[Means for Solving the Problems]
The present inventor has made intensive studies in order to solve the above-mentioned problems of the prior art. As a result, by introducing a peptide or DNA protein complex into a cell having a cell wall mainly composed of peptidoglycan using a transfection reagent, the peptide or DNA protein complex can be easily, safely and efficiently introduced. Was found.
[0016]
That is, the present invention relates to a method for introducing the following peptide, DNA protein complex or metal fine particle.
[0017]
1. A method for introducing a peptide or DNA protein complex into a prokaryotic cell having a cell wall mainly composed of peptidoglycan, wherein the method is performed in the presence of a transfection reagent.
[0018]
2. A method for introducing metal microparticles into a prokaryotic cell having a cell wall mainly composed of peptidoglycan, wherein the method is performed in the presence of a transfection reagent.
[0019]
3. Item 2. The method according to Item 1, wherein the transfection reagent is a reagent containing lipid as a main component.
[0020]
4. Item 2. The method according to Item 1, wherein the transfection reagent is a cationic liposome.
[0021]
5. Item 2. The method according to Item 1, wherein the prokaryotic cell is Escherichia coli.
[0022]
6. Item 3. The method according to Item 2, wherein the metal fine particles are at least one selected from the group consisting of iron, titanium oxide, platinum and gold.
[0023]
7. A transfection kit comprising a peptide, a DNA protein complex or a metal microparticle and a transfection reagent.
[0024]
BEST MODE FOR CARRYING OUT THE INVENTION
The method for introducing a peptide, DNA protein complex or metal microparticle of the present invention is characterized in that a normal transfection reagent is used for prokaryotic cells having peptidoglycan on the cell wall.
[0025]
In the present invention, the prokaryotic cell into which the peptide, the DNA protein complex or the metal microparticle is introduced is a prokaryotic cell having peptidoglycan or a substance similar to the peptidoglycan on the cell wall, for example, bacteria including Escherichia coli, Bacillus subtilis, and cyanobacteria. And the like. Among them, Escherichia coli, cyanobacteria and the like are preferable from the viewpoint of convenience and the like.
[0026]
The type of Escherichia coli is not limited, and examples thereof include those derived from the K12 strain such as DH5α, DH10B, and BL21 (DE3) which are usually used for gene recombination. Also, the type of cyanobacteria is not limited, and examples thereof include Synechococcus, Synechocystis, and Spirulina.
[0027]
In the present specification, the peptide includes, for example, not only ordinary peptides having two or more amino acids but also proteins, enzymes, and the like. The type and preparation method of the peptide are not particularly limited, and can be appropriately selected depending on the purpose of the experiment and the like.
[0028]
In the present specification, a DNA-protein complex refers to a complex of a polynucleotide and a protein such as an enzyme. The types of proteins and polynucleotides are not particularly limited, and combinations thereof can be appropriately selected depending on the purpose of the experiment and the like. For example, a transposon-transposase complex or a complex of a restriction enzyme recognition site and a restriction enzyme can be exemplified.
[0029]
In the present specification, the metal fine particles are not particularly limited as long as they enter prokaryotic cells. For example, at least one selected from the group consisting of iron, titanium oxide, platinum and gold can be used, and among them, metal fine particles such as iron and titanium oxide are preferable. The average particle size of the metal microparticles is not limited as long as it is introduced into prokaryotic cells, for example, about 0.01 μm to about 1.0 μm, preferably about 0.01 μm to about 0.5 μm, more preferably about 0.01 μm To about 0.05 μm.
[0030]
It is necessary to use a transfection reagent when introducing the obtained peptide, DNA protein complex or metal microparticles into prokaryotic cells. As the transfection reagent, a transfection reagent generally used for cultured cells of animals can be used, and preferably, a transfection reagent containing lipid as a main component, and more preferably a transfection reagent containing cationic liposome as a main component. Good.
[0031]
In addition, the cationic liposome refers to, for example, a charged molecule having an atomic group (cation charged molecule) serving as a cation at one end of a molecule mainly composed of a carbon molecular chain having a low degree of unsaturation.
[0032]
Examples of molecules mainly composed of carbon molecular chains include, besides fatty acids having 12 to 20 carbon atoms and 0 to 4 double bonds, a benzene ring having a functional group and a heterocyclic compound containing a nitrogen atom. . Specifically, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, lignoceric acid, palmitooleic acid, oleic acid, linoleic acid, linolenic acid, arachidonic acid, etc., and (S) -2-methyl- 1,4,5,6-tetrahydropyrimidine-4-carboxylic acid ((S) -2-methyl-1,4,5,6-tetrahydropyrimidine-4 carboxylic acid), (S) -2-methyl-5 Examples thereof include hydroxy-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid ((S) -2-methyl-5-hydroxy-1,1,4,5,6-tetrahydropyrimidine-4 carboxylic acid). These can be used alone or in combination of two or more.
[0033]
The cation-charged molecule refers to a molecule charged to a cation, and examples thereof include an amine salt, a quaternary ammonium salt, and a pyridinium salt. These may be used alone or in combination of two or more.
[0034]
The molecule mainly composed of a carbon molecular chain and the cation-charged molecule can be used in any combination, and a method of bonding these can be appropriately selected.
[0035]
Specific transfection reagents containing a cationic liposome as a main component include, for example, DOTAP (N- [1- (2,3-dioleoyloxy) propyl] -N, N, N-trimethylammoniummethyl sulfate), DOSFFER (1,3) -Di-Oleoxyloxy-2- (6-Carboxy-spermyl) -propylamid), Transome (registered trademark), FuGENE (registered trademark) 6, Chariot Kit (registered trademark, manufactured by Active Motief) and the like.
[0036]
When introducing the peptide, DNA protein complex or metal microparticle into prokaryotic cells, the peptide, DNA protein complex or metal microparticle and the transfection reagent may be mixed in advance at room temperature to form a complex.
[0037]
The mixing ratio of the peptide or DNA protein complex and the transfection reagent can be appropriately selected depending on the type of the peptide or DNA protein complex, the type of the transfection reagent used, and the like. For example, about 1 to 1000 μg can be exemplified as a lipid component per 1 μg of the peptide or DNA protein complex.
[0038]
The mixing ratio of the metal fine particles and the transfection reagent can be appropriately selected according to the type of the metal fine particles, the type of the transfection reagent to be used, and the like. For example, about 0.01 to 100 μg can be exemplified as a lipid component per 1 μg of metal fine particles.
[0039]
The method of mixing the peptide, DNA protein complex or metal microparticle with the transfection reagent is not limited as long as the peptide, DNA protein complex or metal microparticle and the transfection reagent form a complex. A solution containing a transfection reagent is put in the case in advance, a necessary amount of a solution containing a peptide, a DNA protein complex or a metal microparticle is added, and the mixture is gently mixed if necessary, for 1 minute or more, preferably 3 to 15 minutes. It may be left on ice for about a minute, for example.
[0040]
Thereafter, the resulting peptide, DNA protein complex or complex of the metal microparticles and the transfection reagent may be brought into contact with the cells. The method is not limited, either. For example, a peptide, DNA protein complex or metal microparticle-transfection reagent complex solution may be added to a cell diluent, mixed gently if necessary, and allowed to stand.
[0041]
The mixing ratio of the peptide, DNA protein complex or metal microparticle-transfection reagent complex and the cell diluent can also be appropriately selected according to the type of cells used and the like. For example, the concentration can be set to about 1 to 100 ng, preferably about 1 to 10 ng, per 1 × 10 ⁹ cells. Further, the metal fine particles can be set to about 1 to 100 ng, preferably about 1 to 10 ng per 1 × 10 ⁹ cells.
[0042]
Thereafter, after culturing the cells in a selection medium, it can be confirmed by a known method that the peptide or DNA protein complex has been introduced into the prokaryotic cells. For example, confirmation by antibiotic resistance and the like, detection of enzyme activity by assay of an introduced protein enzyme, and the like can be mentioned.
[0043]
In addition, the fact that the metal fine particles have been introduced into the prokaryotic cells can be confirmed by direct observation using a known method using, for example, an SEM.
[0044]
Peptide or DNA protein complexes that can be introduced into prokaryotic cells can be used for screening peptide libraries, introducing peptide inhibitors, studying protein half-life, labeling with intracellular antibodies, and the like. -This technique can also be used to create a transgenic transformant into the genome using a transposase complex or the like.
[0045]
In addition, drug delivery becomes possible by introducing metal fine particles into prokaryotic cells. For example, by introducing fine iron particles, prokaryotic cells expressing a pharmacologically active compound (protein or the like) can be directly transferred to the site of action in a living body by magnetic force, and lysed there, thereby exhibiting pharmacological activity.
[0046]
Further, a function not originally possessed by a living body may be given as a catalytic function by metal fine particles, and fusion of a higher-order biological function and catalytic activity may be considered. For example, toxic nitrogen oxides and the like can be oxidized by a photocatalyst and titanium oxide used, and used as nutrients for prokaryotic cells.
[0047]
A mixed solution (complex) containing the above-described peptide, DNA protein complex or metal microparticle and a transfection reagent can be refrigerated or frozen and used when necessary. It can also be used as a transfection kit.
[0048]
【Example】
Examples are shown below to further clarify the features of the present invention.
[0049]
Example 1 <Introduction of β-galactosidase>
A 0.25 μg / μl β-galactosidase solution was dispensed into 3 microtubes by 4 μl each, the first one was kept as it was, the second one was cationic liposome DOTAP Liposomal Transfection Reagent (Roche) 4 μl, and the third one was Chariot Kit 8 μl of Chariot Transfection Reagent (2 mg / ml from Active Motief) was mixed at room temperature and left for 15 minutes.
[0050]
Escherichia coli electrocompetent cell DH5α (TAKARA) was added to each of the above three microtubes, left on ice for 5 minutes, then added with 1 ml of SOC medium warmed to 37 ° C, and shaken at 37 ° C for 1 hour. Cultured.
[0051]
Each of the cell solutions is transferred to a microtube, centrifuged at 5,000 rpm for 1 minute, and the supernatant is removed, followed by centrifugation and washing twice with PBS Buffer (1.5 mM KH ₂ PO ₄ , 150 mM NaCl, 5 mM Na ₂ HPO ₄ ). did.
[0052]
The cells were suspended in an LB medium and incubated for 1 hour at 37 ° C., and then centrifuged again to remove the supernatant, suspended in an LB medium containing 0.05% Xgal, and further incubated for 1 hour.
[0053]
When the cell suspension was observed under a microscope, about 30% of cells were transfected with DOTAP Liposomal Transfection Reagent, and about 20% of Chariot Transfection Reagent were stained blue with β-galactosidase activity. Was. However, no bacterial cells stained blue were observed in those containing no transfection reagent.
[0054]
Example 2 <Introduction of DNA-protein complex>
(1) Tn5 transposase was purified by the method of CRUZ et al. (Journal of Bacteriology, Vol. 175, No. 21, p. 6932-6938, 1993).
[0055]
(2) Kanamycin- resistant Tn5 transposon EZ :: TN ^™ <KAN2> Transposon (EPICENTER TECHNOLOGIES) 15 μg / ml solution 0.4 μl (0.0075 pmol) and Tn5 transposase 0.4 μl (corresponding to 0.1 pmol) are gently tapped. And incubated at 37 ° C. for 10 minutes.
[0056]
(3) Dispense 0.4 μl of the incubated mixture into three microtubes, leave the first one as it is, the second as a cationic liposome DOTAP Liposomal Transfection Reagent (Roche) 0.4 μl, and the third one as Chariot Kit 2.0 ml of Chariot Transfection Reagent (2 mg / ml from Active Motief) was mixed at room temperature and left for 15 minutes.
[0057]
(4) E. coli electrocompetent cell DH5α (TAKARA) was added to each of the above three microtubes, left on ice for 5 minutes, and then 1 ml of SOC medium warmed to 37 ° C was added. The cells were cultured with shaking for 1 hour, applied to an LB medium plate containing kanamycin, and incubated at 37 ° C overnight.
[0058]
For 1 μg of transposon, 4.0 × 10 ³ strains obtained by mixing DOTAP Liposomal Transfection Reagent, and 2.0 × 10 ³ strains obtained by mixing Chariot Transfection Reagent showed kanamycin- resistant transformants. No transformant was obtained without the addition of the injection reagent.
[0059]
(5) No plasmid was present in the transformants obtained at this time, and it was found that these transformants had the gene introduced into the genome by transposase.
[0060]
Example 3 <Introduction of metal fine particles>
Magnetic particles: Streptavidin MagneSphere Paramagnetic Particles (Promega) was collected by centrifugation, and 0.6 mg was suspended in Milli-Q water at 10 μg / μl. Escherichia coli DH5α cells containing pUC19 plasmid exhibiting ampicillin resistance are cultured in an LB medium, collected by centrifugation at OD600 = 0.6, washed three times with Milli-Q water, and adjusted to OD600 = 10.0. Was suspended in Milli-Q water.
[0061]
The above E. coli was dispensed in 50 μl portions into five microtubes and placed on ice. The five microtubes were named a, b, c, d, and e, and each of the microtubes had the following composition.
a: E. coli only b: E. coli + magnetic particles 5 μl
c: 5 μl of Escherichia coli + DOTAP (Roche 1 μg / 1 μl)
d: E. coli + 5 μl of magnetic particles + 5 μl of DOTAP (added separately)
e: Escherichia coli + (5 μl of magnetic particles + 5 μl of DOTAP incubated at 37 ° C. for 5 minutes).
[0062]
Each was left still on ice for 5 minutes, then heat shocked at 42 ° C for 90 seconds, and suspended in 1 ml of 37 ° C SOC culture solution. The mixture was allowed to stand for 1 minute using MagneSphere Technology Magnetic Separation Stands to collect a fraction containing magnetic particles, and the supernatant was removed.
[0063]
The same operation was repeated three times to collect a fraction containing magnetic particles. The collected cells were suspended in 0.1 ml of SOC culture solution, inoculated on an LB plate containing 50 μg / ml of ampicillin, cultured at 37 ° C. overnight, and the number of colonies formed was counted.
[0064]
One colony was formed and b was 87 strains, and it was shown that e was 87 strains, indicating that the bacterial cells were recovered by the magnetic particles. As a result of observing the bacterial cells of e in an enlarged manner, black particles considered to be magnetic particles were observed in the cells, but no black particles were observed in the cells of the cells under other conditions.
[0065]
【The invention's effect】
According to the method of introducing a peptide, a DNA protein complex or a metal microparticle into a prokaryotic cell according to the present invention, when a nucleotide is introduced into a prokaryotic cell having a cell wall composed mainly of peptidoglycan, a transfection reagent is allowed to coexist. It can be introduced simply, safely and efficiently.
[0066]
In addition, the method of the present invention can be performed by anyone because the operation is simple, does not require skilled technology, and does not use a special machine or the like. For example, it can be used for experiments, practical training, and the like in elementary schools, junior high schools, high schools, universities, vocational schools, and the like.

Claims (7)

A method for introducing a peptide or DNA protein complex into a prokaryotic cell having a cell wall mainly composed of peptidoglycan, wherein the method is performed in the presence of a transfection reagent. A method for introducing metal microparticles into a prokaryotic cell having a cell wall mainly composed of peptidoglycan, wherein the method is performed in the presence of a transfection reagent. The method according to claim 1, wherein the transfection reagent is a lipid-based reagent. The method according to claim 1, wherein the transfection reagent is a cationic liposome. The method according to claim 1, wherein the prokaryotic cell is Escherichia coli. The method according to claim 2, wherein the metal fine particles are at least one selected from the group consisting of iron, titanium oxide, platinum, and gold. A transfection kit comprising a peptide, a DNA protein complex or metal microparticles and a transfection reagent.