JPH10150987A - Bacterial type rhodopsin, bacterial type rhodopsin gene, recombinant dna and production of bacterial type rhodopsin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce the subject new protein, comprising a bacterial type rhodopsin having a specific amino acid sequence, having retinal as a chromogen chromophore and useful as a bioelement, etc., such as an image sensor or a biocomputer. SOLUTION: This new bacterial type rhodopsin comprises a bacterial type rhodopsin composed of an amino acid sequence represented by the formula or an amino acid in which one or plural amino acids are deleted, substituted or added in the amino acid sequence represented by the formula and having the bacterial type rhodopsin activities and is a protein having retinal as a chromogen chromophore and useful as a bioelement, etc., such as an image sensor or a biocomputer. The bacterial type rhodopsin is efficiently obtained by providing a bacterial type rhodopsin gene from a highly halophilic bacterium without any light-driven proton pump activities and expressing the gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a bacterial rhodopsin, a gene for the bacterial rhodopsin, a recombinant DNA, and a method for producing bacterial rhodopsin.

[0002]

2. Description of the Related Art Extremely halophilic bacteria require 3 M for growth and survival.
(18%) is an extreme environmental microorganism that requires salt at or near saturation, and is a kind of archaebacteria (Archaebacteria). Halobacterium h.
alobium) has bacteriorhodopsin (b
acteriorhodopsin (hereinafter abbreviated as bR), which forms a purple membrane composed of a retinal protein and a sulfurized glycolipid.
When exposed to light, this purple membrane undergoes a cyclic reaction in which bR returns to its original state through various intermediate states, during which it transports protons (H +) from inside to outside the cell. Having. And bR can be used as a bio device such as an image sensor and a biocomputer, and is useful. (Eg Oesterhelt and Tittor, TIB
S 14, 57-61, 1989).

[0003] Bacterial rhodopsins having a light-driven proton pump function have been known to produce on the cell membrane of halobacterium, haloacura, and halorblum strains of highly halophilic bacteria. For example, Halobacterium halobium
Introduces bR to Haloarcula Baris Mortis (Haloarc
ula vallismortis) is cruxrhodopsin (cruxrho)
dopsin, hereinafter abbreviated as cR), and Halorubrum sodomense, archirodopsin (ar
chaerhodopsin (hereinafter abbreviated as aR).
These are proteins that commonly have retinal as chromogenic chromophores (eg, Mukohata, Bioph
ysical Chemistry 50, 191-201, 1994), but the protein portions differ in their amino acid sequences, have only 60% homology, and can be said to be distinct proteins.

[0004] In addition to the above three genera, several genera are known as highly halophilic bacteria, and various strains have been isolated from nature. Among these, there are obviously strains that do not have a light-driven proton pump function.

[0005]

An object of the present invention is to obtain a bacterial rhodopsin gene from a strain having no light-driven proton pump function and express the gene to produce a novel bacterial rhodopsin. .

[0006]

Means for Solving the Problems As a result of various studies in view of the above object, the present inventors have succeeded in isolating and determining the structure of a novel bacterial rhodopsin gene derived from a halobacterium belonging to the genus Halobacterium. Further, a recombinant DNA obtained by inserting a gene encoding bacterial rhodopsin into vector DNA is obtained, and the recombinant is contained in a strain belonging to the genus Halobacterium. Above, they found that a purple membrane containing bacterial rhodopsin was efficiently produced, and completed the present invention.

That is, the first invention of the present application relates to (a) a bacterial rhodopsin comprising the amino acid sequence represented by SEQ ID NO: 1,
Or (b) a bacterial rhodopsin consisting of a bacterial rhodopsin having an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence (a), and having bacterial rhodopsin activity; The invention of (2) comprises (a) a protein having the amino acid sequence shown in SEQ ID NO: 1, or (b) an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence (a), and A bacterial rhodopsin gene encoding a protein having bacterial rhodopsin activity, and a third invention of the present application is a recombinant DNA comprising the bacterial rhodopsin gene described above inserted into a vector DNA. And the fourth invention of the present application is:
A transformant or a transductant containing the above-mentioned recombinant DNA, and a fifth invention of the present application further comprises culturing the above-described transformant or the transductant in a medium, and removing the bacterial rhodopsin from the culture. And a method for producing bacterial rhodopsin.

Hereinafter, the present invention will be described in detail. The bacterial rhodopsin gene of the present invention can be obtained, for example, as follows. First, Halobacterium sp. H
T [RICM Microorganism Strain Preservation Facility JCM (Japan Collec
The strain has been deposited as JCM9743] in, for example, Archeaea-A Laboratory Manua
l (Cold Spring Harbor Laboratory Press, 1995), the method of Kamekura et al. (J. Bacteriol., 174, 736-74).
2,1992), and the like.
Extract A.

[0009] When comparing the amino acid sequences of known bR, aR and cR, there are two very conserved parts [ie, Asp Trp from No. 85 to No. 91 from the N-terminus of mature bR].
LeuPhe Thr Thr Pro and 208-213 Phe Met Val
(Leu or Ile) Asp (Val or Leu)]. This is
It is present in the C (third) and G (seventh) helices of the transmembrane region of the halobacterium halobium bR whose three-dimensional structure has been elucidated. Based on the nucleotide sequence of the gene encoding the amino acid sequence of the conserved portion, a sense primer and an antisense primer are chemically synthesized.

The highly halophilic bacterium Halobacterium sp. H
Using the chromosomal DNA prepared from T (JCM9743) as a template, a polymerase chain reaction (hereinafter abbreviated as PCR) is performed,
Using the obtained product of about 400 bp as a probe, the bacterial rhodopsin gene is cloned from the chromosomal DNA of Halobacterium sp. HT as follows.

Halobacterium sp. HT (JCM9743)
Chromosomal DNA prepared from various restriction enzymes, for example,
Decomposition with NaeI to obtain a chromosomal DNA fragment mixture. From the DNA fragment mixture thus obtained, for example, by a conventional agarose gel electrophoresis method, preferably 1-2 Kb
A DNA fragment mixture having a size of is obtained and, if necessary, subjected to an alkaline phosphatase treatment or the like, and then, if necessary, further purified, for example, by a purification means such as phenol extraction, and further, for example, by a method such as ethanol precipitation. A concentrated and purified DNA fragment mixture (DNA containing a gene encoding bacterial rhodopsin therein)
Fragments are included).

On the other hand, vector DNAs that can be used for cloning PCR products in the present invention include:
For example, bacteriophage vector DNA, plasmid vector DNA and the like can be mentioned, and pUC119 (manufactured by Takara Shuzo), pT7BlueT (manufactured by Novagen) and the like are preferable. For example, in order to make the plasmid vector pUC119 DNA capable of incorporating the NaeI fragment, the plasmid vector capable of incorporating the NaeI fragment is digested with, for example, SmaI (manufactured by Takara Shuzo Co., Ltd.) and, if necessary, subjected to ethanol precipitation. Obtain DNA. Plasmid vector pT7BlueT can be used as is for cloning PCR products.

Next, a DNA containing a gene encoding the obtained Halobacterium-derived bacterial rhodopsin
The fragment and the plasmid vector DNA capable of incorporating the NaeI fragment obtained as described above were mixed, and this was mixed with, for example, T4 DNA ligase (Boehringer Mannheim)
To obtain a recombinant plasmid DNA.

[0014] The recombinant DNA thus obtained
For example, E. coli K12, preferably E. coli JM109
(Toyobo Co., Ltd.), XL-Blue (Funakoshi Co., Ltd.) or the like can be transformed or transduced to obtain colonies of strains containing recombinant DNA having various gene fragments.

Transformation of Escherichia coli is performed, for example, by using DM Morris.
on method (Methods in Enzymology, 68, 326-331, 1979)
The transduction can be performed, for example, by the method of B. Hohn (Methods
in Enzymology, 68, 299-309, 1979).

A recombinant DN having a DNA fragment containing the bacterial rhodopsin gene was selected from the obtained colonies.
To search for strains containing A, for example, use Example item (3)
The PCR product obtained according to the method described in (1) was labeled with a zygogi sigenin DNA labeling kit (Boehringer Mannheim) as a probe,
tocols in Molecular Biology (WILEY Intersciense, 198
9) Colony hybridization can be performed by the method described above.

In order to obtain a purified recombinant plasmid DNA from the obtained strain (containing the bacterial rhodopsin gene therein), for example, ultracentrifugation CsCl concentration gradient separation method, Qiagen tip (Qiagen) ) Can be used. Using the purified recombinant plasmid DNA, the entire nucleotide sequence of the bacterial rhodopsin gene was analyzed by the method shown in Example item (4) (see SEQ ID NO: 2). The amino acid sequence of the polypeptide translated by the gene having is determined. This amino acid sequence is as shown in SEQ ID NO: 1. The gene encoding the amino acid sequence thus determined is the bacterial rhodopsin gene of the present invention.

In the amino acid sequence of SEQ ID NO: 1, one or more amino acids are deleted, substituted or added, and the bacterial rhodopsin gene encoding the amino acid sequence having bacterial rhodopsin activity is all Included in the present invention.

In order to obtain a bacterial rhodopsin gene encoding the amino acid sequence shown in SEQ ID NO: 1 in which one or more amino acids have been deleted, substituted or added, and which has bacterial rhodopsin activity. Is, for example, a site-specific mutagenesis method that is a well-known technique for generating a point mutation or a deletion mutation in a gene;
A method of selectively cleaving a gene, and then removing or adding a selected nucleotide to ligate the gene; an oligonucleotide mutagenesis method, and the like.

However, the bacterial rhodopsin gene obtained as described above is derived from archaea, a kingdom (Kingdom) different from Escherichia coli, which is a eubacterium. Expression in Escherichia coli is generally not easy, and furthermore, it is difficult to produce bacterial rhodopsin bound to retinal and having a proton pump activity in Escherichia coli. Thus, a bacterial rhodopsin-producing strain is obtained by the following procedure.

On the basis of the nucleotide sequence obtained by the above operation, an oligonucleotide of dozens of bases corresponding to the N-terminal amino acid sequence of bacterial rhodopsin and the last of the nucleotide sequence obtained as described above. A complementary chain of more than a dozen residues was synthesized, and these were used as sense primers and antisense primers, respectively, and PCR was performed using the purified recombinant plasmid DNA obtained above as a template to encode bacterial rhodopsin. D without motor area
Get NA.

On the other hand, Halobacterium halobium is known to produce bR, which is a kind of bacterial rhodopsin, and a gene containing a promoter site encoding the present protein has been cloned. Based on this promoter site, ie, a base sequence several hundred bp upstream of the base corresponding to the N-terminal methionine of bR, a corresponding oligonucleotide was synthesized and used as a primer, and the chromosomal DNA of Halobacterium halobium was used as a template. As PCR
DNA that consists of only the bR promoter region
Get.

The DNA at the promoter site thus obtained is first inserted into a vector DNA. As the vector DNA to be used, for example, plasmid DN
A, bacteriophage DNA, etc., and pWL102, pXLNov-r, etc. are preferred as plasmid vector DNA.

Next, a DNA comprising a region encoding bacterial rhodopsin is ligated downstream of the promoter. Using the obtained recombinant DNA, the bacterial rhodopsin gene is absent, or a highly halophilic bacterium that does not express even if present, for example, contains an insert sequence ISH1 or ISH2 unique to the highly halophilic bacterium inside the bR gene. Each strain is obtained by transforming or transducing a highly halophilic bacterium, Halobacterium halobium IV-8 or L33 (DSM 5735) or the like.

Transformation of highly halophilic bacteria can be performed, for example, by using Cline
(Archaea-A Laboratory Manual, Cold Spring
Harbor Laboratory Press, 1995);
90, 169-172 (1990). Transformants or transductants having the ability to produce bacterial rhodopsin obtained as described above, for example, to produce bacterial rhodopsin using a strain belonging to the genus Halobacterium, are performed as follows. be able to.

First, the cells are grown to a stationary phase in a medium containing 1 μg / ml of novobiocin, and then cultured for 3 to 14 days using a 50 to 100-fold volume of novobiocin-free medium. In order to culture the above microorganism, it may be cultured by a usual solid culture method, but it is preferable to employ a liquid culture method as much as possible.

The medium for culturing the above-mentioned microorganisms may be, for example, a nitrogen carbon source such as yeast extract, peptone or meat extract, or one or more inorganic salts such as potassium chloride, magnesium sulfate or manganese sulfate. 18 to 32% sodium chloride concentration
Is used.

The initial pH of the medium is suitably from 6.0 to 8.0. Cultivation is carried out at 30-42 ° C, preferably around 37 ° C, for 3 hours.
It is preferable to carry out by aeration and stirring deep culture, shaking culture, static culture and the like for up to 14 days.

After completion of the culture, to collect bacterial rhodopsin from the culture, the cells are separated from the culture by, for example, filtration, centrifugation, etc., washed, and the purple membrane is collected from the cells. Is prepared. As a method for this preparation, a method such as Oesterhelt used in preparing bR (Archaea-A
Laboratory Manual, Cold Spring Harbor LaboratoryPr
ess, 1995), the method described by Oesterhelt and Stoeckenius [Methods E
31, Biomembranes, 667-678 (1974)] and the like.

As described above, the purple membrane can be used as it is for collecting the purple membrane from the cells, but the cells obtained by destroying the cells using various disrupting means such as an ultrasonic crusher, a French press, and a dynamill may also be used. Can also be used.

In order to isolate bacterial rhodopsin from the purple membrane thus obtained, a method generally used for membrane protein purification can be used. For example, gel filtration chromatography, ion exchange resin chromatography, sucrose density gradient ultracentrifugation and the like are preferably combined as appropriate.

The bacterial rhodopsin obtained has amino acids 24
This is a peptide having 0 residues. The proton pump activity is measured by the method of Mogi et al. (Proc. Natl. Acad. Sci. USA 85, 4).
148-4152, 1988) by measuring the pH change due to transient proton release and re-adsorption accompanying flash irradiation.

[0033]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described more specifically by way of examples. However, the present invention is not limited to these examples.

[0034]

【Example】

(1) Preparation of Halobacterium Halobacterium sp. HT chromosome Highly halophilic bacterium Halobacterium sp. HT (JCM9743)
Was inoculated into 200 ml of SGC medium (1% casamino acid, 1% yeast extract, 0.3% sodium citrate, 0.2% potassium chloride, 2% magnesium sulfate, 25% sodium chloride) and cultured with shaking at 37 ° C for 5 days. A culture was obtained. This culture is grown at 10,000 rpm
The cells were collected by centrifugation for 10 minutes to obtain cells. From these cells, the method of Kamekura et al. (J. Bacteriol., 174, 736-7
42, 1992) to obtain 500 μg of chromosomal DNA.

(2) Preparation of DNA library 100 μg of the chromosomal DNA was ligated to the restriction enzyme NaeI according to a conventional method.
After digestion by Takara Shuzo Co., Ltd., 10 μg of a fragment of the 1-1.5 kb fraction was obtained by agarose gel electrophoresis. Plasmid vector DNA pUC119 (obtained from Takara Shuzo) 1 μg of Sma
2 μg of the Ie digest and the NaeI digest of the chromosomal DNA obtained above were ligated with 1 unit of T4 DNA ligase (manufactured by Boehringer Mannheim).
Morrison's method (Methods in Enzymology, 68, 326-331, 1
Transformation of Escherichia coli JM109 strain (manufactured by Toyobo Co., Ltd.) according to 979) resulted in 600 colonies, and the nylon membrane filter Hybond was used according to the protocol of Amersham.
-N + (Amersham) was blotted.

(3) Isolation of bacterial rhodopsin gene by colony hybridization Both sense and antisense primers (SEQ ID NOs: 3 and 4, respectively) designed and synthesized as described below and prepared with the above item (1) PCR was performed using the chromosomal DNA as a template.

The product was purified by GENECLEAN II (manufactured by Funakoshi Co., Ltd.) by ordinary agarose gel electrophoresis.
The obtained gene fragment was labeled with dichikogigenin using a DIG-Labeling Kit (manufactured by Boehringer Mannheim) to obtain a probe for colony hybridization. Current Protocols in Molecular Biology (WILEY I
ntersciense, 1989), colony hybridization was carried out, and several positive colony strains were obtained.

(4) Analysis of Bacterial Rhodopsin Gene Of the five isolated positive colonies, a recombinant plasmid DN containing the bacterial rhodopsin gene from one strain was included.
A was prepared by ultracentrifugation and digested with restriction enzymes EcoRI and PstI. As a result, it was found that a chromosomal DNA fragment of about 1.2 kb derived from the genus Halobacterium was inserted. Therefore, the Kilo-Seq
uence dilation kit (Takara Shuzo) and 370 D
The nucleotide sequence was determined using an NA sequencing system (manufactured by Applied Biosystems). The determined 750 bp nucleotide sequence is shown in SEQ ID NO: 2. It is determined to contain a 750 bp open reading frame encoding from the N-terminus to the C-terminus of the predicted bacterial rhodopsin. SEQ ID NO: 1 shows the 250 amino acid sequence of the polypeptide translated from the DNA sequence. This amino acid sequence was compared with the amino acid sequence of known bacterial rhodopsin (aR, bR, and cR) to confirm that it was bacterial rhodopsin.

(5) Amplification of Gene The nucleotide sequence of the halobacterium halobium bR gene (bop) has already been reported (Dunn et al., Proc. Natl. A
cad.Sci.USA 78,6744-6748,1981). This gene has a 360 bp sequence upstream of the open reading frame. This upstream sequence and an open reading frame encoding the N-terminal to the C-terminal of bacterial rhodopsin were ligated.

That is, the bolus of Halobacterium halobium
Primers designed to introduce BamHI and HindIII sites into the nucleotide sequence from bases 1 to 20 and the nucleotide sequence from bases 343 to 363 of p were chemically synthesized (SEQ ID NOS: 5 and 6, respectively). PCR was performed using this primer and the chromosomal DNA of Halobacterium halobium as a template to purify the product. This is called the plasmid vector pT7B
lueT (Novagen) and cloned into E. coli JM109
After amplification by Toyobo, the fragment was cut out with BamHI and HindIII and purified.

On the other hand, from the N-terminal of bacterial rhodopsin to C
A base sequence obtained by binding the terminal 12 residues of the base sequence upstream of the open reading frame (SEQ ID NO: 7) encoding up to the terminal and 9 residues from 1 to 9 of the open reading frame (SEQ ID NO: 2), Primers designed to introduce a HindIII site into the base sequence from bases 70 to 90 of the downstream sequence (SEQ ID NO: 8) of the opening frame were chemically synthesized (SEQ ID NOS: 9 and 1, respectively).
0). This primer and Halobacterium sp. H
Perform PCR using the chromosomal DNA of T (JCM9743) as a template,
The product was purified. This is called plasmid vector pT7BlueT
After cloning into DNA and amplifying in E. coli JM109, H
It was cut out with indIII and purified.

(6) Escherichia co
li)-Construction of shuttle vector between -Halobacterium The plasmid vector DNApXLNov-r was used for the expression of the bacterial rhodopsin gene. This plasmid vector DNA was obtained from Needleman et al. (Brown et al. Biochemistr.
y 34, 12903-12911, 1995). The plasmid vector DNA contains a tetracycline resistance gene and a novobiocin resistance gene, and the cloning site of the plasmid vector DNA is BamH
I and HindIII.

First, plasmid vector DNA, pXLNov
-r was cleaved with BamHI and HindIII, and the BamHI and HindIII fragments purified in the above item (5) were inserted. The obtained plasmid vector DNApXLBH was cleaved with HindIII, and the above purified HindIII-HindIII fragment was inserted. BamHI
And the length of the obtained fragment was measured to determine the orientation of the inserted HindIII fragment. The finally obtained plasmid vector DNA, pXLNovHT9, was used for transformation of Halobacterium halobium L33 (DSM5735). Used for.

(7) Spheroplast formation and transformation Halobacterium halobium L33 (DSM5735) was used as a host cell for transformation. Transformation is performed by
This was performed according to the method disclosed in Gene 90, 169-172 (1990).

1.5 ml of a culture of Halobacterium halobium L33 cultured in a growth medium (sodium chloride 250 g, magnesium sulfate 20 g, trisodium citrate ₂ H ₂ O 3 g, potassium chloride 2 g, yeast extract 10 g, casamino acid 10 g)
Was centrifuged and the supernatant was removed. The cells are suspended in 0.2 ml of spheroplast forming solution and then 20 μl of 0.5 M EDTA
Was added and mixed quickly. Spheroplast formation was completed in about 30-60 minutes at room temperature.

10 μl of the plasmid solution was added to the spheroplast suspension and incubated at room temperature for 5 minutes. 230 μl of the polyethylene glycol solution was mixed rapidly and incubated at room temperature for 20 minutes.

(8) Regeneration of Transformed Spheroplast The transformant solution was diluted with 1 ml of the regenerating solution, and then centrifuged to collect the spheroplast, and 1 m of complete medium (containing 15% sucrose) was collected.
1 and carefully incubated at 37 ° C. overnight. 0.1-0.25 ml of the cell suspension left to stand overnight is added to an agar medium (1
5% sucrose, 2% agar, 1μg / ml novobiocin)
Immediately spread with a spreader. The plate was sealed with vinyl tape and kept at 37 ° C for at least two weeks.

(9) Culture of Transformants and Isolation of Bacterial Rhodopsin A few weeks later, transformed cells, ie, colonies of Halobacterium halobium L33 (pXLNovHT9) were picked up and 2 ml of a test tube medium was collected. , And cultured at 37 ° C for 7 days. In contrast to the fact that the cells culturing the colonies obtained by transforming the plasmid vector DNA, pXLNov-r are beige, the plasmid vector DN
Colonies obtained by transformation with ApXLNovHT9 were purple. Isolation of a purple membrane containing bacterial rhodopsin,
Oesterhelt and Stoecius
kenius) (Methods Enzymol. 31, B
iomembranes, 667-678 (1974)]. And 4.5 mg / ml of purple membrane was obtained. Transformant Halobacterium halobium L33 (pXLN
ovHT9) was deposited on November 8, 1996 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology as FERM P-15940.

Properties of Bacterial Rhodopsin Purified purple membrane exhibited light-driven proton pump activity.
One kind of protein was extracted from the purified purple membrane, and the N-terminal amino acid sequence was as follows. Ala Ala Thr Val Gly Pro Glu Phe Ile Trp Leu Tr
p Ile Gly The Ile Gly Met Thr Leu. That is, it became clear that 10 residues were truncated as compared with the N-terminal amino acid sequence predicted from the nucleotide sequence of the open reading frame. Protein absorption maximum is 25 mM PIPES Good buffer (pH 7.2)
Was 552 nm.

[0050]

According to the present invention, a novel bacterial rhodopsin can be efficiently obtained by acquiring and expressing a bacterial rhodopsin gene from a highly halophilic bacterium having no light-driven proton pump activity.

[0051]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 250 Sequence type: Amino acid Sequence type: Bacterial rhodopsin Sequence: Met Cys Tyr Ala Ala Leu Ala Pro Pro Met Ala Ala Thr Val Gly 1 5 10 15 Pro Glu Phe Ile Trp Leu Trp Ile Gly The Ile Gly Met Thr Leu 20 25 30 Gly Thr Leu Val Phe Val Gly Arg Gly Arg Gly Val Arg Asp Arg 35 40 45 Lys Met Gln Glu Phe Tyr Ile Ile Thr Ile Phe Ile Thr Thr Ile 50 55 60 Ala Ala Ala Met Tyr Phe Ala Met Ala Thr Gly Phe Gly Val Thr 65 70 75 Glu Val Met Val Gly Asn Glu Ala Leu Thr Ile Tyr Trp Ala Pro 80 85 90 Tyr Ala Asp Trp Leu Phe Thr Thr Pro Leu Leu Leu Leu Asp Leu 95 100 105 Ser Leu Leu Ala Gly Ala Asn Arg Asn Thr Ile Ala Thr Leu Ile 110 115 120 Gly Leu Asp Val Phe Met Ile Gly Thr Gly Ala Ile Ala Ala Leu 125 130 135 Ser Ser Thr Pro Gly Thr Arg Phe Ala Trp Trp Ala Ile Ser Thr 140 145 150 Gly Ala Leu Leu Ala Leu Leu Tyr Val Leu Val Gly Thr Leu Ser 155 160 165 Lys Asn Ala Arg Asn Arg Ala Pro Glu Val Ala Ser Leu Phe Gly 170 175 180 Arg Leu Arg Asn Leu Val Ile Ala le u Trp Phe leu Tyr Pro Val 185 190 195 Val Trp Ile Leu Gly Thr Glu Gly Thr Phe Gly Ile Leu Pro Leu 200 205 210 Tyr Trp Glu Thr Ala Ala Pla Met Val Leu Asp Leu Ser Ala Lys 215 220 225 Val Gly Phe Asp Val Ile Leu leu Gln Ser Arg Ser Val Leu Glu 230 235 240 Arg Val Ala Thr Pro Thr Ala Ala Pro Thr 245 250

SEQ ID NO: 2 Sequence length: 750 Sequence type: number of nucleic acid chains: double-stranded Topology: linear Sequence type: chromosomal DNA Origin Organism name: Halobacterium sp. HT Strain name: JCM9743 sequence : ATG TGT TAC GCT GCT CTA GCA CCA CCC ATG GCC GCA ACA GTT GGC CCA 48 GAA TTC ATT TGG TTA TGG ATC GGC ACG ATC GGC ATG ACC CTC GGA ACC 96 CTG GTA TTC GTC GGT CGC GGA CGT GGC GTT CGT GAT CGG ATG CAG 144 GAG TTC TAT ATC ATC ACG ATC TTC ATC ACA ACC ATC GCC GCT GCC ATG 192 TAC TTC GCG ATG GCA ACC GGC TTC GGC GTC ACC GAA GTC ATG GTC GGC 240 AAC GAG GCG CTC ACG ATC TAC TGG GCG CCG TAC GCC GGG CTG TTC 288 ACG ACG CCG CTG CTG TTG CTC GAC CTC TCG CTG CTC GCC GGG GCG AAC 336 CGA AAC ACG ATC GCG ACG CTG ATC GGC CTC GAC GTT TTC ATG ATC GGA 384 ACC GGC GCG ATC GCA GCG CTC TCG TCC ACC CCGT CGG TTC GCC 432 TGG TGG GCG ATC AGC ACC GGT GCT CTG CTC GCC CTG CTG TAC GTC CTC 480 GTC GGG ACG CTC TCC AAG AAC GCG CGC AAC CGG GCC CCC GAG GTC GCA 528 TCG CTG TTC GGG AGA CTC CGC AAC CTG GTT ATC GCG CTG TGG TTC CTC 576 TAC CCG GTG GTC TGG ATC CTC GGC ACG GAA GGG ACG TTC GGC ATC CTT 624 CCG CTG TAC TGG GAA ACC GCG GCG TTC ATG GTG CTC GAC CTC 672 AAG GTC GGA TTC GAC GTG ATC CTG CTC CAG AGC CGC TCC GTC CTG GAG 720 CGG GTC GCG ACG CCG ACG GCT GCC CCG ACC 750

SEQ ID NO: 3 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Sequence characteristics: Synthetic DNA primer Sequence: GAC TGG YTG TTC ACR ACR CC 20

SEQ ID NO: 4 Sequence length: 17 Sequence type: Nucleic acid number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Sequence characteristics: Synthetic DNA primer Sequence: ASG TCR AKS ACC ATG AA 17

SEQ ID NO: 5 Sequence length: 21 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Sequence characteristics: Synthetic DNA primer Sequence: GGG TGG ATC CGT GAA GTC CGC 21

SEQ ID NO: 6 Sequence length: 21 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Sequence characteristics: Synthetic primer Sequence: CAT GCA AGC TTA CCT AAC GAG 21

SEQ ID NO: 7 Sequence length: 399 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Chromosomal DNA Origin Organism: Halobacterium sp. HT Strain: JCM9743 Sequence : GCC GGC GTA GAC CCC CAC CCA GAG GAC GAG CGG CGA CGA CCA CAG CGG 48 ACC NCN GCC CGG NAA CGT CGC TTG TAC TGA CGA CGG ATA GGT GGC GAC 96 GAT CGG AAC GAC GAG CGC GAC ACG AGC AGC AGG CCG CTC TGG ACG 144 AGT TTT TNT GGA ATG CGG CGC TTG ATC AGC GCG TAT CGC TCA ACT CTN 192 AGT CGT TCG TAC GTC GAT TCG CCG AGG AGT GCG TCC GCA ATC GGT TCG 240 TTC GGN TCA GTC GAG GTC ATC GGT TGT GGA GGG GGC GTG ATC GGG 288 CTA TCC GAC CGA ACA CAG AAC ANC CCA AAA TAC GNT GCG GTT TCA TCT 366 TCC ACG TCA AAC TTC TGG ACA TAC ACG GTA CAA TAA GAG TTG TTT CCG 384 TTA GGG TCA AGC AAT 399

SEQ ID NO: 8 Sequence length: 95 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Chromosomal DNA Origin Organism name: Halobacterium sp. HT Strain name: JCM9743 Sequence : TGA GGC CGC TGC ATC GAC TCC CGA CGG ACG GTG CCA GCG ACC GAC GCG 48 ATA GCA GAG ACC CGG ACG CTG TTC CGG TCT GCT CGT GTG CAG CCG GC 95

SEQ ID NO: 9 Sequence length: 21 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Sequence characteristics: Synthetic DNA primer Sequence: GGG TCA AGC TTT ATG TGT TAC 21

SEQ ID NO: 10 Sequence length: 21 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Sequence characteristics: Synthetic DNA primer Sequence: CTG CAC ACA AGC TTA CCG GAA 21

──────────────────────────────────────────────────続 き Continued on front page (51) Int.Cl. ⁶ Identification symbol FI // (C12N 15/09 ZNA C12R 1:01) (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19 )

Claims (5)

[Claims] 1. A bacterial rhodopsin of the following (a) or (b): (a) a bacterial rhodopsin consisting of the amino acid sequence shown in SEQ ID NO: 1 (b) an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence (a), and the bacterial rhodopsin activity is Bacterial rhodopsin having 2. A bacterial rhodopsin gene encoding the following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (b) a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence (a), and having bacterial rhodopsin activity 3. A recombinant DNA obtained by inserting the bacterial rhodopsin gene according to claim 2 into a vector DNA. 4. A transformant or transductant containing the recombinant DNA according to claim 3. 5. A method for producing bacterial rhodopsin, which comprises culturing the transformant or transductant according to claim 4 in a medium, and collecting bacterial rhodopsin from the culture.