JP5544659B2 - Modified photoreceptor channel-type rhodopsin protein - Google Patents

Description

  The present invention relates to a modified photoreceptor channel-type rhodopsin protein, a polynucleotide encoding the protein, an expression vector containing the polynucleotide, and a cell expressing the protein.

Chlamydomonas normally inhabiting freshwater ponds and marshes belong to the green alga, a unicellular eukaryote that has chloroplasts and photosynthesizes. Chlamydomonas has the habit of collecting light (that is, phototaxis) by receiving light in a special membrane region called an eye point and controlling flagellar movement.
The gene sequence of a prokaryotic rhodopsin family protein distributed in the eyes of Chlamydomonas reinhardtii, a Chlamydomonas species, has been elucidated and published (GenBank accession number AF461397). This protein is an example of a photoreceptor channel in the biological world because it transmits ions in response to visible light and controls the membrane potential (Non-Patent Documents 1 and 2).
In Chlamydomonas, there are two types of prokaryotic rhodopsin proteins, channel opsin 1 and channel opsin 2. These are a type of photoreceptor channel, which is a single molecule that has both photosensitivity and ion channel functions. Channel opsin 1 has the property of transmitting H ⁺ in response to light when expressed in frog (Xenopus) oocytes (Non-patent Document 1). It has been reported that channel opsin 2 has a cation permeability such as Na ⁺ and is expressed in cultured mammalian cells (HEK293, BHK) to cause depolarization in response to blue light of 400 to 500 nm. (Non-Patent Document 2).
Photoreceptor channels as described above are expected to be used in various fields including medical care, welfare / care, and information communication.
For example, Patent Document 1 discloses that a nerve cell newly imparted with photosensitivity is created by introducing and expressing channel opsin 2 in a nerve cell using a genetic engineering method. In addition, by applying this technique to in vivo and in vitro systems, attempts have been made to activate a neural cell network by light irradiation (Non-Patent Documents 3 to 6).
Furthermore, it is possible to restore vision by introducing and expressing channel opsin 2 in ganglion cells, bipolar cells and the like using animals in which retinal photoreceptor cells have been degenerated due to various causes ( Non-Patent Documents 7 and 8).
JP 2006-217866 A Nagel et al. , 2002, Science 296, 2395-2398. Nagel et al. , 2003, Proc. Natl. Acad. Sci. USA 100, 13940-13945. Boyden et al. , 2005, Nat. Neurosci. 8, 1263-1268. Li et al. , 2005, Proc. Natl. Acad. Sci. USA 102, 17816-17721. Ishizuka et al. , 2006, Neurosci. Res 54, 85-94. Arenkiel et al. , 2007, Neuron 54, 205-218. Bi et al. 2006, Neuron 50, 23-33. Tomita et al. , 2007, Invest. Ophthalmol. Vis. Sci. 48, 3821-3826.

The above attempts are based on the concept that a photoreceptor channel is introduced and expressed in a eukaryotic cell such as a mammalian cell and used for functional control such as depolarization caused by light irradiation. In such photoreceptor channel applications, the photoreceptor channel is generally desired to have functions such as weak photocurrent inactivation, high conductance, and high frequency response characteristics. Photoreceptor channels have not been found to date.
Also, for example, in an attempt to restore vision of a blind patient by introducing and expressing photoreceptor channels in retinal neurons, it is possible to detect faint light and to respond to white light with low wavelength selectivity. It is desirable to have such functions.
Therefore, it is very useful to provide a photoreceptor channel that has a function that is desirable for use in function control and that is given additional functional properties depending on the intended application.
Then, an object of this invention is to provide the photoreceptor channel type | mold protein by which various functional characteristics were improved or provided.
In the process of analyzing the structure-function relationship of two types of photoreceptor channel-type proteins, channel opsin 1 and channel opsin 2, derived from the green alga Chlamydomonas, the present inventors have established a transmembrane domain between these channel opsin. When various recombinant hybrid apoproteins were produced, it was found that hybrid apoproteins having functional characteristics different from the wild type exist among them. Further, along with this, it was possible to identify transmembrane-containing structural domains involved in various functional characteristics of channel opsin 1 and channel opsin 2, and the present invention was completed.
That is, the present invention includes the following features.
(1) A modified photoreceptor channel-type rhodopsin protein having a plurality of transmembrane domains, wherein at least one of the transmembrane domains is derived from the corresponding channel opsin 1 of Chlamydomonas reinhardtii A transmembrane domain or a variant thereof and / or at least one of the transmembrane domains is a transmembrane domain or variant thereof derived from the corresponding Chlamydomonas reinhardtii channel opsin 2; Said protein.
(2) At least one transmembrane domain is a transmembrane domain derived from Chlamydomonas reinhardtii channel opsin 1 or a variant thereof, and at least one transmembrane domain is The protein according to (1), which is a transmembrane domain derived from channel opsin 2 of Chlamydomonas reinhardtii corresponding to or a variant thereof.
(3) The photoreceptor channel-type rhodopsin protein is channel opsin 1, and at least one transmembrane domain is a transmembrane domain derived from channel opsin 2 of Chlamydomonas reinhardtii or a variant thereof. The protein according to (1) above.
(4) The protein according to (3) above, wherein the photoreceptor channel-type rhodopsin protein is channel opsin 1 derived from Chlamydomonas reinhardtii.
(5) The photoreceptor channel-type rhodopsin protein is channel opsin 2, and at least one transmembrane domain is a transmembrane domain derived from channel opsin 1 of Chlamydomonas reinhardtii or a variant thereof. The protein according to (1) above.
(6) The protein according to (5) above, wherein the photoreceptor channel-type rhodopsin protein is channel opsin 2 derived from Chlamydomonas reinhardtii.
(7) The protein according to any one of (1) to (6) above, comprising the polypeptide shown in any of the following (a) to (l) or a variant thereof.
(A) Polypeptide consisting of amino acids 1-117 of SEQ ID NO: 2 and amino acids 79-315 of SEQ ID NO: 4 (b) Polypeptide consisting of amino acids 1-164 of SEQ ID NO: 2 and amino acids 126-315 of SEQ ID NO: 4 (C) a polypeptide consisting of amino acids 1-184 of SEQ ID NO: 2 and amino acids 146-315 of SEQ ID NO: 4 (d) a polypeptide consisting of amino acids 1-212 of SEQ ID NO: 2 and amino acids 174-315 of SEQ ID NO: 4 (E) a polypeptide consisting of amino acids 1 to 242 of SEQ ID NO: 2 and amino acids 204 to 315 of SEQ ID NO: 4 (f) a polypeptide consisting of amino acids 1 to 276 of SEQ ID NO: 2 and amino acids 238 to 315 of SEQ ID NO: 4 (G) a polypeptide consisting of amino acids 118 to 345 of SEQ ID NO: 2 and amino acids 1 to 78 of SEQ ID NO: 4 (h) Polypeptide consisting of amino acids 165 to 345 of column number 2 and amino acids 1 to 125 of SEQ ID NO: 4 (i) Polypeptide consisting of amino acids 185 to 345 of SEQ ID NO: 2 and amino acids 1 to 145 of SEQ ID NO: 4 (j) A polypeptide consisting of amino acids 213 to 345 of SEQ ID NO: 2 and amino acids 1 to 173 of SEQ ID NO: 4 (k) A polypeptide consisting of amino acids 243 to 345 of SEQ ID NO: 2 and amino acids 1 to 203 of SEQ ID NO: 4 (l) A polypeptide consisting of amino acids 277 to 345 of SEQ ID NO: 2 and amino acids 1 to 237 of SEQ ID NO: 4 (8) A polypeptide consisting of amino acids 1 to 164 of SEQ ID NO: 2 and amino acids 126 to 315 of SEQ ID NO: 4 or a mutation thereof The protein according to (7) above, comprising a body.
(9) The protein according to (7) above, comprising a polypeptide comprising amino acids 1 to 242 of SEQ ID NO: 2 and amino acids 204 to 315 of SEQ ID NO: 4 or a variant thereof.
(10) A polynucleotide encoding the protein according to any one of (1) to (9) above.
(11) An expression vector comprising the polynucleotide according to (10) operably linked to a promoter.
(12) A cell that expresses the protein according to any one of (1) to (9) above.
(13) The cell according to (12), which is a nerve cell.
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2008-076538, which is the basis of the priority of the present application.

FIG. 1 shows (A) amino acid primary structure alignment of Chop1 and Chop2, and (B) transmembrane domain structure of a modified rhodopsin protein in which the C-terminus of the channel opsin gene is replaced with Venus.
FIG. 2 shows the transmembrane domain structure of the hybrid apoprotein produced in the example.
FIG. 3 shows a schematic diagram of the experimental apparatus used in the examples.
FIG. 4 shows a circuit diagram of the booster circuit used in the embodiment.
FIG. 5 shows confocal images of (A) HEK293 cells expressing hybrid Abcdefg and (B) HEK293 cells expressing hybrid Abcdefg .
6A is a HEK293 cells expressing hybrid Abcdefg photocurrent (upper: blue, lower: green light) and the light current of HEK293 cells expressing the hybrid ABCDEFG (upper: blue, lower: green light) the Show. FIG. 6B shows a comparison of the absorption wavelength response characteristics of the hybrid apoprotein according to the G / B ratio (Green / Blue ratio) of the blue light response and the green light response.
FIG. 7A shows the IV relationship of the hybrid Abcdefg photocurrent (black: extracellular Na ⁺ concentration 142 mM, red: extracellular Na ⁺ concentration 20 mM). FIG. 7B shows the IV relationship of the hybrid Abcdefg photocurrent (black: extracellular Na ⁺ concentration 142 mM, red: extracellular Na ⁺ concentration 20 mM). FIG. 7C shows a comparison of the photocurrent reversal potential shift for each hybrid apoprotein.
FIG. 8 shows the whole cell conductance per unit membrane capacity of the photoreceptor channel of each hybrid apoprotein.
FIG. 9A shows the relative size of the irradiation pulse light. FIGS. 9B and C show a comparison of current response to light intensity for hybrid Abcdefg and ABCddefg photocurrents , respectively.
FIG. 10A shows a comparison between the wild-type Chop2 (1-315) abcdefg photocurrent (black) and the hybrid ABCddefg photocurrent (red). FIG. 10B shows the results of quantifying the inactivation strength for each hybrid apoprotein by the ratio of the steady photocurrent to the maximum photocurrent (plateau / peakratio).
FIG. 11A shows a comparison of the rise of photocurrent in hybrid Abcdefg (black) and hybrid ABCddefg (red). FIG. 11B shows a comparison of photocurrent termination for hybrid Abcdefg (black) and hybrid ABcdefg (red). FIG. 11C shows a comparison of the photocurrent ON rate constant and OFF rate constant between the hybrid apoprotein and the wild type apoprotein.
FIG. 12 shows the transmembrane domain structure and primary amino acid structure of the channel opsin wide receiver (chopWR).
FIG. 13 shows the transmembrane domain structure and the primary amino acid structure of channel opsin fast receiver (chopFR).

The present invention relates to a modified photoreceptor channel-type rhodopsin protein (hereinafter also referred to as “modified rhodopsin protein” or “hybrid apoprotein”). Specifically, at least one of a plurality of transmembrane domains having a photoreceptor channel-type rhodopsin protein is derived from a corresponding channel opsin 1 of Chlamydomonas reinhardtii (hereinafter also referred to as “Chop1”). A transmembrane domain or a variant thereof derived from Chlamydomonas reinhardtii channel opsin 2 (hereinafter also referred to as “Chop2”) and / or a variant thereof. It relates to a photoreceptor channel-type rhodopsin protein.
As used herein, “transmembrane-containing domain” refers to a region including a transmembrane structure deduced from homology with bacteriorhodopsin in a photoreceptor channel-type rhodopsin protein.
1. Modified rhodopsin protein
The modified rhodopsin protein of the present invention is a transmembrane domain derived from Chop1, or a variant thereof, corresponding to at least one of the transmembrane domain in the photoreceptor channel rhodopsin protein, and It is characterized by being a transmembrane structure domain derived from Chop2 or a variant thereof corresponding to this.
That is, the modified rhodopsin protein of the present invention is a transmembrane structure domain in which the photoreceptor channel-type rhodopsin protein exhibits preferred functional properties found in Chop1 and / or preferred functional properties found in Chop2. As a result of including a transmembrane-containing domain having a transmembrane structure domain corresponding to this, preferable functional characteristics are maintained.
Since Chop1 and Chop2 have different functional properties, the modified rhodopsin protein of the present invention preferably has both preferred functional properties. Therefore, in the present invention, the photoreceptor channel-type rhodopsin protein preferably has a transmembrane domain having a preferable functional property found in Chop1 and a transmembrane membrane having a preferable functional property found in Chop2. The structural domain is included in the transmembrane structure domain region corresponding to these.
1.1 Photoreceptor channel type rhodopsin protein
As used herein, a photoreceptor channel-type rhodopsin protein refers to an archaeal rhodopsin family protein having both a photoreceptor function and a channel function (hereinafter also referred to as “photoreceptor channel function”). As structurally closely related proteins belonging to archaeal rhodopsin family proteins, Chop1 and Chop2, bacteriorhodopsin derived from halophilic bacteria, halorhodopsin, sensory rhodopsin, proteorhodopsin, opsin protein of green algae, fungi Known opsin proteins with unknown functions are known, and any of them can be used in the present invention.
The preferred photoreceptor channel rhodopsin protein in the present invention is Chop1 or Chop2, or a homologue or variant thereof. The gene sequences and amino acid sequences of Chop1 and Chop2 are known (Chop1: GenBank accession number AF385748, Chop2: GenBank accession number AF461397). The gene sequence and amino acid sequence of Chop1 are shown in SEQ ID NOs: 1 and 2, and the gene sequence and amino acid sequence of Chop2 are shown in SEQ ID NOs: 3 and 4, respectively.
Here, the “homolog” refers to a protein (or nucleic acid) having the same function from different organisms. The homologous protein and the sequence of the nucleic acid encoding it can be searched using algorithms such as BLAST and FASTA by accessing well-known databases such as NCBI and EMBL. Such homologous nucleic acid or gene can be isolated according to a conventional method. For example, a primer for specifically amplifying a gene identified using a database storing base sequence information is designed, chemically synthesized, and by PCR using the primer using genomic DNA extracted from the target organism as a template. The homologue of the desired chop1 or chop2 gene can be amplified and isolated.
The term Chop1 (or Chop2) variant used in the present invention has one to several amino acid substitutions, additions, insertions or deletions in the amino acid sequence of Chop1 (or Chop2), and Chop1 A polypeptide having functional properties equivalent to (or Chop2), and at least 90%, preferably at least 95%, more preferably at least 96%, 97%, 98% or 99% of the amino acid sequence of Chop1 (or Chop2) Polypeptides having sequence homology and functional properties equivalent to Chop1 (or Chop2) are included.
The “several” used in the present specification is an integer of 10 or less, for example, an integer of 2-9, 2-7, 2-5. Further, in the present specification, the value of sequence homology was calculated using software (for example, FASTA, DNASYS, BLAST, etc.) that calculates homology between a plurality (two) of amino acid sequences with default settings. Value.
In this specification, “equivalent functional characteristics” used in relation to Chop1 or Chop2 means that at least one of functional characteristics such as conductance, light absorption wavelength characteristics, inactivation characteristics, and frequency response characteristics of Chop1 or Chop2 is substantially. Are identical.
The mutant of Chop1 (or Chop2) may be naturally occurring or artificially introduced with a mutation. Artificial mutations can be introduced by introducing mutations into the chop1 (or chop2) gene using, for example, site-specific mutagenesis or PCR-based mutagenesis (Proc Natl Acad). Sci USA., 1984 81: 5662; Sambrook et al., Molecular Cloning A Laboratory Manual (1989) Second edition, Cold Spring Harbor Biopress, 95%;
Whether or not the above-mentioned Chop1 (or Chop2) homologue or variant has the same functional characteristics as Chop1 (or Chop2) is determined by, for example, membrane potential recording, membrane current recording, or fluorescent probe using electrophysiological techniques. Can be assayed by examining changes in intracellular ion concentration.
In the present invention, Chop1 or Chop2 is not necessarily the full length, and may be a Chop1 or Chop2 fragment having photoreceptor channel activity. Chop1 or Chop2 has been reported to have photoreceptive activity in the N-terminal region (Chop1: 1-345 (SEQ ID NO: 5); Chop2: 1-315 (SEQ ID NO: 6)) each containing a transmembrane structure, It is preferable to use a polypeptide containing these regions as the fragment.
1.2 Transmembrane domain
Chop1 and Chop2 are each assumed to have seven transmembrane structures because of their similarity to bacteriorhodopsin (see FIG. 1). Further, as described above, it has been reported that the N-terminal region (Chop 1: 1-345, Chop 2: 1-315) including each hypothetical transmembrane structure has photoreceptive channel activity. Thus, a photoreceptor channel-type rhodopsin protein that defines A, B, C, D, E, F, and G as the transmembrane-containing domain including each hypothetical transmembrane structure of Chop1, and holds A to G of Chop1 Or a fragment of it ABCDEFG Is written. Similarly, photoreception that defines a, b, c, d, e, f, and g as transmembrane-containing domain including each of the hypothetical transmembrane structures of Chop2 and holds Chop2-transmembrane domain ag Body channel rhodopsin protein or fragment thereof abcdefg Is written. For example, a photoreceptor channel type rhodopsin protein or fragment thereof in which A to D are derived from Chop1 and e to g are derived from Chop2 ABCDefg Is written.
Chop1 transmembrane domains A to G are the amino acid regions shown below.
Domain A: amino acids 1-117 of the amino acid sequence shown in SEQ ID NO: 2
Domain B: amino acids 118 to 164 of the amino acid sequence shown in SEQ ID NO: 2
Domain C: amino acids 165 to 184 of the amino acid sequence shown in SEQ ID NO: 2
Domain D: amino acids 185 to 212 of the amino acid sequence shown in SEQ ID NO: 2
Domain E: amino acids 213 to 242 of the amino acid sequence shown in SEQ ID NO: 2
Domain F: Amino acids 243 to 276 of the amino acid sequence shown in SEQ ID NO: 2
Domain G: amino acids 277 to 345 of the amino acid sequence shown in SEQ ID NO: 2
In addition, Chop2 transmembrane domains ag are amino acid regions shown below.
Domain a: amino acids 1 to 78 of the amino acid sequence shown in SEQ ID NO: 4
Domain b: amino acids 79 to 125 of the amino acid sequence shown in SEQ ID NO: 4
Domain c: amino acids 126 to 145 of the amino acid sequence shown in SEQ ID NO: 4
Domain d: amino acids 146 to 173 of the amino acid sequence shown in SEQ ID NO: 4
Domain e: Amino acids 174 to 203 of the amino acid sequence shown in SEQ ID NO: 4
Domain f: amino acids 204 to 237 of the amino acid sequence shown in SEQ ID NO: 4
Domain g: amino acids 238 to 315 of the amino acid sequence shown in SEQ ID NO: 4
In the present invention, a variant of the transmembrane domain of Chop1 (or Chop2) (hereinafter also referred to as “mutant transmembrane domain”) is one to several amino acid regions in the transmembrane domain. A polypeptide having amino acid substitutions, additions, insertions or deletions and having functional properties equivalent to an unmodified transmembrane domain, and at least 90% of the amino acid sequence of the transmembrane domain, Preferably, it comprises a polypeptide having at least 95%, more preferably at least 96%, 97%, 98% or 99% sequence homology and functional properties equivalent to an unmodified transmembrane domain. Note that “several” and sequence homology values are defined above.
"Equivalent functional characteristics" used in the context of mutant transmembrane domains are the types of functional characteristics of the transmembrane domain (conductance, light absorption wavelength characteristics, inactivation characteristics, frequency response characteristics, etc.) ) Is substantially identical to the unmodified transmembrane domain, and the properties of the functional properties of the mutant transmembrane domain (conductance intensity, light absorption wavelength range, degree of photocurrent inactivation, frequency) The degree of responsiveness) is substantially the same as the unmodified transmembrane domain.
The mutant transmembrane-containing domain may be naturally occurring or artificially introduced with a mutation. Artificial mutations can be introduced by introducing mutations into a polynucleotide encoding a transmembrane structure domain using, for example, site-specific mutagenesis or PCR-based mutagenesis. (Proc Natl Acad Sci USA., 1984 81: 5662; Sambrook et al., Supra; Ausubel et al., Supra).
1.3 Construction of modified rhodopsin protein
The modified rhodopsin protein of the present invention is a transmembrane domain derived from Chop1, or a variant thereof, corresponding to at least one of the transmembrane domain in the photoreceptor channel rhodopsin protein, and / or Or it is the equivalent transmembrane domain derived from Chop2, or a variant thereof. In other words, in the modified rhodopsin protein of the present invention, at least one transmembrane domain in the photoreceptor channel rhodopsin protein is replaced with the equivalent transmembrane domain derived from Chop1 or a variant thereof. It can also be said that it has been replaced by a transmembrane-containing domain derived from Chop2 or a mutant thereof equivalent thereto. Thereby, the modified rhodopsin protein provided with the preferable functional characteristics of Chop1 or Chop2 can be provided. “The equivalent transmembrane structure domain” is the same as the substituted transmembrane structure domain in the types of functional characteristics (conductance, light absorption wavelength characteristics, inactivation characteristics, frequency response characteristics, etc.). Are the transmembrane structure domains having the same or different functional properties (conductance strength, light absorption wavelength range, degree of photocurrent inactivation, degree of frequency response, etc.). For example, the transmembrane-containing domain of Chop2 corresponding to the domain A of Chop1 is the domain a.
Further, since the Chop1 transmembrane structure domain of Chop1 and the Chop2 transmembrane structure domain have different functional characteristics, various combinations of both the Chop1 transmembrane structure domain and the Chop2 transmembrane structure domain are various combinations. Can be used to provide a lineup of photoreceptor channel-type rhodopsin proteins having various functional properties.
For example, based on the functional properties of Chop1 transmembrane domains A to G and Chop2 transmembrane domains ag, the modified rhodopsin protein of the present invention has increased conductance and increased long wavelength photoresponsiveness. , At least one of functional characteristics such as weak inactivation and high frequency responsiveness.
An increase in conductance is a preferable functional characteristic in that a large membrane potential response and membrane current response are generated even for weak light.
The increase in the long wavelength photoresponsiveness is a preferable functional characteristic in that the wavelength band of light that can be used for activation is expanded and the response to white light is increased.
Weak inactivation is a preferable functional characteristic in that the light input pattern is more accurately reflected in the membrane potential / membrane current and the response is not attenuated by repeated stimulation.
High frequency response is a preferable functional characteristic in that the response to optical information that fluctuates at a high frequency does not attenuate.
When the photoreceptor channel-type rhodopsin protein is Chop1, the modified rhodopsin protein of the present invention replaces at least one of the transmembrane-containing structure domains of Chop1 (for example, domain A), and transmembrane-containing structure of Chop2. It can be created by retaining a domain (eg domain a).
When the photoreceptor channel-type rhodopsin protein is Chop2, the modified rhodopsin protein of the present invention replaces at least one of the transmembrane-containing domains of Chop2 (for example, domain a) and contains the membrane of Chop1 It can be created by retaining a penetrating domain (eg domain A).
Examples of such modified rhodopsin protein created between Chop1 and Chop2 include proteins including the polypeptides exemplified below or variants thereof.
(Abcdefg) A polypeptide comprising amino acids 1 to 117 of SEQ ID NO: 2 and amino acids 79 to 315 of SEQ ID NO: 4
(ABcdefg) A polypeptide comprising amino acids 1 to 164 of SEQ ID NO: 2 and amino acids 126 to 315 of SEQ ID NO: 4
(ABCdefg) A polypeptide comprising amino acids 1 to 184 of SEQ ID NO: 2 and amino acids 146 to 315 of SEQ ID NO: 4
(ABCDefg) A polypeptide comprising amino acids 1-212 of SEQ ID NO: 2 and amino acids 174-315 of SEQ ID NO: 4
(ABCDEfg) A polypeptide comprising amino acids 1 to 242 of SEQ ID NO: 2 and amino acids 204 to 315 of SEQ ID NO: 4
(ABCDEFg) A polypeptide comprising amino acids 1 to 276 of SEQ ID NO: 2 and amino acids 238 to 315 of SEQ ID NO: 4
(ABCDEFG) A polypeptide comprising amino acids 118 to 345 of SEQ ID NO: 2 and amino acids 1 to 78 of SEQ ID NO: 4
(AbCDEFG) A polypeptide comprising amino acids 165 to 345 of SEQ ID NO: 2 and amino acids 1 to 125 of SEQ ID NO: 4
(AbcDEFG) A polypeptide comprising amino acids 185 to 345 of SEQ ID NO: 2 and amino acids 1 to 145 of SEQ ID NO: 4
(AbcdEFG) A polypeptide comprising amino acids 213 to 345 of SEQ ID NO: 2 and amino acids 1 to 173 of SEQ ID NO: 4
(AbcdeFG) A polypeptide comprising amino acids 243 to 345 of SEQ ID NO: 2 and amino acids 1 to 203 of SEQ ID NO: 4
(AbcdefG) A polypeptide comprising amino acids 277 to 345 of SEQ ID NO: 2 and amino acids 1 to 237 of SEQ ID NO: 4
Each variant of the polypeptide includes a polymorphism having one to several amino acid substitutions, additions, insertions or deletions in each amino acid sequence of the polypeptide, and having functional properties equivalent to those of the polypeptide. Peptides, and at least 90%, preferably at least 95%, more preferably at least 96%, 97%, 98% or 99% sequence homology to the amino acid sequence of the polypeptide and the polypeptide A polypeptide having functional properties equivalent to Note that “several”, sequence homology values, and “equivalent functional properties” are defined above.
The mutants may be naturally occurring or artificially introduced with mutations. Artificial mutations can be introduced by, for example, site-directed mutagenesis or PCR-based mutagenesis (Proc Natl Acad Sci USA., 1984 81: 5652; Sambrook et al., Supra). In addition to Ausubel et al., Supra), it may have arisen from a convenient aspect of production, such as an operation to increase the expression efficiency of the polypeptide.
Among the modified rhodopsin proteins created between Chop1 and Chop2, ABCDEfg (Hereinafter, also referred to as “channel opsin wide receiver (chop WR)”) is remarkably superior in conductance, long-wavelength light absorption, weak inactivation, and the like. Na ⁺ High selectivity to ions. The channel opsin / wide receiver is an excellent modified rhodopsin protein that is excellent in the reception of faint light and is excellent in use for introduction into cells deep in tissues, for example.
Among the modified rhodopsin proteins, ABcdefg (Hereinafter also referred to as “channel opsin fast receiver (chop FR)”) is remarkably excellent in frequency response characteristics. Conductance, weakness of inactivation, Na ⁺ Also excellent in selectivity to ions. The channel opsin fast receiver is an excellent modified rhodopsin protein that is excellent in receiving optical information that fluctuates at a high frequency. For example, it is an excellent modified rhodopsin protein as a material for a nerve cell network module that can input information by light.
1.4 Production of modified rhodopsin protein
The modified rhodopsin protein of the present invention can be produced by genetic engineering techniques based on the photoreceptor channel-type rhodopsin protein gene and the sequence information of the chop1 gene and the chop2 gene.
Specifically, first, a polynucleotide encoding the modified rhodopsin protein of the present invention (hereinafter also referred to as “modified rhodopsin gene”) is prepared. The modified rhodopsin gene can be prepared by techniques known to those skilled in the art. For example, a polynucleotide encoding a desired modified rhodopsin protein can be chemically synthesized based on the sequence information of the gene encoding the photoreceptor channel-type rhodopsin protein and the sequence information of the chop1 gene and the chop2 gene. Alternatively, based on the sequence information, a PCR primer that amplifies a desired region of the photoreceptor channel-type rhodopsin gene and a PCR primer that amplifies the desired domain of the chop1 gene and / or the chop2 gene are designed and chemically synthesized, and the target By the PCR using genomic DNA extracted from the organism of the above as a template, the photoreceptor channel type rhodopsin gene region constituting the modified rhodopsin gene and the polynucleotide encoding the Chop1 and / or Chop2 domain region are respectively amplified. You may prepare by connecting these.
Next, the modified rhodopsin gene of the present invention operably linked to a promoter can be maintained in a replica of the host cell, the protein can be stably expressed, and the gene can be stably maintained. The modified rhodopsin protein of the present invention can be produced in the host by transforming the host using the resulting recombinant expression vector incorporated into the expression vector. For recombination techniques, reference can be made to Sambrook et al. (Supra) and Ausubel et al. (Supra).
Expression vectors include, but are not limited to, plasmids derived from Escherichia coli (eg, pET28, pGEX4T, pUC118, pUC119, pUC18, pUC19, and other plasmid DNAs), Bacillus subtilis. Plasmids (eg, pUB110, pTP5, and other plasmid DNAs), yeast-derived plasmids (eg, YEp13, YEp24, YCp50, and other plasmid DNAs), λ phage (λgt11, λZAP, etc.), mammalian plasmids (pCMV) , PSV40), viral vectors (adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, vaccinia virus vectors, etc. Animal virus vectors, and insect virus vectors such as baculovirus vectors, etc.), plant vector (binary vector pBI system, etc.), etc. cosmid vector can be used.
As used herein, “operably linked” refers to a functional between a promoter sequence and a polynucleotide sequence of interest such that the promoter sequence can initiate transcription of the polynucleotide sequence of interest. This is a simple bond.
The promoter is not particularly limited, and a suitable promoter may be selected depending on the host, and any of a constitutive promoter and an inducible promoter known in the art may be used. In the present invention, it is particularly preferable to use a constitutive promoter. For example, promoters that can be used in the present invention include CMV promoter, SV40 promoter, CAG promoter, synapsin promoter, rhodopsin promoter, CaMV promoter, glycolytic enzyme promoter, lac promoter, trp promoter, tac promoter, GAPDH promoter, GAL1 Examples include promoters, PH05 promoters, PGK promoters, thy1 promoters, and the like.
The modified rhodopsin gene is inserted into the expression vector by, for example, preparing or linking a restriction enzyme site flanking the modified rhodopsin gene and inserting it into a restriction enzyme site or a multiple cloning site of an appropriate vector DNA. In addition to the promoter and the modified rhodopsin gene, the expression vector includes enhancers and other cis elements, splicing signals, poly A addition signals, selection markers (drug resistance gene markers such as ampicillin resistance marker, tetracycline resistance marker, LEU1 , An auxotrophic complementary gene marker such as TRP1 and URA3, a dominant selection marker such as APH, DHFR, and TK), a ribosome binding site (RBS), and the like.
To transform a host, use protoplast method, spheroplast method, competent cell method, virus method, calcium phosphate method, lipofection method, microinjection method, gene bombardment method, Agrobacterium method, electroporation, etc. Can be done.
The obtained transformant is cultured under suitable conditions using a medium containing an assimilated carbon source, nitrogen source, metal salt, vitamins and the like. The transformant is usually cultured at 25-37 ° C. for 3-6 hours under aerobic conditions such as shaking culture or aeration-agitation culture. During the culture period, the pH is kept near neutral. The pH is adjusted using an inorganic or organic acid, an alkaline solution, or the like. During the culture, an antibiotic such as ampicillin or tetracycline may be added to the medium according to the selection marker inserted into the recombinant expression vector, if necessary. The host used for transformation is not particularly limited as long as it can express the modified rhodopsin protein of the present invention. Bacteria (E. coli, Bacillus subtilis, etc.), yeast (Saccharomyces cerevisiae, etc.), animal cells ( COS cells, Chinese hamster ovary (CHO) cells, 3T3 cells, BHK cells, HEK293 cells, etc.) and insect cells.
The modified rhodopsin protein of the present invention is fractionated and purified by a general method from a culture obtained by culturing a transformant (culture supernatant, cultured cell, cultured cell, cell or cell homogenate, etc.). And maintaining the activity by ultrafiltration concentration, freeze drying, spray drying, crystallization and the like. Alternatively, the modified rhodopsin protein of the present invention may be provided in the form of cells that express the protein without isolation and purification. In this case, the host cell used for transformation is a host cell suitable for the subsequent use, for example, a nerve cell, preferably a human nerve cell.
Moreover, when using the modified rhodopsin protein of this invention for a medical use, you may provide in the form of this protein expression vector. In this case, it is preferable to use an expression vector excellent in efficiency of introduction into a cell, maintenance of replication in the cell, stability, expression efficiency, and the like. Examples of such vectors include, but are not limited to, viral vectors such as adeno-associated virus vectors, retrovirus vectors, and lentivirus vectors, plasmids that can replicate autonomously, and transposons.
2. Application
Bacteriorhodopsin, one of the photoreceptor channel-type rhodopsins, has been used as a visual recovery (for example, JP-A-2002-363107) and a research tool for basic research (for example, JP-T-2004-521604) since its function has been clarified. Not only for use, but also to photoelectric conversion elements (for example, JP-A-2002-271265, JP-A-2000-67939), optical information storage materials (for example, JP-T-2006-515683), or neural model elements (for example, JP-A-6-295350) It is a substance that has been considered for use. However, since one electron moves per one photon, which is a feature of bacteriorhodopsin, there remains a problem to be solved for use in the above applications.
On the other hand, in the case of channel rhodopsin, since one ion is absorbed and the ion channel is opened, the number of electrons moving is much larger, and as a result, highly efficient photoelectric conversion is expected. On the other hand, wild-type channel rhodopsin has a problem that it is necessary to consider the effect of desensitization.
According to the present invention, it is possible to obtain various lineups of modified rhodopsin proteins that are significantly different from wild-type photoreceptor channel rhodopsin proteins in functional characteristics. Therefore, a modified rhodopsin protein can be selected and used according to the application.
2.1 Visual recovery and therapeutic applicability
In the human eyeball, light is sensed by receptor cells in the retina, and the activity is transmitted to ganglion cells via bipolar cells. The neurites of ganglion cells send nerves to the brain as optic nerves. In retinitis pigmentosa and age-related macular degeneration, visual loss is lost due to degeneration of receptor cells. The former has a genetic background and is blinded at a relatively young age, but it is said that there are 1.5 million patients worldwide. The latter is the biggest cause of blindness in people over the age of 65. In such patients, retinal ganglion cells are healthy. Therefore, retinal bipolar cells and ganglion cells originally have no photosensitivity, but wild-type Chop2 (1-315). abcdefg Research has been carried out to restore vision by introducing and expressing the gene into retinal neurons through genetic engineering methods.
For such purposes, rhodopsin protein requires the following properties: (i) low light sensitivity; (ii) low wavelength selectivity and response to white light; (iii) Weak deactivation of photocurrent. Wild-type Chop2 (1-315) abcdefg Is not excellent in these properties because of its high selectivity to blue light and strong photocurrent inactivation.
In contrast, channel opsin wide receivers, for example, have excellent characteristics such as high conductance, response to a wide wavelength range from blue to green, and almost no photocurrent inactivation. Therefore, it is expected to impart high photosensitivity to cells deep in the tissue.
Therefore, it is expected to be optimal as a material that restores high-resolution vision completely non-invasively and improves the living ability of visually impaired persons accompanying degeneration of retinal photoreceptor cells.
Specifically, the modified rhodopsin protein of the present invention (for example, channel opsin wide receiver), an expression vector containing a polynucleotide encoding the protein, or a eukaryotic cell (for example, optic nerve cell) that expresses the protein is an active ingredient. It can be used as a pharmaceutical composition for restoring visual function.
2.2 Introduction to the nerve cells of the auditory system
It is known that nerve cells of the auditory system perform high frequency activities of several hundred Hz. In order to cause high frequency activity in these neurons by light irradiation, it is necessary that the inactivation of the photocurrent is weak and that the frequency response characteristic is high. Therefore, for example, by using a channel opsin fast receiver (chopFR), it is expected to cause high frequency activity in these nerve cells.
2.3 Availability as a research tool in basic research
In addition to mammals such as mice, rats, and monkeys, various types of animals such as zebrafish, insects, snails, and nematodes are used to study the functions of neurons and the networks they create. In order to study what functions a protein encoded by a gene plays in a living body, it is essential to study the functions of nerve cells and synapses. In developing drugs that affect the nervous system, it is necessary to assay how these chemicals modify the function of nerve cells and synapses. These studies are carried out by stimulating nerve cells in the in vivo or in vitro nervous system and measuring animal behavior and synaptic activity caused thereby. Stimulation of nerve cells is generally performed by bringing a monopolar or bipolar stimulation electrode close to each other and energizing a short electric pulse. However, it is difficult to specify the number, location, and type of nerve cells that are stimulated.
On the other hand, it is attempted to stimulate nerve cells with light pulses because the photoreceptor cells can acquire photosensitivity by introducing and expressing photoreceptor channels in neurons. However, the photocurrent of wild-type channel rhodopsin (eg, Chop2) quickly desensitizes and takes tens of seconds to recover from desensitization. Therefore, it is difficult to stimulate repeatedly at a high frequency.
The photocurrent obtained when expressing the modified rhodopsin protein of the present invention, such as channel opsin wide receiver or channel opsin fast receiver, has high conductance and weak inactivation. Is suitable. Since the channel opsin / wide receiver has a large absorption on the long wavelength side, a larger photocurrent can be induced by using broadband light. Therefore, a large depolarization exceeding the action potential threshold can be surely caused in the nerve cell by the light pulse, and high reliability is expected. Moreover, since the channel opsin fast receiver has a large following frequency, it is suitable for the purpose of causing nerve cells to be activated at a high frequency.
By using these modified rhodopsin proteins, it is expected to promote the study of the functions of nerve cells and networks made by nerve cells and the development of drugs that use these functions in assay systems. In addition, recombinant viral vectors incorporating these modified rhodopsin protein genes and transgenic animals such as mice, rats, zebrafish, Drosophila, nematodes, etc., are expected to be widely applied in research and development. The
2.4 Application to Brain Machine Interface (BMI) and Brain Computer Interface (BCI)
The technology that decodes brain information and uses it for machine control and computer input information, and to input information to the brain is called Brain Machine Interface (BMI) and Brain Computer Interface (BCI). It is. These are technologies that enable new communication through the brain, and in order to realize them, development of a minimally invasive and long-term stable interactive information technology for brain information is indispensable. It is ideal to use light as a bi-directional information medium for realizing high-resolution and high-resolution information input / output in time and space. Further, by using light, mechanical invasion to the brain can be minimized.
The modified rhodopsin protein of the present invention, such as channel opsin wide receiver and channel opsin fast receiver, is expected to be optimal as an information input medium to the brain using light.
2.5 Development of computer elements using neural cell networks
Computers excel at the ability to process information quickly and accurately. In addition, the network can communicate with computers all over the world in real time. The brain, on the other hand, excels in the ability to actively create errors and derive the best answer for the environment and situation from among many options. This ability is the source of inspiration and emotion. Therefore, an attempt has been made to produce a computer that can process information closer to the brain by combining a neuron network with artificial hardware. As a specific method, a silicon substrate or a glass plate is processed, stimulation and measurement electrodes are arranged on a matrix, and cultured neurons and brain slices are set thereon.
Attempts have also been made to use living animal brains for computer information processing. As a method for stimulating neurons, an electrical stimulation method using a metal or semiconductor electrode or a glutamate administration method has been used. The conventional method has a problem that the spatial resolution is low, and further, when a living animal is used, it is invasive because it is necessary to embed a multi-electrode stimulator etc. in the brain, and it is difficult to use it safely for a long time. It was.
On the other hand, photosensitivity can be acquired in a nerve cell by introducing a photoreceptor channel into the nerve cell and expressing it. In the case of a cultured neuron, by using a laser or the like, it becomes possible to simultaneously stimulate individual neurons or even a part of them, and information of a complicated pattern can be sent to a neuron network. This makes it possible to reproduce trial and error information processing performed by the brain even with a simple cultured neuron network. When the cerebral cortex of a living animal is used, multiple points of pattern stimulation are realized with high resolution by light irradiation from the brain surface. When light stimulation is used, no electrical artifacts occur, and it becomes easy to simultaneously measure the activity of individual neurons in real time and feed back to the computer. As a result, it is expected that a computer having excellent brain capabilities such as pattern recognition and self-organization will be created in addition to the capabilities of conventional computers.
The modified rhodopsin protein of the present invention, such as channel opsin wide receiver and channel opsin fast receiver, is expected to be optimal as an information input medium to a neuronal network using light. In addition, there is a prospect that multi-channel information can be input to a neuronal network by utilizing the difference in light absorption characteristics of these modified rhodopsin proteins. For example, it is considered that multi-channel input using a difference in photocurrent wavelength dependency between a channel opsin wide receiver and a channel opsin fast receiver becomes possible.
2.6 Development of micromachine (MEMS) using muscle power
Muscle fibers (muscle cells), which are the smallest unit of muscle, are very small but can generate strong force. It also has very high energy efficiency. Because of these advantages, attempts have been made to utilize biological contraction mechanisms to power micromachines (Song et al., 2000; Bachand and Montemagno, 2000; Hess et al., 2004; Shu et al., 2003; Xi et al., 2005). However, when operating the micromachine, there is a difficult problem of how to control and drive the contraction mechanism in time and space. In muscle, the motor nerves of the spinal cord form almost one-to-one synapses in muscle fibers. When the motor nerves are excited, action potentials are generated at the synaptic site of the muscle fiber through the synapse, which conducts the muscle fiber plasma membrane and depolarizes the T-tubule, thereby causing muscle contraction. Therefore, subtle movements are controlled by controlling the contraction of each muscle fiber at the level of nerve cells. However, it is very difficult to control the activity by letting an artificial micromachine control the nerve. Although it is ideal to stimulate individual muscle cells directly, conventional electrical stimulation methods have limited temporal and spatial controls.
In the production of muscle cells and myoblast clones that incorporate photosensitivity through genetic engineering, and directly controlling muscle contraction with light, the modified rhodopsin proteins of the present invention, such as channel opsin wide receiver and channel opsin fast receiver, It is expected to be optimal as a muscle contraction medium using light. Furthermore, the construction of a system that can freely operate such an optically operated micromachine under a microscope is expected. For example, a micro scissors that cuts an object under a microscope, a micro grabber that grabs the object, a micro conveyor that conveys the object, a micro worm that swims in a liquid, and the like can be considered. These are expected to be used for precision microsurgery under a microscope, for example, surgery that removes cancer cells while protecting blood vessels and nerves in tissues.
EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to the following Example.

(1) Production of Hybrid Molecules In this example, first, 12 types of hybrid apoproteins shown in FIG. 2 were produced from Chop1 (1-345) ABCDEFG and Chop2 (1-315) abcdefg .
These hybrid apoproteins were produced by the overlap extension PCR method. The process will be specifically described below.
The overlap extension PCR method consists of two steps. First, in the first step, a cDNA fragment encoding the N-terminal amino acid residue sequence and a cDNA fragment encoding the C-terminal amino acid residue sequence of the hybrid apoprotein to be prepared are respectively prepared by PCR. At this time, the reverse primer used when preparing the N-terminal fragment and the forward primer used when preparing the C-terminal fragment are designed to have a complementary relationship. For example, when preparing a hybrid Abcdefg, the cDNA fragment of the A domain is a combination of chop1-345-EcoRI-F1 and chop-chR1 primer, and the cDNA fragment of the bcdefg domain is a combination of chop-chF1 and chop2-315-EcoRI-R1. Then, PCR was performed using KOD-Plus-DNA polymerase (Toyobo) using Chop1 (1-345) and Chop2 (1-315) as templates, respectively, and each fragment was prepared. In the second step, the A fragment and the bcdefg fragment are mixed and PCR is performed using KOD-Plus-DNA polymerase. At this time, since one end of the A fragment and the bcdefg fragment contains sequences complementary to each other, the extension of the cDNA occurs between the sense strand of the A fragment and the antisense strand of the bcdefg fragment, and a chimeric molecule called Abcdefg Produced. Hereinafter, similarly, in the production of hybrid ABcdefg, an AB fragment is produced by a combination of chop1-345-EcoRI-F1 and chop-chR2, and a cdefg fragment is produced by a combination of chop-chF2 and chop2-315-BamHI-R1, The ABcdefg fragment was generated by the second PCR. Table 1 summarizes the names and sequences of the primers used for hybrid apoprotein production, and Table 2 summarizes the primer combinations used for the production of each hybrid apoprotein and the cDNA used as the template.
Both ends of the hybrid apoprotein cDNA fragment prepared by overlap extension PCR are cleaved by restriction enzymes EcoRI and BamHI. The cDNA fragment treated with both restriction enzymes was cloned between EcoRI and BamHI of the pVenus-N1 vector (Clontech's pDsRed2-N1 vector with DsRed2 replaced by Venus), and yellow on the C-terminal side of the hybrid apoprotein. A plasmid vector for expression of a mammalian cell that expresses a fusion protein added with a kind of fluorescent protein, Venus, was prepared.
(2) Culture of HEK293 cells HEK293 cells derived from human fetal kidney cells were cultured and maintained on a 60 mm plastic dish (BD Falcon PRIMARIA dish). 90% D-MEM and 10% fetal calf serum were used for the culture solution. Gene transfer treatment was performed when the cells were subcultured to a 4-well plate (NUNC) subjected to collagen coating treatment and became 30-50% confluent. The cells were dispersed by trypsin treatment on the next day after gene introduction, the cells were reseeded on a 12 mm round cover glass that had been subjected to collagen coating treatment, and the function test was performed the next day. One isolated cell was used for the functional assay.
(3) Gene transfer For introduction of the rhodopsin apoprotein gene into HEK293 cells, gene transfer was carried out by the method specified by the manufacturer using Effectene (registered trademark) Transfection Reagent of Qiagen. For gene introduction, 0.2-0.4 μg of rhodopsin apoprotein expression plasmid was used per 12 mm round cover glass.
(4) Experimental apparatus Since rhodopsin apoprotein bound with retinal reacts to the wavelength of light of 400-550 nm, it does not contain blue to green light when cells are transported under a microscope or observed in a bright field. made. A yellow fluorescent lamp (Panasonic FL40S. Y-F) was used for illumination in the laboratory, and light with a wavelength of 520 nm or less was blocked. The background color of the CRT monitor was yellow. The following devices were all installed on an air table to eliminate the effects of vibration. In addition, it was installed in a dark screen to eliminate the effects of room light. An outline of the main apparatus is shown in FIG.
(I) Upright-type epifluorescence microscope (Olympus BH2-RFC): Transmitted light during cell observation blocked light with a wavelength of 490 nm or less through a GIF filter. For the excitation and observation of Venus, a xenon lamp light source was used, and ON / OFF of the light was controlled by an electromagnetic shutter. By combining a filter and a dichroic mirror, cells expressing Chop2-Venus with an excitation light wavelength of 450-490 nm and a fluorescence wavelength of> 515 nm were identified. A 60 × water immersion lens (Olympus LUMplanPl / IR60x) was used as the objective lens.
(Ii) Switching between blue light (477 ± 10 nm) and green light (532 ± 10 nm): A xenon lamp was used as the incident light source. The electromagnetic shutter was driven by a rectangular wave pulse current created by an electrical stimulator (Nihon Kohden SEN-7203) to control light ON-OFF. The blue light bandpass filter (477 ± 10 nm) and the green light bandpass filter (532 ± 10 nm) were switched by a chopper (Olympus OSP-EXCH) placed in the middle of the optical path. The light was introduced into the microscope objective through a dichroic mirror.
(Iii) Blue light emitting diode (LXHL-NB98; Lumileds Lighting Inc.): Pulse-clamped by patch clamp control software (Axon p-Clamp 9.1), digital-to-analog converter (Axon DIGIDATA 1200) and electrical stimulator A rectangular wave pulse current created by (Nippon Kohden SEN-7203) was amplified in a booster circuit (FIG. 4) and turned on. The diode light was introduced into the microscope objective lens via a dichroic mirror.
(Iv) Monochromator (JASCO CAM-230): A xenon lamp light was dispersed and an arbitrary wavelength having a width of 10 nm was taken out. The extracted light was introduced into an epifluorescence microscope through an optical fiber. Light on / off was controlled by a built-in electromagnetic shutter.
(V) Microscope XY drive device (Medical Agent OXY-1): A three-dimensional micromanipulator described later was installed in the fixed part, and the above-mentioned upright epifluorescence microscope was installed in the drive part. The driving in the X-Y2 axis directions was finely controlled by a micrometer.
(Vi) Three-dimensional micromanipulator (Narishige MX-1R): A patch electrode holder, which will be described later, was driven in the three-axis directions of XYZ. Each drive shaft used both coarse and fine movement modes.
(Vii) Measuring chamber: A transparent acrylic plate was processed, and a cover glass was attached to the lower part. Tyrode liquid (see Table 3 below) was circulated through the chamber by a peristaltic pump. A cover glass with cultured cells attached was placed in the chamber.
(Viii) Photodiode (Sharp BS500B): Scattered light was received in front of the microscope objective lens and amplified by a microelectrode amplifier.
(5) Measurement / recording apparatus and data analysis The membrane potential and membrane current of HEK293 cells were measured simultaneously. Moreover, the intensity of irradiated light was measured simultaneously as a photodiode current. These were analog-to-digital converted and recorded on a computer. The following apparatus was used for the measurement.
(I) Patch clamp amplifier (Axon AXOPATCH200A): The membrane current was measured when the membrane potential was kept constant by feedback through the patch electrode (potential fixation experiment).
(Ii) Microelectrode amplifier (Nihon Kohden MEZ-8201): The photocurrent was measured while applying a reverse current of 20 nA to the photodiode. The measured value was expressed as a relative value with respect to the maximum output of the blue light emitting diode.
(Iii) Analog-to-digital converter (Axon DIGIDATA 1200): The current output and voltage output of the patch clamp amplifier and the voltage output of the microelectrode amplifier were converted from analog to digital and output to a computer. A rectangular wave pulse created by a computer was output externally.
(Iv) Computer (Dell Optiplex GXi): Data were acquired using patch clamp control software (Axon p-Clamp 9.1). A square wave pulse was also created. For analysis of the data, Clamp-fit Ver. 9.2 (Axon) and Microsoft Excel were used.
(6) Whole cell patch clamp method The characteristics of wild-type Chop1 and 2, and hybrid apoprotein were confirmed by the whole cell patch clamp method. That is, a glass tube (patch pipette) having a tip diameter of 3 to 4 μm was prepared by heating and pulling a glass tube having a diameter of 1.5 mm (Harvard Appalatas GC150) using a patch pipette puller (Narishige PC-10). Was polished with a microforge (Narishige MF-830). The patch pipette was filled up to the tip of the patch pipette (Table 3) to prepare a patch electrode. The patch electrode was fixed to a patch electrode holder. Cells expressing rhodopsin apoprotein were identified by Venus fluorescence under an upright epifluorescence microscope. While applying positive pressure to the patch electrode, approach the cell, press against the cell surface to create dimples, switch to negative pressure to attach the cell membrane to the glass tube, and apply stronger negative pressure to the glass tube. The film of the attached site was broken, and the inside of the cell and the inside of the glass tube were electrically connected (hole cell mode). The following potential fixation experiment was conducted in the whole cell mode.
(7) Identification of transmembrane domain that controls photoresponse characteristics As a marker for these hybrid apoproteins, a construct cDNA in which Venus, one of the modified green fluorescent protein GFP derived from Aequorea jellyfish, is coordinated to the C-terminal. Was made. When these were introduced and expressed in HEK293 cells, high expression was observed in the plasma membrane in all 12 types of hybrid apoproteins (FIG. 5). That is, it was suggested that the structure as a membrane protein was maintained. Expression cells were identified using the Venus fluorescence as a clue, and the photocurrent was measured with the whole cell patch potential fixed.
The magnitude of the photocurrent of wild-type Chop2 (1-315) abcdefg is maximum at 460 nm depending on the wavelength of the irradiation light. This is explained by the fact that the absorption efficiency of light energy has wavelength dependency. For the purpose of comparing the absorption light wavelength response characteristics of the hybrid photocurrent, the photocurrent induced by blue light (477 ± 10 nm) and green light (532 ± 10 nm) was measured. FIG. 6A shows a comparison of the respective photocurrents between hybrids Abcdefg and ABCdefg . The response of hybrid ABCddefg to green light is increasing. FIG. 6B shows a comparison between the hybrid apoprotein and the wild-type apoprotein by quantifying the absorption wavelength response characteristics using the G / B ratio (Green / Blue ratio) of the blue light response and the green light response. Post. That is, the hybrid Abcdefg showed absorption wavelength response characteristics almost the same as the wild-type channel opsin 2 (1-315) abcdefg . In contrast, in the hybrid ABCcdefg , ABCdefg , ABCDefg , the absorption on the long wavelength side increased. Hybrid ABCDEfg further increased the absorption on the long wavelength side and was almost equal to wild-type channel opsin 1 and ABCDEFG . That is, there is a structure in which the absorption wavelength characteristics are controlled in the transmembrane-containing structure domains B / b and E / e, and it is suggested that when these are b and e, they shift to the longer wavelength side.
(8) Identification of transmembrane domain that controls ion permeability Wild type Chop1 (1-345) Photoreceptive channel expressed in Xenopus oocytes expressing ABCDEFG has high H ⁺ permeability , Na ⁺ has been reported to have little permeability to Na ⁺ (Nagel et al., 2002; Hegemann et al., 2005). In contrast, wild-type Chop2 (1-315) abcdefg has been reported to have high Na ⁺ permeability (Nagel et al., 2003; Ishizuka et al., 2006). For the purpose of identifying the structure controlling Na ⁺ permeability, the whole cell patch solution is Na ⁺ -glutamate (Na ⁺ , 100 mM), and the extracellular solution Na ⁺ is 142 mM and 20 mM (N-methyl-D). The IV relation between the holding potential and the photocurrent in the case of -glucamine ion substitution) was examined.
FIG. 7A shows the IV relationship of hybrid Abcdefg . With the external solution Na ⁺ 142 mM, the polarity was reversed at about 10 mV, but when the external solution Na ^{+ was} 20 mM, the reversal potential was about −10 mV. That is, it shifted to about 20 mV minus side. This change is significantly less than wild-type Chop2 (1-315) abcdefg . FIG. 7B shows the IV relationship of hybrid ABCddefg . The inversion potential was shifted by 30 mV or more. FIG. 7C compares the shift of the reversal potential of the photocurrent for each hybrid apoprotein. Also in the hybrid ABCdefg and ABCDefg , the inversion potential shift was significantly small. Therefore, in these hybrid opsin proteins, there is a possibility that the Na ⁺ selectivity of permeated ions is reduced. On the other hand, in the IV relation of hybrid ABCdefg , ABCDEFfg , ABCDEFg , ABCDEFG , the inversion potential shift is more strongly dependent on the external solution Na ⁺ , and is significantly different from wild-type Chop2 (1-315) abcdefg. There was no difference. That is, the permeation ion selectivity is controlled by a plurality of transmembrane-containing domains, which suggests that the transmembrane-containing domains A / a, B / b, and C / c have important functions.
Further, the photocurrents of hybrid ABCDEFg and wild-type Chop1 (1-345) ABCDEFG tended to be very weak compared to the others. Therefore, in FIG. 8, the whole cell conductance per unit film capacity was compared. The whole cell conductance per unit membrane capacity is proportional to the single channel conductance of the photoreceptor channel and its expression density. The whole cell conductance per unit membrane capacity showed the maximum in the hybrid ABCDEfg .
(9) Identification of transmembrane structure domain controlling kinetic characteristics FIG. 9A shows the relative intensity of blue LED irradiation light (470 ± 25 nm). In HEK293 cells expressing hybrid Abcdefg , photocurrent depending on the intensity of irradiation light was measured (FIG. 9B). The photocurrent peaked in a few milliseconds but was found to deactivate quickly. This photoresponsive property is similar to wild-type Chop2 (1-315) abcdefg . On the other hand, the photocurrent of hybrid ABcdefg is hardly inactivated (FIG. 9C). FIG. 10A is a comparison of the photocurrent when the blue LED light with the maximum illuminance is irradiated for 1 second, together with the magnitude of the maximum photocurrent. It has been shown that the photocurrent of hybrid ABcdefg has a large ratio of stationary photocurrent and maximum photocurrent relative to wild-type Chop2 (1-315) abcdefg . Therefore, the strength of inactivation was quantified using this ratio (plateau / peakratio). A comparison of this between the hybrid apoprotein and the wild type apoprotein is shown in FIG. 10B. That is, hybrid Abcdefg was weakly inactivated as compared to wild-type Chop2 (1-315) abcdefg , but was found to be more inactivated than hybrid ABCddefg . Hybrid ABCDEfg further weakens inactivation. That is, there is a structure that controls inactivation in the transmembrane structure domains A / a, B / b, and E / e, and when these are a, b, and e, strong inactivation is caused. Is suggested.
FIG. 11A compares the speed of photocurrent activation (ON) for hybrid Abcdefg and hybrid ABCddefg . FIG. 11B compares the speed of photocurrent deactivation (OFF). It was confirmed that both the photocurrent ON and OFF were faster in the hybrid ABcdefg . FIG. 11C shows a comparison of the constant of constant ON and OFF of photocurrent between the hybrid apoprotein and the wild type apoprotein. It is suggested that when the transmembrane structure domain B / b is B, ON and OFF are both faster, and when the transmembrane structure domain E / e is E, both ON and OFF are slower.
(10) Characterization of hybrid rhodopsin protein Modified Rhodopsin Protein with Increased Conductance The above example suggests the presence of a structure controlling conductance in the transmembrane domain F / f. Therefore, a novel rhodopsin protein with increased conductance is created by setting the structure of this transmembrane-containing structure domain to f. Among the hybrid rhodopsin proteins investigated to date, the maximum conductance per unit membrane capacity can be obtained by using ABCDEfg .
2. Modified Rhodopsin Protein with Increased Absorption on the Long Wavelength Side According to the above example, existence of a structure controlling absorption wavelength characteristics in transmembrane-containing domain B / b, E / e, F / f, G / g Is suggested. Among the hybrid rhodopsin proteins investigated to date, ABCDEFg has the highest absorption on the long wavelength side, but ABCDEfg is excellent in practical use when the conductance is taken into consideration.
3. Wild-type Chop2 (1-315) abcdefg has been used as a means to photostimulate nerve cells and the like, although weak inactivation has been used. Repeated photostimulation has the disadvantage that the photocurrent decreases. In order to obtain the same amount of photocurrent, it was necessary to provide an interval of 10 seconds or more. According to the above embodiment, there is a structure that controls the inactivation in the transmembrane-containing structure domains A / a, B / b, and E / e, and when these are A, B, and E, the inactivation is It was suggested that it became weak. Among the hybrid rhodopsin proteins examined so far, the use of ABCDEfg provides the photocurrent with the weakest inactivation.
4). Modified rhodopsin protein with high frequency response characteristics When nerve cells are repeatedly photostimulated, it becomes important how much frequency the photocurrent that can follow the blinking of light can be caused. The magnitude of the following frequency is equal to the ON speed constant of the photocurrent. The magnitude of the ON speed constant depends on the transmembrane structure domains B / b and E / e, and is small in the combination of BE and be, and large in the combination of Be. Among the hybrid rhodopsin proteins examined so far, a photocurrent with high frequency response characteristics can be obtained by using ABCdefg , ABCdefg , ABCDefg or the like. Taking into account the ON speed constant and selectivity for Na ⁺ , ABCdefg is the best.
5. Modified rhodopsin protein excellent in practicality Among the modified rhodopsin proteins produced in the above examples, ABCDEfg (chop WR) has a conductance, a large absorption on the long wavelength side, a weak inactivation, etc. Remarkably superior. Moreover, the selectivity with respect to Na ^<+> ion is also high. Therefore, the channel opsin wide receiver is excellent in applications intended to receive faint light.
Among the modified rhodopsin proteins prepared in the above examples, ABCdeg (chop FR) is remarkably excellent in frequency response characteristics. It is also excellent in conductance, weak inactivation, and selectivity for Na ⁺ ions. Therefore, the channel opsin fast receiver is excellent in applications intended to accept optical information that fluctuates at a high frequency.

According to the present invention, it is possible to provide a photoreceptor channel type protein with various functions improved or imparted.
In addition, by including the transmembrane domain of channel opsin 1 and the transmembrane domain of channel opsin 2 in various combinations, a photoreceptor channel-type protein specialized for a specific function can be provided. it can.
Since the use of the modified rhodopsin protein of the present invention is via light, it is completely non-invasive to tissues such as retina, nerve, and brain. Moreover, the novel photoreceptor channel-type rhodopsin protein by this invention can be constructed | assembled as what is superior to the conventional channel rhodopsin protein in temporal and spatial resolution. Therefore, it is a very useful material in fields including medical care, welfare / nursing care, and information communication.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
[Sequence Listing]

Claims (10)

A modified photoreceptor channel-type rhodopsin protein having a plurality of transmembrane domains, wherein the modified photoreceptor channel-type rhodopsin protein comprises:
Chlamydomonas reinhardtii at least one含膜transmembrane structural domain or the hydrated transmembrane structural domains derived from the channel opsin 1 of Chlamydomonas reinhardtii The equivalent of含膜through structural domains in the channel opsin 2 or a homologue or variant from A polypeptide having 1-10 amino acid substitutions, additions, insertions or deletions in the amino acid region and having functional properties equivalent to the unmodified transmembrane domain, or the transmembrane structure the amino acid sequence of domains, at least 90% sequence homology, and if it were replaced with a polypeptide having the hydrated transmembrane structural domain equivalent functional properties of unmodified, or,
Chlamydomonas reinhardtii at least one含膜transmembrane structural domain or the hydrated transmembrane structural domains derived from the channel opsin 2 of the corresponding Chlamydomonas reinhardtii thereto含膜through structural domains in the channel opsin 1 or a homologue or variant from A polypeptide having 1-10 amino acid substitutions, additions, insertions or deletions in the amino acid region and having functional properties equivalent to the unmodified transmembrane domain, or the transmembrane structure Substituted with a polypeptide having at least 90% sequence homology to the amino acid sequence of the domain and having functional properties equivalent to the unmodified transmembrane domain,
And, compared to the wild-type channel opsin 1 or channel opsin 2 of Chlamydomonas reinhardtii, at least it has the one of the functional properties of a weak inactivation and high frequency response, the homolog or variant, from Chlamydomonas reinhardtii Having 1 to 10 amino acid substitutions, additions, insertions or deletions in the amino acid region of channel opsin 1 or channel opsin 2, and equivalent to channel opsin 1 or channel opsin 2 from unmodified Chlamydomonas reinhardtii A polypeptide having functional properties or an amino acid sequence of an amino acid region of channel opsin 1 or channel opsin 2 derived from Chlamydomonas reinhardtii, The above protein, which is a polypeptide having at least 90% sequence homology and having functional properties equivalent to channel opsin 1 or channel opsin 2 derived from unmodified Chlamydomonas reinhardtii . A polypeptide having any one of the following (a) to (l) or an amino acid region of the polypeptide having 1 to 10 amino acid substitutions, additions, insertions or deletions, and equivalent to the polypeptide polypeptide having the functional characteristics of, or to the amino acid sequence of the polypeptide has at least 90% sequence homology, and comprises a polypeptide having the polypeptide equivalent functional properties claim 1 Symbol The listed protein.
(A) a polypeptide consisting of amino acids 1-117 of SEQ ID NO: 2 and amino acids 79-315 of SEQ ID NO: 4 (b) a polypeptide consisting of amino acids 1-164 of SEQ ID NO: 2 and amino acids 126-315 of SEQ ID NO: 4 (C) Polypeptide consisting of amino acids 1-184 of SEQ ID NO: 2 and amino acids 146-315 of SEQ ID NO: 4 (d) Polypeptide consisting of amino acids 1-212 of SEQ ID NO: 2 and amino acids 174-315 of SEQ ID NO: 4 (E) a polypeptide consisting of amino acids 1 to 242 of SEQ ID NO: 2 and amino acids 204 to 315 of SEQ ID NO: 4 (f) a polypeptide consisting of amino acids 1 to 276 of SEQ ID NO: 2 and amino acids 238 to 315 of SEQ ID NO: 4 (G) a polypeptide comprising amino acids 118 to 345 of SEQ ID NO: 2 and amino acids 1 to 78 of SEQ ID NO: 4 (h) SEQ ID NO: A polypeptide consisting of amino acids 165 to 345 of 2 and amino acids 1 to 125 of SEQ ID NO: 4 (i) a polypeptide consisting of amino acids 185 to 345 of SEQ ID NO: 2 and amino acids 1 to 145 of SEQ ID NO: 4 (j) SEQ ID NO: Polypeptide consisting of amino acids 213 to 345 of 2 and amino acids 1 to 173 of SEQ ID NO: 4 (k) polypeptide consisting of amino acids 243 to 345 of SEQ ID NO: 2 and amino acids 1 to 203 of SEQ ID NO: 4 (l) SEQ ID NO: 2. A polypeptide comprising amino acids 277 to 345 of 2 and amino acids 1 to 237 of SEQ ID NO: 4 A polypeptide comprising amino acids 1 to 164 of SEQ ID NO: 2 and amino acids 126 to 315 of SEQ ID NO: 4 or a substitution, addition, insertion or deletion of 1 to 10 amino acids in the amino acid region of the polypeptide, A polypeptide having functional characteristics equivalent to the polypeptide, or a polypeptide having at least 90% sequence homology to the amino acid sequence of the polypeptide and having functional characteristics equivalent to the polypeptide 3. The protein of claim 2 , comprising. A polypeptide consisting of amino acids 1 to 242 of SEQ ID NO: 2 and amino acids 204 to 315 of SEQ ID NO: 4 or a substitution, addition, insertion or deletion of 1 to 10 amino acids in the amino acid region of the polypeptide, A polypeptide having functional characteristics equivalent to the polypeptide, or a polypeptide having at least 90% sequence homology to the amino acid sequence of the polypeptide and having functional characteristics equivalent to the polypeptide 3. The protein of claim 2 , comprising. A polynucleotide encoding the protein according to any one of claims 1 to 4 . An expression vector comprising the polynucleotide of claim 5 operably linked to a promoter. The cell which expresses the protein of any one of Claims 1-4 . The cell according to claim 7, which is a nerve cell.   A polypeptide comprising amino acids 1 to 164 of SEQ ID NO: 2 and amino acids 126 to 315 of SEQ ID NO: 4 or a substitution, addition, insertion or deletion of 1 to 10 amino acids in the amino acid region of the polypeptide, A polypeptide having functional characteristics equivalent to the polypeptide, or a polypeptide having at least 90% sequence homology to the amino acid sequence of the polypeptide and having functional characteristics equivalent to the polypeptide The protein of claim 1 comprising.   A polypeptide consisting of amino acids 1 to 242 of SEQ ID NO: 2 and amino acids 204 to 315 of SEQ ID NO: 4 or a substitution, addition, insertion or deletion of 1 to 10 amino acids in the amino acid region of the polypeptide, A polypeptide having functional characteristics equivalent to the polypeptide, or a polypeptide having at least 90% sequence homology to the amino acid sequence of the polypeptide and having functional characteristics equivalent to the polypeptide The protein of claim 1 comprising.