JP6959626B2 - Proton pump and its use - Google Patents

Description

The present invention relates to a proton pump in a living body and its use.

Methods of ion transport inside and outside the cell membrane include active transport, which transports ions in a certain direction using energy, and passive transport, which transports ions by the difference in ion concentration inside and outside the membrane and the membrane potential. An ion transporter that carries out active transport is called an ion pump, and an ion transporter that carries out passive transport is called an ion channel.

A proton pump that actively transports protons (hydrogen ions) is involved in the synthesis and energy conversion of ATP (adenosine triphosphate) at the cell membrane in the living body. That is, the proton pump transports protons on the cytoplasmic side to the outside of the cell. As a result, ATP synthase works by utilizing the generated proton concentration gradient, and ATP is synthesized. ATP is a molecule that drives various life phenomena such as moving muscles and exciting nerves.

Due to the importance of such proton active transport control, the present inventors have been conducting research on proton transport so far. In addition, as described in Non-Patent Document 1, the present inventors use one amino acid of Anabaena sensory rhodopsin (ASR), which acts as an optical sensor in algae bacteria (aspartic acid at position 217 to glutamate). It has been found that when a complex of a substituted protein and retinal is expressed in Escherichia coli and irradiated with light, proton transport from the outside of the cell to the cytoplasmic side is performed. The proton transport by the complex of the mutant protein and retinal described in Non-Patent Document 1 was confirmed in Escherichia coli using an artificial protein in which an amino acid was mutated. I don't know if it's done.

Engineering an inward plot transport from bacterial sensor rhodopsin. J. Am. Chem. Soc. (2009) Kawanabe, A. et al. et al.

The proton pump has a direction of transporting protons on the cytoplasmic side to the outside of the cell. Therefore, it was thought that a proton pump that transports protons outside the cell, which interferes with ATP synthesis, to the cytoplasm side does not exist in nature because it is contrary to a biological phenomenon.

On the other hand, a proton pump that actively transports protons outside the cell to the cytoplasm side is an experimental tool that controls the increase in the amount of protons in the cell by introducing, for example, a gene expressing the proton pump into the cell using a vector. In addition, it is expected to be applied to a therapeutic method in which a proton pump is specifically expressed in cancer cells to bring the cancer cells to a low pH state and kill them, so that the added value is high. In general, it is known that the proton pump does not orient in the opposite direction to the cell membrane, so that it is difficult to apply the proton pump that transports the proton from the cytoplasmic side to the outside of the cell to the above-mentioned treatment method. It is considered.

A main object of the present invention is to provide a proton pump that actively transports protons from the outside of a cell to the cytoplasmic side.

According to the present invention, the following means are provided.
[1] A proton pump having a complex of a chromophore composed of retinal or a retinal derivative and a photoreceptive protein, and actively transporting hydrogen ions from the outside of the cell to the cytoplasm side by light energy.
[2] The photoreceptive proteins are described in (A) and (B) below.
(A) PoXeR protein In the amino acid sequence of (B) PoXeR protein, one or more amino acids are deleted, substituted, inserted and / or added, and hydrogen ions are extracellularly generated by light energy in the complex with the chromophore. The proton pump according to the above [1], which is one of the proteins having a proton pump activity that actively transports the protein from the cell to the cytoplasmic side.
[3] The proton pump according to the above [2], wherein the photoreceptive protein has an amino acid sequence satisfying at least one of the following a) to d).
a) Substitute 119th asparagic acid (hereinafter referred to as D) in the amino acid sequence of PoXeR protein with glutamic acid (hereinafter referred to as E), asparagine (hereinafter referred to as N), or glutamine (hereinafter referred to as Q) b) PoXeR protein Replace E at position 158 with Q, D, or N in the amino acid sequence of c) Replace D at position 216 with E, N, or Q in the amino acid sequence of PoXeR protein d) E at position 219 in the amino acid sequence of PoXeR protein Is replaced with Q, D, or N [4] One or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of the PoXeR protein, and a complex with a chromophore consisting of retinal or a retinal derivative. A protein with proton pump activity that actively transports hydrogen ions from the outside of the cell to the cytoplasm side by light energy.
[5] The protein according to the above [4], which has an amino acid sequence satisfying at least one of the above a) to d).
[6] A gene encoding the protein according to the above [4] or [5].
[7] An expression vector that retains a coding region encoding the protein according to the above [4] or [5].
[8] A transformant into which the expression vector of the above [7] has been introduced.
[9] A kit containing the gene according to the above [6] or the expression vector according to the above [7].
[10] A kit for controlling intracellular hydrogen ion concentration by light energy, which comprises an expression vector carrying a gene encoding a PoXeR protein or a coding region encoding a PoXeR protein.

Since the proton pump of the present invention transports protons from the outside of the cell to the cytoplasm side by active transport by light energy, the amount of protons on the cytoplasm side or the outside of the cell can be controlled by adjusting the amount of light energy. In addition, by using mammalian cells, it is possible to develop them for medical purposes.

Schematic diagram of the retinal-PoXeR protein complex. The figure which shows the result of the pump activity assay of the retinal-PoXeR protein complex in Escherichia coli cells. Absorption spectrum of retinal-PoXeR protein complex. The figure which shows the result of the pump activity assay when NaCl, CsCl, and Na ₂ SO _{4 were added to the outside of a cell.} (A) shows the result of the retinal-PoClR protein complex, and (b) shows the result of the retinal-PoXeR protein complex. The figure which shows the result of the electrophysiological experiment in the mammalian cell of the retinal-PoXeR protein complex. The figure which shows the result of the pump activity assay of the complex of retinal and mutant PoXeR protein in Escherichia coli cells.

Hereinafter, embodiments of the present invention will be described with reference to the drawings. The present invention is not limited to the following embodiments, and changes, modifications, and improvements can be made without departing from the scope of the invention.

The proton pump of the present invention has a complex (chromophore-protein complex) of a chromophore composed of retinal or a retinal derivative and a photoreceptive protein.

(Chromophore)
The chromophores, retinal and retinal derivatives, isomerize from the trans form to the cis form by absorbing light, causing structural changes in the protein portion of the chromophore-protein complex. Examples of the retinal include all trans retinal, 11-cis retinal, 13-cis retinal, 9-cis retinal and the like.

The retinal derivatives are preferably 3,4-dehydroretinal, 13-ethylretinal, 9-dm-retinal, 3-hydroxyretinal, 4-hydroxyretinal, naphthylretinal, 3,7,11-trimethyl-dodeca-2, 4,6,8,10 pentaenal, 3,7 dimethyl-deca-2,4,6,8-tetraenal, 3,7-dimethyl-octa-2,4,6-trienal, 6-7 rotation inhibition (rotation-) It is selected from the group consisting of blocked) retinal, 8-9 rotation blocking retinal, and 10-11 rotation blocking retinal.

(Photoreceptive protein)
A photoreceptive protein is a protein that receives light as a stimulus in a state of forming a complex with a chromophore. In the chromophore-protein complex, one of the photoreceptive proteins is bacteriorhodopsin. One of the forms of proteins constituting the chromophore-protein complex, which functions as a proton pump that transports protons outside the cell to the cytoplasm side by using light energy, includes PoXeR protein existing in nature. By using the PoXeR protein derived from the genome of an organism existing in nature, it is possible to obtain an artificial proton pump that is structurally stable and whose function is optimized.

The PoXeR protein is a protein reported by Parvularcula oceani as rhodopsin of unknown function in the non-patent literature (Mar. Genomics 24, 211-213 (2015) Tang, K. et al.). The amino acid sequence of PoXeR is according to the National Center for Biotechnology Information (NCBI), ACCESS No. It is stored in the database as WP_051881467.

Examples of the photoreceptive protein constituting the chromophore-protein complex include variants of the PoXeR protein. It is unlikely that all amino acids in the PoXeR protein are involved in proton transport. Proton pump activity in which one or more amino acids of the wild-type PoXeR protein are deleted, substituted, inserted and / or added, and hydrogen ions are actively transported from the outside of the cell to the cytoplasm side by light energy in the complex with the chromophore. According to the complex with the protein, it is possible to maintain or improve the active transport function of protons to the cytoplasm side.

For example, amino acids other than the transmembrane region are unlikely to be involved in proton transport. Therefore, even if one or more amino acids in the region other than the transmembrane region of the PoXeR protein, that is, the extracellular region and / or the intracellular region are deleted, substituted, inserted and / or added, proton active to the cytoplasm side is performed. It is thought that the transport function will be maintained. In fact, the present inventors are a protein in which six histidines are added to the C-terminal side, which is the intracellular region of the PоXeR protein (homologity with the PoXeR protein is 97%), and the active proton from the outside of the cell to the cytoplasmic side. We are confirming the transportation function.

When the photoreceptive protein is a variant of the PoXeR protein, the amino acid mutation in the amino acid sequence of the PoXeR protein may be any of deletion, substitution, insertion and addition, and two or more of these. It may be a combination. The total number of amino acid mutations is not particularly limited as long as it has a proton pump activity that actively transports hydrogen ions from the outside of the cell to the cytoplasm side by light energy in the complex with the chromophore, but it is preferably 1 or more and 10 or less. It is more preferably 1 or more and 6 or less.

Amino acid substitutions are preferably by conservative substitutions. By "conservative substitution" is meant that the substitution of an amino acid residue is carried out by an amino acid residue having a side chain of similar nature. Specifically, for example, amino acids having a basic side chain (eg, lysine, arginine, histidine), amino acids having an acidic side chain (eg, aspartic acid, glutamic acid), amino acids having an uncharged polar side chain (eg, asparagine, glutamine). , Serin, threonine, tyrosine, cysteine), amino acids with non-polar side chains (eg glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (eg threonine, valine) , Isoleucine), amino acids with aromatic side chains (eg, tyrosine, phenylalanine, tryptophan) are substituted in groups with side chains of similar nature. Further, even if it does not depend on conservative substitution, it is preferably applied if it is possible to actively transport hydrogen ions from the outside of the cell to the cytoplasm side by light energy in the complex with the chromophore.

As a variant of PoXeR protein, it has 80% or more identity with the amino acid sequence of wild-type PoXeR protein, and in the complex with the chromophore, hydrogen ions are actively activated from the outside of the cell to the cytoplasmic side by light energy. Examples include proteins with proton pump activity to transport. The identity is preferably 85% or more, more preferably 90% or more, still more preferably 95% or more. The identity of the amino acid sequence can be determined by using an analysis program such as BLAST.

The photoreceptive protein preferably has an amino acid sequence satisfying at least one of the following a) to d) in that a protein complex having a higher proton transport efficiency from the outside of the cell to the cytoplasmic side can be obtained. ..
a) Substitute aspartic acid (hereinafter referred to as D) at position 119 in the amino acid sequence of the PoXeR protein with glutamic acid (hereinafter referred to as E), asparagine (hereinafter referred to as N), or glutamine (hereinafter referred to as Q).
b) Substitute E at position 158 with Q, D, or N in the amino acid sequence of the PoXeR protein.
c) Substitute D for E, N, or Q in the 216th amino acid sequence of the PoXeR protein.
d) Replace E with Q, D, or N in the 219th amino acid sequence of the PoXeR protein.

A protein having an amino acid sequence satisfying at least one of the above a) to d) is an artificial protein having a higher proton transport efficiency from the outside of the cell to the cytoplasm side than a protein derived from the genome of a naturally occurring organism. Therefore, it is preferable in that the applicable range of the proton pump can be expanded. The protein may consist of an amino acid sequence satisfying one of the above a) to d), or may consist of an amino acid sequence in which two or more of these are combined. Further, as long as it has a proton pump activity that actively transports hydrogen ions from the outside of the cell to the cytoplasmic side by light energy in a complex with a chromophore composed of retinal or a retinal derivative, at least one of the above a) to d). One or more amino acids may be deleted, substituted, inserted and / or added to a portion other than the amino acid position.

(Complex of retinal and PoXeR protein)
FIG. 1 is a schematic diagram of a complex 10 of retinal 17, which is one of the preferred forms of a chromophore-protein complex, and a wild-type or mutant PoXeR protein (hereinafter, also referred to as “PoXeR protein 11”). Is shown. When expressed in the host, the complex 10 is present on the cell membrane such that the N-terminal side 13 is located on the cell outer side 5 and the C-terminal side 15 is located on the cytoplasmic side 7, as shown in FIG. Specifically, the PoXeR protein 11 penetrates the cell membrane 3 and is arranged so that the N-terminal side 13 of the PoXeR protein 11 is in contact with the cell outer side 5 and the C-terminal side 15 is in contact with the cytoplasmic side 7. In complex 10, retinal 17 is attached to PoXeR protein 11. Reference numeral 12 indicates the mutation position of the amino acid when the PoXeR protein 11 is a mutant type. FIG. 1 shows the position of the 216th amino acid from the N-terminal side of the PoXeR protein.

When light irradiation is started by the light source 31, retinal 17 is isomerized by absorbing light, and a structural change of PoXeR protein 11 occurs. As a result, the proton 21 existing on the cell outer side 5 passes through the PoXeR protein 11 and is transported to the cytoplasmic side 7.

FIG. 3 shows the absorption spectrum of the retinal-PoXeR protein complex when expressed in Escherichia coli. In order for the retinal-PoXeR protein complex to carry out proton transport, according to FIG. 3, it is preferable to irradiate light having a wavelength of 500 nm to 615 nm, and particularly preferably to irradiate light (green light) in the vicinity of 570 nm. ..

(Expression vector)
The expression vector of the present invention is a vector having a coding region encoding a protein consisting of an amino acid sequence satisfying at least one of the above a) to d). These vectors can be used as experimental tools that control the increase in the amount of protons in cells by being introduced into cells and expressing the gene of interest, or by expressing a proton pump specifically for cancer cells to express cancer cells. It can be used when applied to a therapeutic method or the like that causes death in a low pH state.

In the expression vector, the type of vector into which the gene is inserted is not limited to this, but is not limited to Escherichia coli-derived vectors (for example, pET28, pGEX4T, pUC118, pUC119, pUC18, pUC19, and other vectors. DNA), vectors derived from Bacillus subtilis (eg pUB110, pTP5, and other vector DNA), vectors derived from yeast (eg YEp13, YEp24, YCp50, and other vector DNA), λ phage (λgt11, λZAP) Etc.), mammalian plasmids (pCMV, pSV40), virus vectors (adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, vaccinia virus vector, animal virus vector such as Sendai virus vector, baculovirus vector, etc. Insect virus vector, etc.), plant vector (binary vector pBI system, etc.), cosmid vector, etc. can be used.

The gene is usually expressibly linked downstream of the promoter in a suitable vector. The type of promoter used is not particularly limited, and various promoters can be used depending on the host to be transformed. Further, the expression vector of the present invention may have an enhancer, a poly A addition signal, a splicing signal, a ribosome binding sequence, a selectable marker and the like, if desired.

(Transformant)
The transformant of the present invention is the one into which the above expression vector has been introduced. The vector introduction method can be appropriately selected according to the type of vector and host, and specific examples thereof are not limited to this, but for non-viral vector systems, the lipofection method and electroporation are used. A method, a microinjection method, a protoplast method, or the like can be used, and a virus vector system can be introduced by infecting a virus. The host is not particularly limited as long as it can express a gene encoding a protein consisting of an amino acid sequence satisfying at least one of the above a) to d). By using mammalian cells as a host, for example, it is possible to develop a therapeutic method in which a proton pump is specifically expressed in a cancer cell and the cancer cell is brought to a low pH state to be killed. Examples of mammalian cells include humans, cows, mice, rats, Chinese hamsters and the like.

(kit)
The kit of the present invention contains a gene encoding a protein consisting of an amino acid sequence satisfying at least one of the above a) to d), or a vector into which the gene is inserted. The kit of the present invention may contain other components other than these genes or vectors, either integrally or separately from the genes or vectors. Examples of such other components include solutions, buffers, reagents, salts and the like. The kits of the present invention include, for example, studying the mechanism of hydrogen ion concentration gradient in animal cells and plant cells, cancer treatment kits, alternative kits for proton pump inhibitors, tools for controlling neural activity, neurotransmitters in synaptic vesicles. It can be used for control of.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

(Gene construction, protein expression and purification)
The PoXeR gene was prepared by gene synthesis of a codon-optimized gene for expression in Escherichia coli, and a 6 × His tag for purification was added to the C-terminal side. This was introduced into the pET21a (+) vector. The amino acid mutant was prepared by using the QuikChange method. Wild-type and mutant proteins are 0.21 mM isopropyl β-D-thiogalactopyranoside in a suspension of E. coli C43 (DE3) strain transformed with the PoXeR gene, along with 10 μM total transretinal (sigma-aldrich). Was induced at 37 ° C. for 4 hours for expression. After that, the purification of PoXeR was based on the method of Inoue et al. (Inoue et al., Nat.Commun., 4, 1678 (2013)) in the past. Escherichia coli cells were disrupted by a French press, and membrane fragments were collected by ultracentrifugation under the condition of 125,000 × g. Solubilized in 5, mM imidazole, 50 mM MES (pH 6.5). Further, ^{after purification using Co 2+} -NTA affinity chromatography, the recovered fraction was dialyzed against 0.1% DDM, 100 mM NaCl, 50 mM Tris-HCl (pH 8.0) to remove imidazole.

(Example 1)
First, the ion transport of the retinal-PoXeR protein complex was investigated. A wild-type PoXeR protein was used (the same applies to Examples 2 and 3). FIG. 2 shows the results of a pump activity assay for the retinal-PoXeR protein complex in E. coli cells.

The light-driven pump activity assay with the retinal-PoXeR protein complex in Escherichia coli cells was performed as follows.
Escherichia coli expressing PoXeR was recovered by centrifugation (4,800 × g, 3 minutes), suspended and washed with _{100 mM salt (NaCl, CsCl or Na 2} SO _4). For this Escherichia coli cell, 7.5 mL of a suspension in which the cell concentration was adjusted so that the turbidity at 660 nm became 2, was used for the measurement. After placing the suspension in a dark condition, a long-pass glass filter is used to irradiate light from a 1-kW tungsten halogen projector lamp that cuts light on the wavelength side shorter than 500 nm, and the photo-induced pH change that occurs at that time is measured by a pH meter. The pump activity of the retinal-PoXeR protein complex was assayed by measurement using. The same measurement was also carried out under the condition that 10 μM Carbonyl cyanide m-chromopheneyl hydrazine (referred to as CCCP in FIG. 2, hereinafter referred to as CCCP) was added.

In FIG. 2, the vertical axis represents the pH outside the cell and the horizontal axis represents the time (seconds). Light irradiation started at 0 s and ended at 150 s. The solid line shows when CCCP is not added, and the broken line shows when CCCP is added. Here, CCCP has a function of increasing the permeability of the cell membrane of protons. Therefore, when CCCP is added, the pH on the outside of the cell and the pH on the cytoplasm side are close to each other when there is no difference in the charge due to ions other than protons inside and outside the cell.

-Results and discussion In Escherichia coli expressing the retinal-PoXeR protein complex, as shown by the solid line in FIG. 2, the pH outside the cells increased when irradiated with light in the absence of CCCP. It is considered that this is because the protons on the outside of the cell are transported to the cytoplasm side, so that the protons on the outside of the cell are reduced and the pH is increased. When CCCP was added, the pH outside the cells hardly changed during irradiation with light, as shown by the broken line in FIG. When CCCP was added, it is considered that the change in the amount of protons was small because the protons transported to the cytoplasm side permeated the cell membrane and returned to the cytoplasm side due to the influence of CCCP. These results suggest that the retinal-PoXeR protein complex transports protons from the outside of the cell to the cytoplasmic side by light energy.
Proteins derived from the genomes of naturally occurring organisms are generally considered to be structurally stable and functionally optimized proteins adapted to the environment during evolution. Therefore, it is considered that the proton pump using PoXeR protein exhibits a stable proton transport function when applied to experimental tools and therapeutic methods.

(Example 2)
Next, the ion transport of the retinal-PoXeR protein complex when NaCl, CsCl, and Na ₂ SO _{4 were added to the outside of the cell was investigated.} The result is shown in FIG. FIG. 4 (a) ^{shows the results of the retinal-PoClR protein complex having the function of transporting Cl −} inward for comparison, and FIG. 4 (b) shows the results of the retinal-PoXeR protein complex. .. In FIG. 4, the vertical axis represents the pH outside the cell and the horizontal axis represents the time (seconds). The graph shows the results when _{NaCl, CsCl, and Na 2} SO ₄ are added to the outside of the cell in order from the top.

If the protein complex has the function of transporting any of the ions constituting NaCl, CsCl, and Na ₂ SO ₄ from the outside of the cell to the inside of the cell, the graph of the experimental result in which three kinds of substances are added is different. Is thought to appear. In addition, it is considered that the effect on the transport function when NaCl, CsCl, and Na ₂ SO _{4 are present extracellularly can be confirmed.} In FIG. 4, for each of NaCl, CsCl, and Na ₂ SO _{4, the} time when CCCP is not added is shown by a solid line, and the time when CCCP is added is shown by a broken line. Further, in the graph, NaCl, CsCl, and Na ₂ SO ₄ are displayed in order from the top, but the value of each pH at 0 s, which is the start time of light irradiation, is the same, and the difference in pH depending on the presence or absence of CCCP is displayed. Shown.

First, regarding the retinal-PoClR protein complex, in the absence of CCCP, when NaCl and CsCl were added as shown by the solid line in FIG. 4 (a), the pH outside the cells increased with light irradiation. This is because Cl ⁻ was transported to the cytoplasmic side and the cytoplasmic side became electrically negative, so that the protons on the outside of the cell (H ⁺ ) were electrically transferred to the cytoplasmic side, and as a result, the protons on the outside of the cell were transferred. It is probable that this was due to the decrease in pH and the increase in pH. Further, when CCCP was added, as shown by the broken line in FIG. 4A, the pH outside the cells became higher than that when CCCP was not added. It is considered that this is because the addition of CCCP increased the permeability of the cell membrane and the protons on the outside of the cell moved to the cytoplasm side.

On the other hand, when adding _{over Na} 2 SO _4, the "+ CCCP" and no difference in the change of extracellular pH in "-CCCP", Cl extracellularly ^- indicates that the ion transport is not performed because there is no rice field.

For the retinal-PoXeR protein complex, as shown by the solid line in FIG. 4 (b), in the absence of CCCP, when NaCl, CsCl, and Na ₂ SO ₄ were added, the pH outside the cell was determined by irradiation with light. Has become higher. On the other hand, in the presence of CCCP, as shown by the broken line in FIG. 4 (b), the pH outside the cells hardly changed even when irradiated with light. This is because in the absence of CCCP, the protons on the outside of the cell are transported to the cytoplasm side, so that the protons on the outside of the cell decrease and the pH becomes high, and in the presence of CCCP, the protons transported to the cytoplasm side are transferred to the cell membrane. It is considered that this is because the cells permeated and returned to the cytoplasmic side.

In this way, in Escherichia coli expressing the retinal-PoXeR protein complex, when NaCl, CsCl, and Na ₂ SO ₄ were added to the outside of the cell and irradiated with light, there was a difference in the graph of the experimental results in which three kinds of substances were added. It hardly appeared. It was also confirmed that the extracellular pH of "-CCCP" increased with respect to the extracellular pH of "+ CCCP". These results suggest that the retinal-PoXeR protein complex transports protons from the outside of the cell to the cytoplasmic side using light energy as a driving source. In addition, since the function was confirmed even when NaCl, CsCl, and Na ₂ SO ₄ were added to the outside of the cell, the retinal-PoXeR protein complex was not affected by the environmental state outside the cell, and was transferred from the outside of the cell to the cytoplasm side. It was confirmed that it has a function of transporting protons.

(Example 3)
The retinal-PoXeR protein complex was expressed in mammalian cells and the ion transport of the retinal-PoXeR protein complex was analyzed. In the experiment using Escherichia coli in Example 1, the intracellular potential could not be changed, and the intracellular potential could be measured only at one level in the normal state. However, if it can be expressed in mammalian cells, It is possible to change the intracellular potential, and it is possible to investigate whether the proton transport method is active transport or passive transport. Further, by using mammalian cells, for example, it is possible to develop a therapeutic method in which a proton pump is specifically expressed in a cancer cell and the cancer cell is brought to a low pH state to be killed.

(Expression in mammalian cells)
The PoXeR gene optimized for human codon was prepared by gene synthesis and cloned between the HindIII site and the BamHI site of the pEGFP vector. For expression, ND7 / 23 cells purchased from DS Pharma Biomedical were used and cultured in a high glucose DMEM medium (Wako Pure Chemical Industries, Ltd.) at 37 ° C. in a 5% CO ₂ environment. Transfection of the plasmid containing the PoXeR gene into ND7 / 23 cells was performed using Lipofectamine 2000 (Invitrogen). Further, 1 μM total transretinal (sigma-aldrich) was added to the medium obtained by transfecting the cells. Protein expression was confirmed using a fluorescence microscope (IX-73, Olympus). As a result, the expression in ND7 / 23 cells, which are mammalian cells, was confirmed. From this result, it is considered that the expression in human cells is possible.

(Electrophysiology experiment)
Next, the retinal-PoXeR protein complex was expressed in mammalian cells (ND7 / 23 cells), and the intracellular potential was changed to confirm the active transport of protons from the retinal-PoXeR protein complex. The measurement was performed by the whole cell patch clamp method, specifically as follows.
-Measurement method The whole cell patch clamp measurement was performed using an amplifier (Axopatch 200B). Continuous light irradiation was performed by introducing light from OSG L12194-00-39070 (Hamamatsu Photonics) into an inverted microscope (IMT-2, Olympus) using a light guide. The irradiation light was controlled using a mechanical shutter (LS6S, Vincent Associates). The glass pipette was molded using a micropipette pillar (P-97, Sutter Instrument) and fire-finished using a microforge (MF-830, NARISHIGE). The pipette resistance was about 1.5-2.5 MΩ. Pipette electrodes were operated using a micromanipulator (PCS-5000, Burleigh instruments). The current was recorded at 10 kHz and filtered by an internal circuit in the amplifier at 2 kHz. Data storage and shutter opening and closing were performed by software pClamp 10 using Digidata 1550. The obtained data was analyzed using software Clampfit 10.
140 mM NaCl, 2 mM MgCl ₂ , 2 mM CaCl ₂ , 2 mM KCl, 10 mM HEPES-NaOH (pH 7.2) was used as the standard external solution. On the other hand, 110 mM NaCl, 2 mM MgCl ₂ , 1 mM CaCl ₂ , 5 mM KCl, 10 mM EGTA, and 10 mM HEPES-NaOH (pH 7.2) were used as the standard internal solution. The osmotic pressure of the solution was adjusted to 300 mOsm by adding an appropriate amount of sucrose.

-Results and discussion Fig. 5 shows the results of electrophysiological experiments on mammalian cells of the retinal-PoXeR protein complex. FIG. 5A is a measurement result of the current of the cell membrane when irradiated with light (570 nm) with the outside pH of the cell set to 7.2. The parameter was the voltage on the cytoplasmic side, and the voltage on the cytoplasmic side was measured as 20 mV, 0 mV, -20 mV, -40 mV, and -60 mV, respectively.
As shown in FIG. 5 (a), when the outside of the mammalian cell expressing the retinal-PoXeR protein complex was irradiated with light, an electric current was generated and the value dropped once. In addition, a stable current flowed while irradiating with light. The stable current value (absolute value) was the largest when the voltage on the cytoplasm side was -60 mV and the smallest when the voltage was 20 mV. Further, the lower the voltage value (in the order of -60 mV, -40 mV, -20 mV, 0 mV, 20 mV), the larger the current value (absolute value) at the time of stabilization. This indicates that the lower the intracellular voltage, the larger the current flows. When the light irradiation was stopped, the difference in the current value due to the voltage on the cytoplasmic side disappeared, and the current value returned to the original value without the light irradiation.

FIG. 5B shows the current measurement results when light (570 nm) was applied to mammalian cells (ND7 / 23 cells) expressing the retinal-PoXeR protein complex. The horizontal axis is the voltage on the cytoplasmic side and the vertical axis is the vertical axis. Is the current value at the time of stabilization when irradiated with light. Black circles are the results when the extracellular pH is 7.2, and white circles are the results when the extracellular pH is 9.0. The experimental line with black circles is shown by a solid line, and the experimental line with white circles is shown by a broken line. Further, in FIG. 5 (b), using the experimental value of pH 7.2, the theoretical value at pH 9.0 when the retinal-PoXeR protein complex is assumed to be a channel is shown by a broken line (broken line above). Shown. As shown by the dashed line (dashed line above), when the retinal-PoXeR protein complex is a channel, it is expected that the increase in extracellular pH will reduce proton transport.

As shown in FIG. 5 (b), in mammalian cells expressing the retinal-PoXeR protein complex, the outside pH of the cells was 7.2 (solid line) and 9.0 (broken line), which were almost the same regardless of the voltage value. It showed the same current value. This indicates that the retinal-PoXeR protein complex has the same transport capacity without being affected by the pH (proton amount) outside the cell, and instead of passive transport, which transports by an ion concentration gradient, active transport is performed. It turned out that I was doing it. That is, it can be said that the PoXeR protein has a function as a proton pump that actively transports protons from the outside of the cell to the cytoplasm side by light energy.

Further, in the result of FIG. 5B, the intersection of the solid line which is the approximate line of the black circle (pH 7.2) and the current value of 0 pA is 164 mV, and the broken line and the current which are the approximate lines of the white circle (pH 9.0). The intersection with the value of 0 pA was 150 mV. In addition, from the result of FIG. 5 (b), the voltage on the cytoplasmic side ranges from -75 mV to 150 mV, and the opposite active transport of protons by the PoXeR protein by light energy in the mammalian cell (from the cytoplasmic side to the outside of the cell). It has been demonstrated that active transport) takes place.
Since the voltage range of -75 mV to 150 mV covers the voltage that can be generated in the cell, it can be said that it can be applied to various cell types in all states. For example, a gene that expresses a proton pump can be introduced using a vector to control the increase in the amount of protons in the cell.

(Example 4)
Next, a mutant of PoXeR protein was prepared and the proton transport ability was measured. FIG. 6 shows the results of a pump activity assay when the retinal-PoXeR protein complex was expressed in E. coli cells using wild-type or mutant PoXeR protein. In FIG. 6, (a) shows the time change of the extracellular pH when irradiated with light, and (b) is the rate of pH change (ΔpH / s) until the extracellular pH reaches its peak in the absence of CCCP. Is shown. The pump activity assay was performed in the same manner as in Example 1. The light irradiation was started at 0 seconds and 500 seconds on the horizontal axis scale of FIG. 6A, and the light irradiation time was 150 seconds.

The mutant is to replace aspartic acid (D) and glutamic acid (E) contained in the α-helix of the wild PoXeR protein with other amino acids (glutamine (Q), aspartic acid, glutamic acid, asparagine (N)). A total of 11 variants shown in FIG. 6 were prepared. The display in FIG. 6 corresponds to the following.
PoXeR WT: WT of PoXeR protein
E3Q: Replace the third E from the N-terminal side of the PoXeR protein with Q E35D: Replace the 35th E from the N-terminal side of the PoXeR protein with D E35Q: Replace the 35th E from the N-terminal side of the PoXeR protein with Q Substitution D74E: Replace the 74th D from the N-terminal side of the PoXeR protein with E D108E: Replace the 108th D from the N-terminal side of the PoXeR protein with E D108N: Replace the 108th D from the N-terminal side of the PoXeR protein with N Replaced with D119N: Replaced the 119th D from the N-terminal side of the PoXeR protein with N E158Q: Replaced the 158th E from the N-terminal side of the PoXeR protein with Q D216E: Replaced the 216th D from the N-terminal side of the PoXeR protein with Q Replaced with E D216N: Replaced 216th D from N-terminal side of PoXeR protein with N E219Q: Replaced 219th E from N-terminal side of PoXeR protein with Q

In this example, the expression level of the retinal-protein complex was quantified so that the difference in protein expression level did not affect the proton transport capacity, and the rate of pH change immediately after light irradiation was determined by that of the retinal-protein complex. The ion transport activity of wild-type and mutant proteins was compared by the rate of change per expression level.
(Quantification of expression level of retinal-protein complex in E. coli cells)
The expression level of the retinal-protein complex in Escherichia coli was quantified with reference to the method of Inoue et al. (Inoue et al., Nat. Commun., 4, 1678 (2013)). The cells were collected under the conditions of 4,800 × g and 4 ° C., and then suspended in a solvent of 100 mM NaCl and 50 mM Tris-HCl (pH 8.0) to adjust the volume to 3 mL. 200 μL of 1 mM lysozyme solution was added, and the mixture was slowly mixed at room temperature for 1 hour. E. coli cells were crushed by ultrasound and solubilized with 3.0% DDM. For the mutants to which the final concentration of hydroxylamine (HA) was added and the absorption wavelength was shifted to the short wavelength side by the long-pass glass filter (D74E and D108E in FIG. 6), the wavelength was 460 nm, and for the other mutants, the wavelength was shorter than 500 nm. The absorption change representing the bleaching process of the retinal-protein complex by HA, which was generated at that time by irradiating the light of a 1-kW tungsten halogen projector lamp with the side light cut off, was absorbed by ultraviolet visible absorption with an integrating sphere unit attached. It was measured using a photometer. Then, the amount of substance of the retinal-protein complex contained in Escherichia coli was calculated from the amount of absorption of retinal oxime (molar absorption cross section at 360 nm: ε = 33,600 M-1 cm-1) generated at that time.

-Results and discussion As shown by the solid line in FIG. 6 (a), the pH of the outside cells of Escherichia coli expressing the complex using the wild-type and mutant PoXeR proteins increased when irradiated with light. Regarding the rate of change in pH (ΔpH / s), as shown in FIG. 6 (b), the rate of change in pH decreased due to mutation, but all mutants have proton transport ability. Was there. In addition, the pH change rate of the mutants of E3Q, D119N, E158Q, D216 E , and E219Q was higher than that of the wild type (PoWeR WT). From these results, it was possible to increase the proton transport capacity by mutating the amino acid residues at positions 3, 119, 158, 216, and 219 in the amino acid sequence of PoXeR protein. In particular, mutants in which the 119th, 158th, 216th, and 219th amino acid residues were mutated greatly improved the proton transport activity from the outside of the cell to the cytoplasmic side.

From the above, aspartic acid and glutamic acid located at the 119th, 158, 216, and 219th positions from the N-terminal side of the protein, which are artificial proteins having a higher transport function than the proteins derived from the genomes of living organisms existing in nature, are used. A substituted variant was found (Fig. 6).

The chromophore-protein complex of the present invention has high added value because the intracellular hydrogen ion concentration can be manipulated by light energy. The chromophore-protein complex, gene, expression vector and transformant of the present invention can be utilized, for example, as follows.
(1) Use for research on hydrogen ion concentration gradient mechanism It can be used as basic research on the mechanism of hydrogen ion concentration gradient. For example, it is sold as a reagent kit or the like as DNA containing the PoXeR gene for genetic manipulation of animal cells used in basic research.
(2) Utilization in cancer treatment The chromophore-protein complex of the present invention is incorporated into cancer cells and sold as a therapeutic agent or reagent kit that kills cancer cells with light.
(3) Use of proton pump inhibitor as an alternative treatment Express wild-type or mutant PoXeR in patient cells to alleviate proton transport by the proton pump in the body.
(4) Use as a tool for controlling neural activity by making full use of the RGB3 colors of the light source As one of the tools for controlling neural activity using the RGB3 colors of the light source, wild-type or mutant PoXeR protein is used as green light (G). It is used as a proton pump for cytoplasm using. For example, the PoXeR gene is integrated into iPS cells and the like and used for treatment. Examples of tools include R: a chloride pump derived from highly halophilic archaea (known substance), G: a complex of PoXeR protein and retinal (the present invention), and B: channelrhodopsin derived from algae (known substance) for three RGB colors. An example of using is given.
(5) Utilization in the control of neurotransmitters in synaptic vesicles It is used as a reagent capable of specifically expressing PoXeR in synaptic vesicles.

3 Cell membrane 5 Cell outer 7 Cytoplasmic side 10 Complex 11 PoXeR protein 12 Mutation position 13 N-terminal side 15 C-terminal side 17 Retinal 21 Proton 31 Light source

Claims (8)

A proton pump having a complex of a chromophore composed of retinal or a retinal derivative and a photoreceptive protein and actively transporting hydrogen ions from the outside of the cell to the cytoplasmic side by light energy. The photoreceptive protein is a PoXeR protein. In the amino acid sequence of, one or more amino acids are deleted, substituted, inserted and / or added, and a proton pump activity that actively transports hydrogen ions from the outside of the cell to the cytoplasmic side by light energy in the complex with the chromophore A proton pump having an amino acid sequence satisfying at least one of the following a) to d).
a) Substitute aspartic acid (hereinafter referred to as D) at position 119 in the amino acid sequence of PoXeR protein with aspartic acid (hereinafter referred to as N) b) Glutamic acid (hereinafter referred to as E) at position 158 in the amino acid sequence of PoXeR protein (hereinafter referred to as E) Substituted with Q) c) Replaced D at position 216 with E in the amino acid sequence of PoXeR protein d) Replaced E at position 219 with Q in the amino acid sequence of PoXeR protein. One or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of the PoXeR protein, and hydrogen ions are transferred from the outside of the cell to the cytoplasmic side by light energy in a complex with a chromophore consisting of retinal or a retinal derivative. A protein having a proton pump activity that actively transports to, and having an amino acid sequence satisfying at least one of the following a) to d).
a) Replace D at position 119 with N in the amino acid sequence of PoXeR protein b) Replace E at position 158 with Q in the amino acid sequence of PoXeR protein c) Replace D at position 216 with E in the amino acid sequence of PoXeR protein d) Replace E at position 219 with Q in the amino acid sequence of PoXeR protein A gene encoding the protein according to claim 2. An expression vector that retains a coding region encoding the protein according to claim 2. A transformant into which the expression vector of claim 4 has been introduced. A kit comprising the gene according to claim 3 or the expression vector according to claim 4. A kit for controlling the intracellular hydrogen ion concentration by light energy, which comprises the gene according to claim 3 or the expression vector according to claim 4. It is a method of actively transporting hydrogen ions from the outside of cells to the cytoplasm side by light energy, and has a complex of a chromophore composed of retinal or a retinal derivative and a photoreceptive protein.
The photoreceptive proteins are described in (A) and (B) below.
(A) PoXeR protein In the amino acid sequence of (B) PoXeR protein, one or more amino acids are deleted, substituted, inserted and / or added, and hydrogen ions are extracellularly transferred by light energy in the complex with the chromophore. A method of actively transporting hydrogen ions from the outside of a cell to the cytoplasmic side by light energy, which comprises using any of the proteins having proton pump activity for active transporting from the cell to the cytoplasmic side.

Images