JP4863280B2 - Novel fluorescent protein and gene encoding it - Google Patents

Description

  The present invention relates to a novel fluorescent protein and a gene encoding the same. Specifically, the present invention relates to a novel fluorescent protein derived from newly discovered marine plankton exhibiting luminescent ability and a coding gene that can be used for recombinant expression of such fluorescent protein.

  Green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria or its variant protein can be expressed recombinantly in heterologous cells, particularly in various mammalian cells. And the resulting recombinant protein exhibits fluorescent properties in the host cell. Using this characteristic, as an in vivo fluorescent marker protein that can be expressed in animal cells in the fields of biochemistry, cell physiology, and medicine, A. Victoria-derived GFP and homologues thereof have been used for various objects and uses (Reference 1: Lippincott-Schwartz, J. GH Patterson, Science vol. 300, 87-91 (2003); Reference 2: Tsien, RY, Annu. Rev. Biochem. Vol. 67, 509-544 (1998)).

  In addition, A. In addition to GFP derived from Victoria, a GFP-like protein has been cloned from a Candaria phylum (Class Hydrozoa), and further, Candaria anteater (Class Antozoa). ), A GFP-like protein has been cloned. Regarding the GFP-like proteins found in these Candaria antomozoa, it may constitute a fluorescent protein family with a common origin in terms of biological evolution. (Ref. 3: Reference is made to YA Labas et al., Proc. Natl. Acad. Sci. USA vol. 99, 4256-4261 (2002)).

  A. As for GFP derived from victoria, research on the mechanism necessary for exerting its fluorescence properties is in progress. First, when the translated GFP polypeptide is folded into a natural three-dimensional structure, it undergoes cyclization of the internal tripeptide site that forms its fluorophore, followed by oxidation, resulting in a mature GFP having fluorescent properties. It has been elucidated. Furthermore, A.I. In the deduced amino acid sequence of wild-type GFP derived from victoria, it has also been confirmed that the 65th to 67th SYG is an internal tripeptide site forming a fluorophore. For example, in Y66H-GFP in which the 66th Tyr is mutated to His, the fluorescence causes a blue shift with respect to the green fluorescence of wild-type GFP, and shows blue fluorescence with a maximum wavelength of 448 nm. It has been reported. Furthermore, in S65T-GFP in which the 65th Ser is mutated to Thr, the maximum wavelength of the fluorescence is 510 nm, which shows a slight red shift with respect to the green fluorescence of wild-type GFP. It has also been reported that in S65T-GFP, the cyclization of the internal tripeptide: TYG site and subsequent fluorophore formation by oxidation proceed significantly faster than SYG of wild-type GFP.

  In addition to the introduction of mutations into the 65th to 67th SYG sites, A.I. In the wild-type GFP derived from Victoria, when the 203rd Thr is replaced with His, Phe, Tyr, and T203H, T203F, T203Y are introduced, the maximum fluorescence wavelength is about 530 nm and a remarkable red shift occurs. It is also reported that it is produced as a yellow fluorescent protein (YFP). In addition, FGFPL mutation-introduced EGFP (“enhanced” GFP), which replaces the 64th Phe adjacent to the 65th to 67th SYG sites with Leu, is compared with wild-type GFP. It has been reported that the maturation process accompanied by the formation of is significantly improved (see Reference 4: BP Comack et al., Gene vol. 173, 33-38 (1996)).

  Thus, A.I. Many attempts have been made to use GFP-like proteins derived from various marine animals belonging to Candaria, including GFP derived from Victoria, as in vivo fluorescent marker proteins that can be expressed in animal cells. It has been broken. On the other hand, it is known that many marine organisms, particularly zooplanktons, exhibit bioluminescence. Therefore, A. From the viewpoint of biological evolution, a novel fluorescent protein constituting another type of protein family having a different origin from the fluorescent protein family to which GFP derived from victoria belongs is expected. That is, the search for a novel fluorescent protein family that can be used as an in vivo fluorescent marker protein that can be expressed in a host animal cell is awaited.

When the fluorescent protein is used as an in vivo fluorescent marker protein that can be expressed in a host cell, photoexcitation is performed from outside the host cell in order to observe the fluorescence. The wavelength used for this photoexcitation is selected as a light absorption band located on the shorter wavelength side (higher energy side) than the fluorescence wavelength indicated by the fluorescent protein. The fluorescence intensity obtained from the fluorescent protein depends on the product of the molar absorption coefficient ε (cm ⁻¹ · M ⁻¹ ) and the fluorescence quantum yield η at the excitation wavelength. In practice, while monitoring the fluorescence intensity obtained from the fluorescent protein, the excitation spectrum is measured to determine the maximum peak wavelength. For example, A.I. In the excitation spectrum of GFP derived from Victoria, a main peak at 396 nm and a sub-peak at 475 nm are shown. On the other hand, A. In the excitation spectrum of YFP obtained by modifying Victoria-derived GFP, a maximum peak is shown at about 520 nm.

  When two types of in vivo fluorescent marker proteins are used in a host cell, two types of fluorescent proteins having different fluorescence wavelengths and excitation wavelengths are required. From this point of view, A. It is desired to provide a novel fluorescent protein that emits distinguishable fluorescence when used in combination with a fluorescent protein that emits green fluorescence, such as GFP derived from victoria. Specifically, the maximum wavelength of fluorescence is A.I. V. is found on the longer wavelength side than the maximum wavelength of green fluorescence exhibited by GFP or the like derived from Victoria; It is desired to provide a novel fluorescent protein having a different origin in terms of bioevolution from the fluorescent GFP-like protein family to which Victoria-derived GFP belongs.

The present invention solves the above-mentioned problems, and an object of the present invention is derived from zooplankton belonging to a phylum different from Cnaria, and at least the maximum wavelength of fluorescence is longer than 510 nm. The object is to provide a novel fluorescent protein that exists on the wavelength side and exhibits yellow fluorescence or yellow-green fluorescence and that can be recombinantly expressed in heterologous cells, and a gene encoding the same.

In order to solve the above-mentioned problems, the present inventors have searched for luminescent plankton that exhibits luminescence / fluorescence in the yellow or green-yellow region from zooplankton that inhabit the ocean. Specifically, a number of luminescent plankton were found in the process of classification of zooplankton present in deep ocean waters collected off the coast of Japan and Toyama Bay. Furthermore, among these luminescent planktons, zooplankton belonging to a phylum that is taxonomically different from the Cnidaria phylum (Cndaria), and expresses a protein exhibiting yellow fluorescence or yellow-green fluorescence in the body. Sorted out things. In the screening process, among the planktons of crustaceans, one type of Red Ceppoda (red podapod) that appears red when visually observed under white light irradiation exhibits high-brilliance yellow-green fluorescence under ultraviolet light irradiation. I found out.

  The present inventors tried to classify Red Copepoda (red rodent) expressing a yellow-green fluorescent protein found in this selection process, Arthropoda phylum, Greater jaw (Mandibulata subphyllum), crustacea (Crustacea class), and podoptera (Coppoda subclass), which were found to be consistent with the morphological features of the Aetideidae family and the Bradyidius genus. Whether or not the species has already been reported has not been determined at this stage. After that, as a result of further taxonomic identification based on more detailed comparison, it was concluded that it belongs to Chiridius poppei.

  Subsequently, with respect to the yellow green fluorescent protein derived from Red Copepoda, an attempt was made to specify the base sequence of the gene encoding it and to determine the deduced amino acid sequence. First, total RNA was extracted from the Red Copepoda, the contained mRNA was purified, and the corresponding cDNA was synthesized using reverse transcriptase according to a standard method.

  The present inventors have previously reported A.I. In the fluorescent GFP-like protein family to which the GFP derived from V. victoria belongs, in many cases, the collected cDNA is translated into a peptide chain in E. coli, and the translated peptide chain is folded and a luminophore is formed. In view of the fact that it is expressed as a fluorescent protein, a synthesized cDNA is inserted into a general-purpose cloning vector: pBluescript II SK to construct a cDNA library. It was investigated. At that time, when the target yellow-green fluorescent protein is expressed, it is irradiated with light of near ultraviolet light (wavelength range 330 nm to 400 nm) or purple / blue to blue green (wavelength range 400 nm to 500 nm). , Yellow-green fluorescence should be observed, and screening was performed using the presence or absence of yellow-green fluorescence as a selection criterion. As a result, colonies in which yellow-green fluorescence was clearly observed were selected, and then secondary screening was performed on the selected colonies to isolate clones. Analysis of the nucleotide sequence of the cDNA fragment inserted in the isolated clone vector, elucidation of the full-length amino acid sequence of the desired red green fluorescent protein derived from Red Copepoda and the nucleotide sequence of the gene encoding it did. Furthermore, the present inventors have determined that the full-length amino acid sequence of the yellow-green fluorescent protein derived from Red Copepoda Comparison with the full-length amino acid sequence of GFP derived from Victoria confirms that the sequence similarity is low and that it is a novel fluorescent protein having a bioevolutionally different origin from the fluorescent GFP-like protein family Thus, the present invention has been completed.

That is, the fluorescent protein derived from the amphipod according to the present invention is:
Taxonomically, a fluorescent protein derived from a rodent that is classified as Chiridius poppei,
The full-length amino acid sequence of the fluorescent protein is
MTTFKIESRI HGNLNGEKFE LVGGGVGEEG
RLEIEMKTKD KPLAFSPFLL SHCMGYGFYH 60
FASFPKGTKN IYLHAATNGG YTNTRKEIYE
DGGILEVNFR YTYEFNKIIG DVECIGHGFP 120
SQSPIFKDTI VKSCPTVDLM LPMSGNIIAS
SYARAFQLKD GSFYTAEVKN NIDFKNPIHE 180
SFSKSGPMFT HRRVEETHTK ENLAMVEYQQ
VFNSAPRDM 219
It is a fluorescent protein characterized by being (the amino acid sequence described in SEQ ID NO: 1).

In addition, a gene encoding a fluorescent protein derived from the apopod according to the present invention is:
A gene comprising DNA encoding the amino acid sequence set forth in SEQ ID NO: 1. For example, the base sequence of DNA encoding the amino acid sequence set forth in SEQ ID NO: 1 is
ATG ACA ACC
TTC AAA ATC GAG TCC CGG ATC CAT GGC AAC CTC AAC GGG 48
GAG AAG TTC
GAG TTG GTT GGA GGT GGA GTA GGT GAG GAG GGT CGC CTC 96
GAG ATT GAG
ATG AAG ACT AAA GAT AAA CCA CTG GCA TTC TCT CCC TTC 144
CTG CTG TCC
CAC TGC ATG GGT TAC GGG TTC TAC CAC TTC GCC AGC TTC 192
CCA AAG GGG
ACT AAG AAC ATC TAT CTT CAT GCT GCA ACA AAC GGA GGT 240
TAC ACC AAC
ACC AGG AAG GAG ATC TAT GAA GAC GGC GGC ATC TTG GAG 288
GTC AAC TTC
CGT TAC ACT TAC GAG TTC AAC AAG ATC ATC GGT GAC GTC 336
GAG TGC ATT
GGA CAT GGA TTC CCA AGT CAG AGT CCG ATC TTC AAG GAC 384
ACG ATC GTG
AAG TCG TGT CCC ACG GTG GAC CTG ATG TTG CCG ATG TCC 432
GGG AAC ATC
ATC GCC AGC TCC TAC GCT AGA GCC TTC CAA CTG AAG GAC 480
GGC TCT TTC
TAC ACG GCA GAA GTC AAG AAC AAC ATA GAC TTC AAG AAT 528
CCA ATC CAC
GAG TCC TTC TCG AAG TCG GGG CCC ATG TTC ACC CAC AGA 576
CGT GTC GAG
GAG ACT CAC ACC AAG GAG AAC CTT GCC ATG GTG GAG TAC 624
CAG CAG GTT TTC
AAC AGC GCC CCA AGA GAC ATG TAG 660

It may be a gene characterized by (base sequence described in SEQ ID NO: 2).

In addition, a gene encoding a fluorescent protein derived from the apopod according to the present invention is:
A cDNA prepared from mRNA of a fluorescent protein derived from the rodent, which is contained in the coding region of the amino acid sequence shown in SEQ ID NO: 1, and the base sequence of the cDNA is
AGAACACTCA GTGTATCCAG TTTTCCGTCC
TACTACAAAC 40
ATG ACA ACC TTC AAA ATC GAG TCC CGG
ATC CAT GGC AAC CTC AAC GGG 88
GAG AAG TTC GAG TTG GTT GGA GGT GGA
GTA GGT GAG GAG GGT CGC CTC 136
GAG ATT GAG ATG AAG ACT AAA GAT AAA
CCA CTG GCA TTC TCT CCC TTC 184
CTG CTG TCC CAC TGC ATG GGT TAC GGG
TTC TAC CAC TTC GCC AGC TTC 232
CCA AAG GGG ACT AAG AAC ATC TAT CTT
CAT GCT GCA ACA AAC GGA GGT 280
TAC ACC AAC ACC AGG AAG GAG ATC TAT
GAA GAC GGC GGC ATC TTG GAG 328
GTC AAC TTC CGT TAC ACT TAC GAG TTC
AAC AAG ATC ATC GGT GAC GTC 376
GAG TGC ATT GGA CAT GGA TTC CCA AGT
CAG AGT CCG ATC TTC AAG GAC 424
ACG ATC GTG AAG TCG TGT CCC ACG GTG
GAC CTG ATG TTG CCG ATG TCC 472
GGG AAC ATC ATC GCC AGC TCC TAC GCT
AGA GCC TTC CAA CTG AAG GAC 520
GGC TCT TTC TAC ACG GCA GAA GTC AAG
AAC AAC ATA GAC TTC AAG AAT 568
CCA ATC CAC GAG TCC TTC TCG AAG TCG
GGG CCC ATG TTC ACC CAC AGA 616
CGT GTC GAG GAG ACT CAC ACC AAG GAG
AAC CTT GCC ATG GTG GAG TAC 664
CAG CAG GTT TTC AAC AGC GCC CCA AGA
GAC ATG TAG 700
AATGTGGAAC GAAACCTTTT TTTCTGATTA
CTTTCTCTGT TGACTCCACA 750
TTCGGAACTT GTATAAATAA GTTCAGTTTA
AA 782

It may be a gene characterized by (base sequence described in SEQ ID NO: 3).

In addition, the present invention also provides an invention of a plasmid vector that holds cDNA prepared from the mRNA derived from the above-mentioned podidae,
That is, the plasmid vector according to the present invention is
A plasmid vector comprising a cDNA prepared from mRNA of a fluorescent protein derived from the rodent, which is contained in the coding region of the amino acid sequence set forth in SEQ ID NO: 1,
The cDNA base sequence is
AGAACACTCA GTGTATCCAG TTTTCCGTCC
TACTACAAAC 40
ATG ACA ACC TTC AAA ATC GAG TCC CGG
ATC CAT GGC AAC CTC AAC GGG 88
GAG AAG TTC GAG TTG GTT GGA GGT GGA
GTA GGT GAG GAG GGT CGC CTC 136
GAG ATT GAG ATG AAG ACT AAA GAT AAA
CCA CTG GCA TTC TCT CCC TTC 184
CTG CTG TCC CAC TGC ATG GGT TAC GGG
TTC TAC CAC TTC GCC AGC TTC 232
CCA AAG GGG ACT AAG AAC ATC TAT CTT
CAT GCT GCA ACA AAC GGA GGT 280
TAC ACC AAC ACC AGG AAG GAG ATC TAT
GAA GAC GGC GGC ATC TTG GAG 328
GTC AAC TTC CGT TAC ACT TAC GAG TTC
AAC AAG ATC ATC GGT GAC GTC 376
GAG TGC ATT GGA CAT GGA TTC CCA AGT
CAG AGT CCG ATC TTC AAG GAC 424
ACG ATC GTG AAG TCG TGT CCC ACG GTG
GAC CTG ATG TTG CCG ATG TCC 472
GGG AAC ATC ATC GCC AGC TCC TAC GCT
AGA GCC TTC CAA CTG AAG GAC 520
GGC TCT TTC TAC ACG GCA GAA GTC AAG
AAC AAC ATA GAC TTC AAG AAT 568
CCA ATC CAC GAG TCC TTC TCG AAG TCG
GGG CCC ATG TTC ACC CAC AGA 616
CGT GTC GAG GAG ACT CAC ACC AAG GAG
AAC CTT GCC ATG GTG GAG TAC 664
CAG CAG GTT TTC AAC AGC GCC CCA AGA
GAC ATG TAG 700
AATGTGGAAC GAAACCTTTT TTTCTGATTA
CTTTCTCTGT TGACTCCACA 750
TTCGGAACTT GTATAAATAA GTTCAGTTTA
AA 782

It is a plasmid vector pBlescriptII SK-NFP (FERM BP-08681) which is (the base sequence described in SEQ ID NO: 3).

Furthermore, the present invention provides a method for using a gene encoding a fluorescent protein derived from an apopod according to the present invention for recombinant expression of the fluorescent protein derived from the anthropod in an in vitro culture system of a mammalian cell. Also provide an invention,
That is, the use of DNA having the base sequence set forth in SEQ ID NO: 2 according to the present invention is:
In an in vitro culture system of mammalian cells, a fluorescent protein derived from a rodent having the amino acid sequence of SEQ ID NO: 1 as a full-length amino acid sequence is recombinantly expressed in the mammalian cells, and an in vivo fluorescent marker -Use of DNA having a base sequence described in SEQ ID NO: 2 as a base sequence encoding a peptide chain having the amino acid sequence described in SEQ ID NO: 1, which is intended to be used as a protein. For example, the mammalian cell may be a human-derived cell line that can be cultured in vitro.

FIG. 1 (a) shows the outline of Red Copepoda (red podapod), which is the origin of the fluorescent protein of the present invention, observed under a microscope under white light irradiation, and FIG. 1 (b) shows Dark. A region emitting yellow-green fluorescence in the internal organ of the Red Copepoda observed with a fluorescence microscope under irradiation of Reader light (wavelength range: 420 nm to 500 nm) is shown. FIG. 2 shows a fluorescent gene derived from Red Copepoda, in which a gene (673 bp) encoding a fluorescent protein derived from Red Copepoda according to the present invention is inserted into a commercially available plasmid pET101 / D-TOPO (manufactured by Invitrogen). It is a figure explaining the structure of protein expression vector: pET101-NFP. FIG. 3 shows a gene (688 bp) encoding a fluorescent protein derived from Red Copepoda according to the present invention in a commercially available GST-tagged fusion protein expression plasmid vector: pGEX-6P-1 (manufactured by Amersham Biosciences). GST-tagged fluorescence in which a fluorescent protein derived from Red Copepoda is linked to the C-terminus of glutathione S-transferase (GST), a fusion partner, via a linker sequence that is inserted and provided with a cleavage site by endopeptidase Factor Xa 1 shows the structure of a protein expression vector: pGEX6P1-NFP. FIG. 4 shows SDS-related proteins in the soluble fraction (cytoplasmic component) and the insoluble fraction (membrane component) in Escherichia coli transformed with an expression vector of fluorescent protein derived from Red Copepoda: pBluescript II SK-NFP. The PAGE analysis results are shown, and a new band with a molecular weight of 25 kDa found in the soluble fraction (cytoplasmic component) of transformed E. coli is shown. FIG. 5 shows an expression vector of a fluorescent protein derived from Red Copepoda: transformed Escherichia coli carrying pET101-NFP in large quantities, and an anion exchange column: HiTrap DEAE FF (manufactured by Amersham Biosciences) from a soluble fraction (cytoplasmic component). The results of SDS-PAGE analysis of the fluorescent protein recombinant purified and recovered by gel filtration: HiLoad 16/60 Superdex 200 pg (manufactured by Amersham Biosciences) are shown. FIG. 6 shows a visual result of a fluorescent protein solution sample derived from Red Copepoda having the purification purity shown in FIG. 5 under irradiation with white light and yellow-green fluorescence under irradiation with Dark Reader light (wavelength range: 420 nm to 500 nm). The observation result which shows is shown. FIG. 7 shows an excitation spectrum measured with a fluorescent protein solution sample derived from Red Copepoda having the purification purity shown in FIG. 5 while monitoring the fluorescence intensity at a wavelength of 500 nm, and fluorescence measured while being excited at a wavelength of 508 nm. The measurement result of a spectrum is shown. FIG. 8 shows GST-tagged fluorescent protein expression vector: E. coli transformed with pGEX6P1-NFP (middle), negative control host E. coli (upper left), and cloning vector: derived from Red Copepoda SK in pBluescript II SK. An isolated clone (positive control; upper right) in which cDNA encoding a fluorescent protein is inserted is cultured on each medium, and each colony is observed under irradiation with Dark Reader light (wavelength range: 420 nm to 500 nm). Show. FIG. 9 shows a comparison between the fluorescent protein derived from Red Copepoda according to the present invention and the amino acid sequence of the fluorescent protein derived from Copepoda published by EVRΩGEN. FIG. 10 shows the results of SDS-PAGE analysis showing the purification process of a Red Copepoda-derived fluorescent protein recombinant expression product according to the present invention. FIG. 11 shows the fluorescence properties (measured at a wavelength of 517 nm) exhibited by the fluorescent protein recombinant expressed product of the Red Copepoda derived fluorescent protein recombinant expressed product according to the present invention after incubation treatment at various temperatures. The stability of the fluorescence intensity) with respect to the treatment temperature. FIG. 12 shows the fluorescence characteristics exhibited by the fluorescent protein recombinant expressed product after subjecting the Red Copepoda-derived fluorescent protein recombinant expressed product according to the present invention to incubation treatment in the presence of various reagents (drugs). (The fluorescence intensity measured at a wavelength of 517 nm) shows the stability against the action of various drugs. FIG. 13 shows the fluorescent properties (wavelengths) exhibited by the fluorescent protein recombinant expressed product after subjecting the red protein recombinant expressed product derived from Red Copepoda according to the present invention to incubation at pH in various buffers. The fluorescence intensity measured at 517 nm) is shown to be stable with respect to pH during processing. FIG. 14 shows the fluorescent properties (excitation) of the fluorescent protein recombinant expression product obtained by subjecting the fluorescent protein recombinant expression product derived from Red Copepoda according to the present invention to incubation at pH in various buffer solutions. The fluorescence spectrum measured under excitation at a wavelength of 507 nm) with respect to the pH during processing. FIG. 15 shows a commercially available coding protein (660 bp) of a fluorescent protein derived from Red Copepoda, which is used for recombinant expression in a HeLa cell of a recombinant recombinant protein derived from Red Copepoda according to the present invention. It is a figure explaining the structure of the expression vector of this fluorescent protein derived from Red Copepoda inserted in the cloning site of plasmid pcDNA3.2 / V5-GW / D-TOPO (made by Invitrogen). FIG. 16 shows the expression of a CMV-derived promoter after 18 hours of culturing in a recombinant HeLa cell produced by injecting the expression vector of the fluorescent protein derived from Red Copepoda shown in FIG. 15 into HeLa cells. It is a fluorescence image printout which shows the result of having observed the fluorescence in the HeLa cell by the fluorescent protein recombinant expression body expressed by control under a fluorescence microscope. FIG. 17 shows a fluorescent protein recombinant expression product derived from the red fluorescent protein recombinant expression product derived from Red Copepoda according to the present invention after various irradiation times and irradiation with ultraviolet light with a wavelength of 302 nm. The stability of the fluorescence characteristic (fluorescence intensity measured at a wavelength of 517 nm) against ultraviolet light irradiation is shown. FIG. 18 shows the intensity of fluorescence intensity in cells caused by NFP-Human cytoplasmic β-Actin (1128 bp, 375aa) fusion protein expressed in HeLa cells into which a fusion protein expression vector with fluorescent protein NFP has been introduced. It is a fluorescence image printout which shows the result of having observed the localization of distribution under a fluorescence microscope.

The fluorescent protein of the present invention derived from the amphipod can be recombinantly expressed in a heterologous host cell as a mature protein having its natural fluorescent properties. In addition, the fluorescence of the fluorescent protein derived from the recombined species that is recombinantly expressed is yellow-green fluorescence having a maximum wavelength of 518 nm and covering a yellow region (570 nm to 590 nm). It can be distinguished from green fluorescence such as GFP derived from victoria. Therefore, both can be used as two types of in vivo fluorescent marker proteins that show distinctly different fluorescence in host cells.

Hereinafter, the fluorescent protein derived from the anthropoid of the present invention will be described in more detail.

First, the zooplankton which is the origin of the fluorescent protein of the present invention is a crustacean plankton found in deep sea water collected from the Sea of Japan, off Toyama Bay, and a depth of 321 m. As shown in FIG. 1 (a), the form is a kind of Red Copepoda (red poda) that appears red when viewed under a microscope under white light irradiation. This Red Copepoda is taxonomically classified into Arthropoda phylum, Mandibulata subphylum, Crustacea (Crustacea class), Cypridae (Croppoda subclass, of the inferred as a kind of Copepoda, then, a result of our more detailed taxonomic identification based on comparison it is concluded to belong to Chiridius poppei. When observed with a fluorescence microscope under irradiation of ultraviolet light, for example, Dark Reader light (wavelength range: 420 nm to 500 nm), yellow-green fluorescence is emitted in the internal organs of the Red Copepoda as shown in FIG. An emitting area is observed.

  As a result of examining the region emitting yellow-green fluorescence observed in the body of the Red Copepoda in more detail, the present inventors have not caused by parasitic or adhesion of bacteria producing fluorescent proteins. It was concluded that it was due to a fluorescent protein derived from Red Copepoda itself. Actually, it was considered to collect the plankton and proceed with the isolation of the fluorescent protein. However, the amount of plankton obtained is not sufficient, and it is difficult to recover the protein amount sufficient for the amino acid sequence analysis. It was judged. Therefore, based on the results of amino acid sequence analysis, a degenerate probe that encodes a part of the amino acid sequence is prepared, and the fluorescent protein gene is cloned from genomic DNA by probe hybridization. Judged that it was difficult.

  Therefore, the present inventors tried to select a variety of mRNAs that can be translated into the fluorescent protein among the various remaining mRNAs with the expression of the protein in the body of Red Copepoda. Specifically, an expression cloning method was applied for the purpose of preparing a cDNA library from mRNA and selecting those capable of expressing a fluorescent protein using this cDNA library.

  First, a total RNA was extracted from the Red Copepoda using a commercially available RNA extraction reagent; TRIZOL reagent (manufactured by Invitrogen), and then a commercially available purification kit; Oligotex-dT30 <SUPER> mRNA Purification Kit (manufactured by TAKARA). Thus, poly (A) + mRNA used for translation of various proteins was purified. Further, from the purified mRNA, the corresponding cDNA was synthesized and amplified using a commercially available cDNA preparation kit: cDNA Synthesis kit (manufactured by Stratagene). The prepared cDNA was inserted into a cloning vector: pBluescript II SK to prepare a cDNA library.

  Use the region derived from the PCR amplification primer base sequence at both ends in the prepared cDNA. The 5 ′ end of the cDNA is the Blunt end, and the 3 ′ end is the digested end of the Xho I restriction enzyme. . On the other hand, in the multi-cloning site of cloning vector: pBluescript II SK, the Xho I restriction enzyme site was enzymatically digested, the other cleavage ends were made Blunt ends, and the cDNA fragment was ligated, A cDNA fragment was inserted into the site.

  Conventionally reported A.I. Victoria-derived GFP or the like is expressed in Escherichia coli from cDNA prepared from mRNA, and it has been found that the translated peptide chain becomes mature GFP. Similarly, a vector constituting the prepared Red Copepoda-derived cDNA library was introduced into Escherichia coli, and the inserted cDNA was expressed, and the presence or absence of fluorescence in the encoded protein was confirmed. Under the condition that approximately 300,000 colonies are generated, one colony that fluoresces was found under irradiation with Dark Reader light (wavelength range: 420 nm to 500 nm). One colony selected by this primary screening was subjected to secondary screening under the same conditions, and clones were isolated.

The introduced vector was recovered from the isolated clone, and the nucleotide sequence of the inserted cDNA was determined. The cloning vector used: Based on the known base sequence of pBluescript II SK, the sequencing to the cDNA inserted between its Blunt-Xho I sites was advanced. As a result, the full length: 782 bp and the ORF (translation frame): 660 bp were specified as the cDNA base sequence derived from mRNA used for the expression of the fluorescent protein derived from Red Copepoda in E. coli. The entire base sequence and the amino acid sequence deduced from ORF: 219 amino acids are shown below.
AGAACACTCA GTGTATCCAG TTTTCCGTCC
TACTACAAAC 40

ATG ACA ACC TTC AAA ATC GAG TCC CGG
ATC CAT GGC AAC CTC AAC GGG 88
MTTFKIESRIHGNLNG
1 5 10 15
GAG AAG TTC GAG TTG GTT GGA GGT GGA
GTA GGT GAG GAG GGT CGC CTC 136
EKFELVGGGVGEEGRL
20 25 30
GAG ATT GAG ATG AAG ACT AAA GAT AAA
CCA CTG GCA TTC TCT CCC TTC 184
EIEMKTKDKPLAFSPF
35 40 45
CTG CTG TCC CAC TGC ATG GGT TAC GGG
TTC TAC CAC TTC GCC AGC TTC 232
LLSHCMGYGFYHFASF
50 55 60
CCA AAG GGG ACT AAG AAC ATC TAT CTT
CAT GCT GCA ACA AAC GGA GGT 280
PKGTKNIYLHAATNGG
65 70 75 80
TAC ACC AAC ACC AGG AAG GAG ATC TAT
GAA GAC GGC GGC ATC TTG GAG 328
YTNTRKEIYEDGGILE
85 90 95
GTC AAC TTC CGT TAC ACT TAC GAG TTC
AAC AAG ATC ATC GGT GAC GTC 386
VNFRYTYEFNKIIGDV
100 105 110
GAG TGC ATT GGA CAT GGA TTC CCA AGT
CAG AGT CCG ATC TTC AAG GAC 424
ECIGHGFPSQSPIFKD
115 120 125
ACG ATC GTG AAG TCG TGT CCC ACG GTG
GAC CTG ATG TTG CCG ATG TCC 472
TIVKSCPTVDLMLPMS
130 135 140
GGG AAC ATC ATC GCC AGC TCC TAC GCT
AGA GCC TTC CAA CTG AAG GAC 520
GNIIASSYARAFQLKD
145 150 155 160
GGC TCT TTC TAC ACG GCA GAA GTC AAG
AAC AAC ATA GAC TTC AAG AAT 568
GSFYTAEVKNNIDFKN
165 170 175
CCA ATC CAC GAG TCC TTC TCG AAG TCG
GGG CCC ATG TTC ACC CAC AGA 616
PIHESFSKSGPMFTHR
180 185 190
CGT GTC GAG GAG ACT CAC ACC AAG GAG
AAC CTT GCC ATG GTG GAG TAC 664
RVEETHTKENLAMVEY
195 200 205
CAG CAG GTT TTC AAC AGC GCC CCA AGA
GAC ATG TAG 700
QQVFNSAPRDM *
210 215
AATGTGGAAC GAAACCTTTT TTTCTGATTA
CTTTCTCTGT TGACTCCACA 750
TTCGGAACTT GTATAAATAA GTTCAGTTTA AA 782

In addition, based on the above base sequence, PCR primers were prepared, and total RNA was extracted from 31 Red Copepoda, and RT-PCR was performed using RNA as a template to show the corresponding base sequence and molecular weight. It was confirmed that an amplification product was obtained, and it was actually verified that it encodes the fluorescent protein NFP (Namerikawa Fluorescent Protein) derived from the Red Copepoda. Moreover, the fluorescent protein NFP (Namerikawa Fluorescent Protein) derived from Red Copepoda is a yellow-green fluorescent protein derived from Chiridius poppei, and scientifically, it has been abbreviated as Cp YGFP (Chiridius poppei Yellowish Green Fluoresent Protein). .

In addition, based on the above base sequence, as a PCR forward primer and reverse primer,
Forward primer: pET-UP1 (28mer)
5-CACCATGACAACCTTCAAAATCGAGTCC
Reverse primer: SalI-LP1 (35mer); the corresponding base sequence is added to introduce a restriction enzyme SalI cleavage site at the 3 ′ end.
5-CTCGTCGACCTACATGTCTCTTGGGGCGCTGTTGA
A PCR amplification product was obtained using the vector recovered from the isolated clone as a template. As shown in FIG. 2, a PCR amplification product 673 bp containing this ORF (translation frame) is inserted into a commercially available plasmid pET101 / D-TOPO (manufactured by Invitrogen), and the expression vector of the fluorescent protein NFP: pET101-NFP Was made.

On the other hand, the fluorescent protein derived from Red Copepoda is linked to the C-terminal of the fusion partner glutathione S-transferase (GST) via a linker sequence having a cleavage site by endopeptidase Factor Xa. -Tagged fluorescent protein expression vector: pGEX6P1-NFP was prepared. That is, as a PCR forward primer and reverse primer,
Forward primer: GST-UP1 (43mer); a partial sequence of ATCGAAGGCCGC encoding the cleaved amino acid sequence of the protease: Facter Xa with respect to the pET-UP1 (28mer), and a restriction enzyme Eco RI cleavage site at the 5 ′ end The corresponding base sequence GAATTC has been added to introduce
5-CGAATTCATCGAAGGCCGCATGACAACCTTCAAAATCGAGTCC

5-CACC ATGACAACCTTCAAAATCGAGTCC
Reverse primer: SalI-LP1 (35mer); the corresponding base sequence is added to introduce a restriction enzyme SalI cleavage site at the 3 ′ end.
5-CTCGTCGACCTACATGTCTCTTGGGGCGCTGTTGA
A PCR amplification product was obtained using a vector recovered from the isolated clone as a template. Once the PCR amplification product was incorporated into pCR4 Blunt-TOPO (manufactured by Invitrogen), clone selection was performed using a selection marker. About each selection clone, after culture | cultivation, the plasmid: pCR4Blunt-NFP contained was refine | purified, The molecular weight size and the base sequence of the inserted DNA fragment were confirmed. Next, as shown in FIG. 3, the Eco RI / SalI fragment of the 688 bp inserted DNA containing ORF (translation frame) was converted into a commercially available GST-tagged fusion protein expression plasmid vector: pGEX-6P-1 (Amersham). Biosciences) to produce a GST-tagged fluorescent protein NFP expression vector: pGEX6P1-NFP.

  Escherichia coli was transformed with the fluorescent protein NFP expression vector: pET101-NFP shown in FIG. The resulting transformed Escherichia coli was subjected to clone selection using the ampicillin resistance gene as a selection marker. In addition, the expression of the inserted gene was induced from the vector-derived promoter using IPTG, and the presence or absence of the expression of the fluorescent protein was confirmed 2 hours and 4 hours after the induction. After induction of IPTG expression, the cultured bacterial cells of the transformed strains whose fluorescent protein expression has been confirmed were disrupted, and then separated by centrifugation (15,000 rpm; 18,800 × g), soluble fraction (cytoplasmic component) and insoluble fraction SDS-PAGE analysis was performed on the proteins contained in each minute (membrane component). As a result, a new band having a molecular weight of 25 kDa was found in the soluble fraction (cytoplasmic component) of transformed E. coli. That is, the molecular weight of the fluorescent protein derived from Red Copepoda is estimated to be 24.7 kDa from the above-mentioned deduced amino acid sequence, and the new band with a molecular weight of 25 kDa shown in the SDS-PAGE analysis result of FIG. 4 corresponds. .

  The fluorescent protein expression vector derived from the Red Copepoda: transformed Escherichia coli carrying pET101-NFP was cultured in large quantities, and separation and purification of the fluorescent protein recombinant were attempted. As a result of estimating (calculating) the isoelectric point (pI) of the fluorescent protein recombinant based on the deduced amino acid sequence in advance, it was calculated that pI = 6.50. Referring to the results, the soluble fraction (cytoplasmic component) was applied to an anion exchange column: HiTrap DEAE FF (manufactured by Amersham Biosciences), elution conditions: A buffer 20 mM Tris-HCl pH 7.6 B buffer 1M NaCl in A buffer, The fluorescent protein recombinant was recovered in a fraction of 4.8-8.8% B buffer in a linear gradient 0-20% B buffer (0-200 mM NaCl concentration). Next, the collected fraction was concentrated in advance under the conditions of VIVASPIN20 MW 10,000 cut. This concentrated sample was subjected to gel filtration: HiLoad 16/60 Superdex 200 pg (manufactured by Amersham Biosciences), elution conditions: A buffer 20 mM Tris-HCl pH 7.6, and the fluorescent protein as a fluorescent fraction having a molecular weight of 100 kDa or less. The recombinant was purified and recovered. As a result of SDS-PAGE analysis at this stage, the target fluorescent protein recombinant was almost purified as shown in FIG. As shown in FIG. 6, it is confirmed that the purified protein solution sample at this stage emits yellow-green fluorescence when irradiated with Dark Reader light (wavelength range: 420 nm to 500 nm). Of course, no fluorescence was observed in the control sample in which the soluble fraction (cytoplasmic component) of the host E. coli was subjected to the same purification treatment. Actually, the fluorescence spectrum and the excitation spectrum were measured using the purified protein solution sample at this stage. As shown in FIG. 7, a maximum peak was found at a wavelength of 507 nm in the excitation spectrum measured while monitoring the fluorescence intensity at a wavelength of 500 nm. On the other hand, in the fluorescence spectrum measured while being excited at a wavelength of 508 nm, fluorescence having a maximum peak at a wavelength of 518 nm and covering the yellow region (wavelength region of 570 nm to 590 nm) has been confirmed.

Moreover, Escherichia coli was transformed using the GST-tagged fluorescent protein expression vector: pGEX6P1-NFP shown in FIG. The obtained transformed Escherichia coli, host Escherichia coli (negative control), and cloning vector: isolated clone (positive control) in which the cDNA encoding the fluorescent protein derived from Red Copypoda is inserted in pBluescript II SK Each was cultured on a medium. As shown in FIG. 8, each colony was observed under irradiation with Dark Reader light (wavelength range: 420 nm to 500 nm). As a result, the obtained colonies of transformed Escherichia coli emitted fluorescence, and the GST-tagged fluorescent protein It was confirmed that expression was made. In addition, as a fusion protein linked to another protein through an appropriate linker sequence, cyclization of an internal tripeptide site that forms a fluorophore from a translated peptide chain even when recombinantly expressed, Through subsequent oxidation, it is confirmed that a mature fluorescent protein having fluorescent properties is obtained.

On the other hand, the present inventors searched for the presence or absence of a report on a fluorescent protein derived from Copepoda in addition to the fluorescent protein derived from Red Copepoda of the present invention. It has been found that a green fluorescent protein expression vector containing is commercially available from EVRΩGEN under the trade name Cop-Green ^™ . The Copepoda-derived fluorescent protein recombinant expression CopGFP expressed from the commercially available green fluorescent protein expression vector exhibits a maximum peak at a wavelength of 502 nm, exhibits green fluorescence, and the excitation spectrum has a wavelength. The product catalog shows that it has a maximum peak at 482 nm.

In addition, the amino acid sequence of the Copepoda-derived fluorescent protein encoded in Cop-Green ^™ published by EVRΩGEN, and the base sequence obtained by converting the ORF codon to the corresponding humanized codon are shown below.
Sequence
of the humanized version of the CopGFP's open reading frame

ATG CCC GCC
ATG AAG ATC GAG TGC CGC ATC ACC GGC ACC CTG AAC
GGC 48
MPAMKIECRITGTLNG 16

GTG GAG TTC
GAG CTG GTG GGC GGC GGA GAG GGC ACC CCC GAG CAG
GGC 96
VEFELVGGGEGTPEQG 32

CGC ATG ACC
AAC AAG ATG AAG AGC ACC AAG GGC GCC CTG ACC TTC AGC 144
RMTNKMKSTKGALTFS 48

CCC TAC CTG
CTG AGC CAC GTG ATG GGC TAC GGC TTC TAC CAC TTC GGC 192
PYLLSHVMGYGFYHFG 64

ACC TAC CCC
AGC GGC TAC GAG AAC CCC TTC CTG CAC GCC ATC AAC AAC 240
TYPSGYENPFLHAINN 80

GGC GGC TAC
ACC AAC ACC CGC ATC GAG AAG TAC GAG GAC GGC GGC GTG 288
GGYTNTRIEKYEDGGV 96

CTG CAC GTG
AGC TTC AGC TAC CGC TAC GAG GCC GGC CGC GTG ATC GGC 336
LHVSFSYRYEAGRVIG 112

GAC TTC AAG
GTG GTG GGC ACC GGC TTC CCC GAG GAC AGC GTG ATC TTC 384
DFKVVGTGFPEDSVIF 128

ACC GAC AAG
ATC ATC CGC AGC AAC GCC ACC GTG GAG CAC CTG CAC CCC 432
TDKIIRSNATVEHLHP 144

ATG GGC GAT
AAC GTG CTG GTG GGC AGC TTC GCC CGC ACC TTC AGC CTG 480
MGDNVLVGSFARTFSL 160

CGC GAC GGC
GGC TAC TAC AGC TTC GTG GTG GAC AGC CAC ATG CAC TTC 528
RDGGYYSFVVDSHMHF 176

AAG AGC GCC
ATC CAC CCC AGC ATC CTG CAG AAC GGG GGC CCC ATG TTC 576
KSAIHPSILQNGGPMF 192

GCC TTC CGC
CGC GTG GAG GAG CTG CAC AGC AAC ACC GAG CTG GGC ATC 624
AFRRVEELHSNTELGI 208

GTG GAG TAC
CAG CAC GCC TTC AAG ACC CCG ATC GCA TTC GCC
TGA 669
VEYQHAFKTPIAFA * 223

Comparing the red protein-derived fluorescent protein according to the present invention with the amino acid sequence of the copepoda-derived fluorescent protein published by EVRΩGEN, as shown in FIG. When homologous amino acid residues are included, considerable homology has been found. However, there is a clear difference between the fluorescence spectra exhibited by both.

  These crustacean-derived fluorescent proteins show considerable homology and can be assumed to constitute a novel family of fluorescent proteins. The tripeptide site involved in the formation of the fluorophore is presumed to be a GYG site. Furthermore, in these two types of crustacean-derived fluorescent proteins, mature proteins that have undergone fluorination and oxidation of fluorophores are presumed to exhibit similar three-dimensional structures. Copepoda-derived fluorescent protein recombinant expression CopGFP expressed from an expression vector commercially available from EVRΩGEN has been shown to exhibit fluorescence as a monomer. In consideration of homology, the Red Copepoda-derived fluorescent protein according to the present invention can be similarly in the form of a monomer when recombinantly expressed in mammalian cells, and can be used as an in vivo fluorescent marker protein. It is predicted.

  The vector: pBluescript II SK-NFP in which the gene (cDNA) encoding the fluorescent protein derived from Red Copepoda according to the present invention is inserted into the cloning vector: pBluescript II SK multi-cloning site is based on the Budapest Treaty. International Deposit (2004) under the accession number FERM BP-08681, at the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (Chuo 6-1, 1st East, Tsukuba, Ibaraki, Japan, postal code 305-8586) March 31).

When the Red Copepoda-derived fluorescent protein according to the present invention is recombinantly expressed in mammalian cells as an in vivo fluorescent marker protein, A. A technique for substituting the coding region of victoria-derived GFP or an artificially modified expression system thereof can be used. In addition to mammalian cells, a fluorescent protein derived from Red Copepoda can be similarly expressed recombinantly in hosts such as bacteria, yeast, fungi, and insect cells that are capable of recombinant expression of conventional GFP. . When used for such recombinant expression, it is preferable that the gene encoding the fluorescent protein derived from Red Copepoda is converted into a codon frequently used in the host, if necessary, and then inserted into an expression vector. Of course, no mutation is introduced into the amino acid sequence itself encoded by the gene due to codon conversion. Upon insertion into the expression vector, the coding gene that has been previously codon-converted is digested with restriction enzymes in the non-coding regions at both ends and fragmented. When performing this restriction enzyme digestion, if there is no appropriate restriction enzyme site, it is possible to introduce a desired restriction enzyme site by introducing a mutation into the base sequence of the non-coding region by site-directed mutagenesis. it can.

Hereinafter, the present invention will be specifically described with reference to examples. The specific examples shown here are examples of the best mode according to the present invention, but the present invention is not limited to these specific examples.

(Collecting crustacean plankton that produces a novel fluorescent protein)
The inventors have described A.I. GFP-like protein family derived from Candaria hydra (class Hydrozoa) and flower ant (class Anzozoa) typified by GFP from Victoria For the purpose of discovering a new fluorescent protein family that has no origins, animals that have newly collected deep ocean water from the 321m depth of the Sea of Japan, off Toyama Bay, and are producing fluorescent proteins Searched for sex plankton.

  In the search process, it has been confirmed that many luminescent plankton exist in the sampled deep ocean water. Among them, among the crustacean plankton, the fluorescence caused by the fluorescent protein was found in the body organ, and further, the fluorescence exhibiting yellow fluorescence or yellow-green fluorescence was selected.

In the sorting process, as shown in FIG. 1 (a), the form of Red Copepoda (red poda) that appears red under microscopic observation under white light irradiation is ultraviolet light, for example, Dark. When observed with a fluorescence microscope under irradiation of Reader light (wavelength range: 420 nm to 500 nm), as shown in FIG. 1 (b), it was found that a region emitting yellow-green fluorescence was shown in the internal organ of the Red Copepoda. . This Red Copepoda is taxonomically classified into Arthropoda phylum, Mandibulata subphylum, Crustacea (Crustacea class), Cypridae (Croppoda subclass, It was inferred that it was a kind of pinniped . As a result of further detailed taxonomic identification, based on the identification by assistant attorney Atsushi Yamaguchi who belongs to the Graduate School of Fisheries Sciences, Hokkaido University Graduate School of Fisheries Science, this Red Copepoda is based on Metazoa (animal kingdom) and Arthropoda. (Arthropoda), Crucacea (Crustacea), Maxillopoda (Copepoda), Copepoda (Copepoda), Neocopododa (Copepoda), Gymnoplea (Coleoptera), Calanoida aetae ), It was concluded that it belongs to Chiridius poppei, which is classified as Chiridius.

(Cloning of a gene encoding the fluorescent protein NFP derived from Red Copepoda)
Collecting total RNA From the sampled deep ocean water, 300 collected Red Copepoda were drained, suspended in a total of 3 mL of TRIZOL reagent, frozen and stored at -80 ° C.

  The frozen Red Copepoda individuals were thawed at room temperature, and 3 mL of TRIZOL reagent was further added. This suspension was transferred to a 15 mL Teflon homogenizer container and applied to a 10-degree crusher to crush the outer shell and body cells. The obtained cell debris was transferred to a 15 mL falcon tube and centrifuged (11,000 rpm) at 2-8 ° C. for 10 minutes. The supernatant (first extracted component) was collected in another 15 mL falcon tube.

  To the remaining pellet pellet, 1 mL of TRIZOL reagent was added and resuspended. This resuspension was transferred to a glass homogenizer container and subjected to homogenization again. The reprocessed solution was transferred to another 15 mL Falcon tube, 4 mL of TRIZOL reagent was added, and then centrifuged (11,000 rpm) at 2-8 ° C. for 10 minutes. The obtained supernatant (second extracted component) was collected and combined with the supernatant of the previous stage (first extracted component) to obtain a total of 10 mL of extracted components, and each 5 mL was dispensed into a 15 mL Falcon tube.

  In the dispensed tube, 0.2 mL of chloroform (1 mL per tube) was added per 1 mL of the TRIZOL reagent, and then shaken well to disperse both liquid phases. After leaving still at room temperature for 2-3 minutes, it centrifuged at 2-8 degreeC for 10 minutes (11,000 rpm). The separated aqueous phase (about 3 mL) was aliquoted into another tube.

  To the collected aqueous phase (about 3 mL), 0.5 mL of isopropanol (2.4 mL per tube) per 1 mL of the original TRIZOL reagent was added and mixed well. After leaving still at room temperature for 5 minutes, it centrifuged at 1-8 rpm at 2-8 degreeC for 10 minutes. After removing the supernatant, 5 mL of absolute ethanol was added to the alcohol precipitation fraction per tube and stored at −20 ° C.

  1 mL of 75% ethanol (5 mL per tube) per 1 mL of the original TRIZOL reagent was added to the ethanol precipitation pellet in the tube, vortexed, dispersed and mixed. The dispersion / mixture was centrifuged (11,000 rpm) at 2-8 ° C. for 10 minutes. After removing the supernatant, the RNA precipitate pellet was left at room temperature for 10 minutes to evaporate the remaining solvent and dried. One of the two dried total RNA samples purified from the two aliquots was stored dry at -80 ° C.

The remaining dried total RNA sample was added with 400 μL of RNase-free water, allowed to stand for 10 to 20 minutes, and redissolved. A part of the lysate was collected, and absorbances OD ₂₆₀ , OD ₂₈₀ , and OD ₃₂₀ (background absorption) at wavelengths of 260 nm, 280 nm, and 320 nm were measured. Based on the results, the RNA-containing concentration was calculated from the absorbance OD ₂₆₀ according to a conventional method. At the same time, gel electrophoresis was performed under non-denaturing conditions, and the presence or absence of contaminants was analyzed to confirm the purity of the contained RNA. Table 1 shows the evaluation results of the RNA-containing concentration with respect to the obtained total RNA sample.

From the purified total RNA solution (RNA-containing concentration: 1.14 μg / μL) 200 μL prepared in the purification of poly (A) + mRNA, the poly (A) + mRNA contained therein is a commercially available purification kit; Oligotex− It isolate | separated and refine | purified using dT30 <SUPER> mRNA Purification Kit (made by TAKARA).

  To 200 μL of the total RNA solution, 200 μL of the hybridization buffer: 2 × Binding buffer attached to the kit was added, and a total of 400 μL of the solution was homogenized. To this RNA solution, 20 μL of Oligotex-dT30 dispersion was added and mixed well. The solution in the tube was heated to 70 ° C., held for 3 minutes, and then allowed to cool at room temperature for 10 minutes. The poly (A) + mRNA poly (A) end and the Oligotex-dT30 Oligo-dT probe part Hybridization with was carried out. Centrifugation (15,000 rpm) was performed for 5 minutes to separate Oligotex-dT30 as a precipitate fraction. Supernatant containing RNA components that were not hybridized with Oligotex-dT30 was removed.

  The precipitated fraction was dispersed in 350 μL of the washing buffer attached to the kit and transferred to a centrifuge column tube. The supernatant was removed by centrifugation (15,000 rpm) for 30 seconds. Further, the same washing operation was performed using the same amount of washing buffer.

  50 μL of DEPC-water (aqueous solution) attached to the kit, which had been heated to 70 ° C. in advance, was added to the precipitate fraction that had been washed twice, and this mixture was transferred to another tube for a centrifugal column. Centrifugation (15,000 rpm) was performed for 30 seconds, and a supernatant containing poly (A) + mRNA released from the Oligotex-dT30 probe was collected. To the precipitate fraction, 50 μL of DEPC-water (aqueous solution) heated in advance to 70 ° C. was added again, and the release operation from the probe was performed, and the supernatant was collected. The collected supernatants were combined to form a solution containing a total of 100 μL of purified mRNA.

  To the solution containing the purified mRNA, 10 μL of 3M sodium acetate aqueous solution and 100 μL of 100% isopropanol were added and mixed well. Then, it was left to stand at −20 ° C. for 10 minutes to precipitate the contained mRNA in alcohol. Centrifugation (14,000 rpm) was performed for 30 minutes, the precipitated mRNA was collected in the precipitate fraction, and the supernatant was removed. Furthermore, 1 mL of 75% ethanol was added to the precipitated fraction of the precipitated mRNA and mixed well. Centrifugation (14,000 rpm) for 5 minutes was performed to separate the precipitated mRNA fraction, and the supernatant was removed.

  The purified mRNA precipitate obtained was redissolved in 11 μL of DEPC-water (aqueous solution). A sample for partial evaluation was taken, and the remaining (10.5 μL) purified mRNA sample solution was frozen and stored at −80 ° C. In addition, RNA containing concentration was evaluated using the sample for evaluation. Further, in each purification step, the removed supernatant and the purified mRNA precipitate were subjected to gel electrophoresis under non-denaturing conditions to confirm the purification process. Table 2 shows the evaluation results of the RNA-containing concentration for the obtained purified mRNA sample.

Synthesis of cDNA using mRNA as a template Using a commercially available cDNA synthesis kit: cDNA Synthesis Kit (Stratagene), cDNA was synthesized using purified mRNA as a template.

First, single-stranded cDNA (first strand) was synthesized by the following procedure.
Reverse transcription buffer attached to the kit: 10 × 1st strand buffer 5 μL, methyl dNTP mixture 3 μL, linker-primer mixture 2 μL, RNase Block Ribonuclease Inhibitor solution 1 μL, water 6 μL
7.44 μL of the purified mRNA solution (mRNA amount 5 μg) was once heat-treated at 70 ° C. for 3 minutes to eliminate the higher-order structure, rapidly cooled by ice cooling, added, and gently mixed. It was kept at room temperature for 10 minutes to allow the polymerase to bind to the 3 ′ end of the mRNA. Attached reverse transcriptase: 1.5 μL of StrataScript Riverse transcriptase solution was added, gently mixed, and subjected to an enzyme reaction at 42 ° C. for 1 hour.

Subsequently, using the synthesized single-stranded cDNA (first strand) as a template, the complementary strand (second strand) was synthesized by the following procedure.

With respect to 50 μL of the obtained enzyme reaction solution,
Buffer for DNA synthesis attached to the kit: 10 × 2nd strand buffer 20 μL, 2nd strand dNTP mixture 6 μL, and distilled water (DDW) 111 μL were sequentially added, and RNaseH solution 2 μL (enzyme concentration 1.5 U / μL) was further added as an RNase. ), DNA pol. I Solution 11 μL (enzyme concentration 9.0 U / μL) was added and gently mixed. The remaining mRNA was digested with RNase H, while the prepared single-stranded cDNA (first strand) was used as a template from the upstream primer, DNA pol. The synthesis of the complementary strand (second strand) is advanced by I. The enzyme reaction solution was kept at 16 ° C. for 2.5 hours, the complementary strand (second strand) was extended to form double-stranded cDNA, and then cooled with ice to stop the enzyme reaction.

Smoothing of double-stranded cDNA ends Both ends of the double-stranded cDNA were smoothed according to the following procedure.

  To the reaction solution containing the double-stranded cDNA, 23 μL of blunting dNTP mixture and 2 μL of cPfu enzyme solution (enzyme concentration 2.5 U / μL) were added. The reaction solution was vortexed and mixed uniformly, and then maintained at 72 ° C. for 30 minutes for enzyme treatment.

  After 30 minutes, 200 μL of phenol was added to the reaction solution, vortexed and mixed. The liquid phase was separated by centrifuging (15,000 rpm) for 2 minutes, and the upper aqueous layer was separated. To the separated aqueous layer, 200 μL of chloroform was added, subjected to vortexing and mixed. The liquid phase was separated by centrifuging (15,000 rpm) for 2 minutes, and the upper aqueous layer was separated.

To the separated aqueous layer, 20 μL of 3M sodium acetate aqueous solution and 400 μL of absolute ethanol were added, and mixed well by vortexing to precipitate the contained cDNA in ethanol. The ethanol-precipitated cDNA precipitate was centrifuged for 60 minutes (15,000 rpm) and separated into precipitate fractions. The supernatant was removed, and 500 μL of 70% ethanol was added to the remaining fraction of the cDNA precipitate and mixed well. Centrifugation (15,000 rpm) was performed for 2 minutes to separate the cDNA precipitate into a precipitate fraction, and the supernatant was removed. The recovered pellet of cDNA precipitate was dried.

The dry pellet of the cDNA precipitate was redispersed in 9 μL of Eco RI adapter solution, kept at 4 ° C. for 1 hour. To this solution, 4.5 μL of a commercially available Ligation reaction solution: Ligation High was added and kept at 16 ° C. overnight (16 hours), and Eco RI adapter was ligated to the cDNA end. To the reaction solution, 186.5 μL of distilled water was added and diluted to a total volume of 200 μL. Further, 200 μL of phenol was added, and vortexed and mixed well. The liquid phase was separated by centrifuging (15,000 rpm) for 5 minutes, and the upper aqueous layer was separated. To the separated aqueous layer, 200 μL of chloroform was added, subjected to vortexing and mixed. The liquid phase was separated by centrifuging (15,000 rpm) for 5 minutes, and the upper aqueous layer was separated.

To the separated aqueous layer, 10 μL of 3M sodium acetate aqueous solution and 200 μL of 100% isopropanol were added, and mixed well by vortexing to precipitate the contained cDNA in alcohol. The alcohol-precipitated cDNA precipitate was separated into precipitate fractions by centrifugation (15,000 rpm) at 4 ° C. for 60 minutes. The supernatant was removed, and 500 μL of 70% ethanol was added to the remaining fraction of the cDNA precipitate and mixed well. Centrifugation (15,000 rpm) was performed for 2 minutes to separate the cDNA precipitate into a precipitate fraction, and the supernatant was removed. The recovered pellet of cDNA precipitate was dried.

The recovered cDNA precipitate pellet was well redispersed in a mixture of 20 μL of distilled water, 3 μL of T4 PNK buffer, 3 μL of 50% glycerol, and 3 μL of 75 mM ATP solution. The reaction solution for T4 PNK enzyme was cooled to -20 ° C. After thawing, 1 μL of T4 Polynucleotide Kinase enzyme solution from a commercially available enzyme solution set (manufactured by TAKARA) was added and maintained at 37 ° C. for 1 hour to carry out the enzyme reaction. After the phosphorylation of the double-stranded cDNA to the 5 ′ end was completed, heat denaturation treatment was performed by heating and maintaining at 70 ° C. for 30 minutes to complete the reaction.

Xho I digestion treatment of double-stranded cDNA The double-stranded cDNA subjected to the above-mentioned end blunting treatment was digested with Xho I according to the following procedure.

  End-blunted double-stranded cDNA solution 30 μL, commercially available restriction enzyme buffer solution (manufactured by TAKARA): 10 × H Buffer 11.5 μL, water (DDW) 70.5 μL, commercially available restriction enzyme solution kit ( 3 μL of Xho I restriction enzyme solution (manufactured by TAKARA) (enzyme concentration: 10 U / μL) is mixed to prepare a total of 115 μL of reaction solution. After enzymatic digestion at 37 ° C. for 2 hours, 200 μL of phenol was added, vortexed and mixed well. The liquid phase was separated by centrifuging (15,000 rpm) for 5 minutes, and the upper aqueous layer was separated. To the separated aqueous layer, 115 μL of chloroform was added, subjected to vortexing and mixed. The liquid phase was separated by centrifuging (15,000 rpm) for 5 minutes, and the upper aqueous layer was separated.

  Next, 115 μL of the collected aqueous layer containing the enzymatically digested cDNA fragment was applied to S-300 spin column, which was equilibrated by adding 3 volumes of 1 × STE buffer. Centrifugation (1,500 rpm; 400 × g) for 2 minutes recovered 105 μL of the aqueous layer to be separated. 200 μL of absolute ethanol was added to the recovered aqueous layer, and the mixture was allowed to stand at −20 ° C. for 1 hour to precipitate a cDNA fragment. The precipitate of the cDNA fragment was separated into a precipitate fraction by centrifugation (15,000 rpm) at 4 ° C. for 60 minutes. The operation of adding 900 μL of 70% ethanol to the precipitate fraction of the cDNA precipitate and washing it twice was repeated, and then the recovered pellet of the cDNA fragment precipitate was dried.

The recovered pellet of the cDNA fragment precipitate was redispersed in 6.0 μL of TE buffer to obtain a solution. A part (1.0 μL) was collected and the concentration of the contained cDNA was evaluated. The cDNA concentration was 404.1 ng / μL, and the 5 ′ end was Blunt end, and the 3 ′ end was Xho I-digested solution of a double-stranded cDNA fragment, giving a total of 5.0 μL.

Cloning vector: formation of the insertion site for the Blunt-end / Xho I cleavage in the multi-cloning site of pBluescript II SK. A solution of SK (+) (concentration 500 ng / μL) was prepared. This vector solution 6 μL (vector amount 3 μg), 10 × H Buffer 3 μL, Xho I restriction enzyme solution 1 μL (enzyme concentration 10 U / μL), water (DDW) 20 μL, a total of 30 μL reaction solution is maintained at 37 ° C. for 3 hours. The vector is then enzymatically digested at the Xho I site. The main fragment of the enzyme-digested vector was separated and purified by the MinElute method, and recovered as 26 μL of eluate in Election Buffer.

  To this 26 μL eluate, 3 μL of CIAP buffer (One-phor-All buffer) and 1 μL of CIAP enzyme solution were added, and a total of 30 μL of the reaction solution was maintained at 37 ° C. for 30 minutes, and only the Xho I site was subjected to CIAP treatment. did. The main fragment of the treated vector was separated and purified by the MinElute method and recovered as 26 μL of eluate in Election Buffer.

  To the recovered 26 μL eluate, add 3 μL of 10 × H Buffer and 1 μL of Eco RV restriction enzyme solution (enzyme concentration: 15 U / μL), hold at 37 ° C. overnight (14 hours), and remove the Xho I digested end of the vector. One was treated with Eco RV. As a result, the vector: pBluescript II SK (+) is cleaved to have an insertion site for the Blunt-end / Xho I cleavage site.

The reaction solution after the Eco RV treatment was applied to a 0.7% agarose gel and electrophoresed to cut out a DNA fragment band having a desired size. The excised gel was treated with Ultra free DA and centrifuged (7,000 rpm) for 10 minutes to recover a liquid layer containing the vector DNA fragment. Next, from this solution, the vector DNA fragment was separated and purified by the MinElute method, and separated as 20 μL of the eluate in the Elution Buffer. The DNA fragment in this vector DNA fragment solution was once ethanol precipitated and redispersed in 5 μL of Elution Buffer to prepare a vector DNA fragment solution having a DNA concentration of 94.5 ng / μL.

Construction of cDNA library A vector in which the insertion site of the aforementioned Blunt-end / Xho I cleavage site is formed: pBluescript II SK (+) fragment, 5 ′ end is Blunt end, 3 ′ end is digested with Xho I A double-stranded cDNA fragment was ligated to construct a cDNA library.

Vector DNA fragment solution 0.53 μL (DNA amount 50 ng), end-treated double-stranded cDNA fragment solution 1 μL (DNA amount 200 ng), Ligation High 0.765 μL were mixed and kept at 16 ° C. overnight (14 hours). Both DNA fragments were ligated. In the obtained plasmid vector, cDNA prepared from mRNA recovered from Red Copepoda is inserted, and a cDNA library is constructed.

Introduction of plasmid vector into host Escherichia coli The constructed cDNA library was introduced into the host Escherichia coli by Electroporation method, and a transformant was selected. The plasmid vector was introduced into the host E. coli by the electroporation method according to the following procedure.

5 μL of Strata Clean Resin solution was added to 2.295 μL of Ligation solution containing the prepared cDNA library, and the mixture was mixed well by vortexing for 15 seconds. Centrifugation allowed the Resin to settle and the supernatant was collected in a separate tube. Again, 5 μL of Resin solution was added to the supernatant and vortexed for 15 seconds to mix well. Moreover, it centrifuged, sedimented Resin, and collect | recovered supernatants to another tube.

  Each Resin sediment remaining in the two tubes was washed with 2.5 μL of water (DDW). Centrifugation was performed to precipitate Resin, and the liquid layer containing the plasmid vector recovered by washing was separated.

The previous supernatant and the collected liquid layer were combined to obtain a solution containing a total of 10 μL of plasmid vector. Once again, the mixture was centrifuged to settle slightly contaminated Resin, and the supernatant was collected in another tube. From the collected supernatant, the enzyme used for the ligation reaction is removed to become a plasmid DNA solution.

The TOP10 strain and TenBlue strain used for host Escherichia coli are thawed at cryogenic temperature in a cryopreserved Component Cell. 5 μL of the plasmid DNA solution subjected to the Resin adsorption treatment was added to 40 μL of a thawed host cell Escherichia coli Competent Cell suspension. Commercial Electroporation Equipment: E.I. Using E. coli Pulser (manufactured by Bio-rad), electroporation was injected into the host in a pulse voltage; 1.7 kV, 0.1 mm gap cuvette. The time constant of the pulse used was set to 4.1 μs for the cDNA library / TOP10 strain system and 4.0 μs for the cDNA library / TenBlue strain system. After the vector injection treatment, 955 μL of the medium component SOC preheated to 37 ° C. was added to 45 μL of the host E. coli suspension, and the mixture was cultured with shaking at 37 ° C. for 1.5 hours.

  Thereafter, 5 μL was collected from 1000 μL of the obtained culture solution, 100 μL of the medium component SOC was added, seeded on a 9 cmφ petri dish, and cultured at room temperature (20 ° C.) for 2 days. To 995 μL of the remaining culture solution, 428.57 μL of 50% glycerol was added, and the mixed solution (containing a final concentration of 15% glycerol) was stored frozen at −80 ° C.

In the petri dish culture, the number of colonies that showed ampicillin resistance was counted. As a result, 1425 colonies were found in the cDNA library / TOP10 strain system and 700 colonies were in the cDNA library / TenBlue strain system. Therefore, the density of the transformant contained in the culture solution is 2.8 × 10 ⁵ cfu / mL (1.1 × 10 ⁷ cfu / μg vector) in the cDNA library / TOP10 strain system. / TenBlue strain system corresponds to 1.4 × 10 ⁵ cfu / mL (5.6 × 10 ⁶ cfu / μg vector).

On the other hand, using the colony PCR method, the ratio of transformants having a vector into which a cDNA fragment was inserted (cDAN library efficiency) was evaluated. Vector: The presence or absence of cDNA fragment insertion between the T7 promoter site located upstream of the multi-cloning site of pBluescript II SK (+) and the T3 promoter site located downstream of the multi-cloning site Confirmed in the procedure.

colony RCR is
As a forward primer, corresponding to the base sequence of the T7 promoter site,
T7 primer: GTAATACGACTCACTATAGGGC
As a reverse primer, complementary to the base sequence of the T3 promoter site
TTAATTGGGAGTGATTTCCC
T3 primer: AATTAACCCTCACTAAAGGG
PCR amplification was performed using a commercially available DNA synthase: KOD Dash DNA polymerase using the vector DNA contained in the clone as a template. Table 3 shows the temperature conditions of the PCR reaction used and the reaction solution composition.

Ten colonies were randomly selected from the colonies on the petri dish, and the cells of each colony were suspended in 70 μL of water (DDW). This cell suspension was treated at 95 ° C. for 5 minutes. A solution containing the vector recovered from the cells was used as a colony solution in the reaction solution.

After completion of the PCR reaction, 3 μL was collected from 10 μL of the reaction solution containing the amplification product, and electrophoresed on a 0.7% gel, and the presence or absence of the PCR amplification product corresponding to the cDNA fragment and its size range were examined. PCR amplification products corresponding to the cDNA fragments were found in 5 colonies out of 10 randomly selected colonies with respect to the cDNA library prepared above. Therefore, the ratio of those having a vector having a cDNA fragment inserted in the transformed strain was actually determined to be about 50%.

Assuming that the type of gene encoding a protein derived from Red Copepoda contained in a clone library prepared by clone selection having a cDNA encoding a fluorescent protein derived from Red Copepoda by Expression cloning method is 3 × 10 ⁴ . On the other hand, when a total number of colonies of 3 × 10 ⁵ cfu is generated, colonies having a vector into which a cDNA fragment has been inserted are expected to have 1.5 × 10 ⁵ cfu when the ratio is 50%. Assuming that the type of coding gene contained in the cDNA library is 3 × 10 ⁴ and contained at the same frequency, about 5 colonies are expected for each coding gene. The variation in the existence frequency mainly reflects the abundance ratio of the original mRNA, but at least 2 to 3 colonies of the transformant in which the cDNA encoding the target fluorescent protein is inserted are found. It was estimated.

In addition, it has been reported that many of the GFPs known so far are produced as mature GFP having fluorescence when expressed in host E. coli. Red Copepoda-derived fluorescent protein may also be produced as a mature fluorescent protein having fluorescence when expressed in host E. coli.

Based on the above considerations, a transformant holding a cDNA library is cultured on a plurality of petri dishes using medium LB / Car under the condition that the total number of colonies is 1.5 × 10 ⁵ or more. Colony formation was performed.

  The culture solution supplemented with glycerol, which was stored frozen at −80 ° C., was thawed, and the culture medium component SOC was added to make a total culture solution of 2000 μL. Among them, 1 μL of the culture solution was collected, added to 100 μL of the medium component SOC, seeded on a 9 cmφ petri dish, and cultured at 37 ° C. overnight (14 hours). The entire amount of the remaining culture solution was seeded on a medium LB / Car prepared in a total of 11 15 cmφ petri dishes and cultured at 37 ° C. overnight (14 hours).

On a 9 cmφ petri dish, 153 colonies were generated from 1 μL of the culture solution. On the other hand, in a total of 11 15 cmφ petri dishes, at least the total number of colonies reached 1.5 × 10 ⁵ or more. Accordingly, the total number of colonies generated is determined to be 153 × 2000, approximately in the range of 3.0 × 10 ^{5 to} 1.5 × 10 ⁵ .

  As a result of examining the presence or absence of fluorescent colonies for a total of 11 15 cmφ petri dishes, one fluorescent colony was found under the ultraviolet irradiation of Dark Reader. The fluorescent colony was taken out, and a solution suspended in 5 mL of medium LB / Car was diluted 1/10 with the same medium. 100 μL of this bacterial cell diluted solution was inoculated on a medium LB / Car prepared in a 15 cmφ petri dish, and a secondary screening for fluorescent colonies was performed. After culturing at 37 ° C. overnight (14 hours), colonies formed on a 15 cmφ petri dish were observed under the dark reader irradiation of ultraviolet light, and 80 to 90% of the colonies showed fluorescence.

In this secondary screening, in particular, four fluorescent positive colonies were randomly picked from a plurality of colonies showing clear fluorescence (fluorescent positive). Each of these fluorescent positive colonies was suspended in 5 mL of medium LB / Car and cultured at 37 ° C. for 9 hours. Each of the obtained four cultures of fluorescent positive colonies was cryopreserved as a clone culture solution (containing 15% glycerol).

Replication and purification of the plasmid vector introduced into the selected clones From the four selected clones, the introduced plasmid vector was replicated and purified by the following procedure.

  In a system in which a cDNA library was introduced using TOP 10 host E. coli, four colonies (clones) selected by the two-stage screening as colonies emitting fluorescence were suspended in 5 mL of medium LB / Car. Culturing was carried out at 0 ° C. for 8 hours. The culture solution was centrifuged (5000 × g), and the cells were collected.

  From the sorted cells, the plasmid was separated and purified using a commercially available plasmid purification kit: QIAGEN plasmid purification kit (manufactured by QIAGEN). To the sorted cells, 0.375 mL of the P1 solution attached to the purification kit was added and dispersed well by vortexing. To this cell dispersion, 0.375 mL of the attached P2 solution was added and mixed. It left still for 5 minutes at room temperature (20 degreeC). Next, 0.525 mL of the attached N3 solution was added and mixed. After the sterilization treatment, centrifugation (11,000 rpm) was performed at 4 ° C. for 15 minutes, and a soluble fraction (supernatant) of plasmid DNA was separated and collected.

  The soluble fraction (supernatant) containing plasmid DNA was applied to the QIAprep 4 column of the same purification kit. The liquid layer was removed by centrifugation (15,000 rpm) at 4 ° C. PB 0.5 mL was added for washing, followed by PE 0.75 mL for washing. Finally, the washing solution was removed by centrifugation (15,000 rpm) at 4 ° C. for 1 minute. The plasmid adsorbed on the QIAprep of the purification kit was eluted with 30 μL of Elute Buffer (EB) and collected. Of 30 μL of the solution containing the purified plasmid, 2 μL was taken, and 98 μL of distilled water was added to make a 50-fold diluted solution.

  Table 4 shows the results of evaluating the DNA concentration contained in the liquid containing the purified plasmid for each clone.

Nucleotide sequence analysis of cDNA fragments in selected clones From the results of colony RCR, the four clones have been found to have cDNA fragments with the same base length in the plasmid vector they hold. First, the cDNA portion inserted into this plasmid vector was PCR amplified by the following procedure.

About the liquid containing the plasmid vector collect | recovered and refine | purified from the selection clone, the density | concentration adjustment was performed so that the DNA density | concentration might be 500 ng / microliter. Next, a DNA sample preparation kit for commercially available sequencing: BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq polymerase, using this purified plasmid vector as a template, a sample for nucleotide sequence analysis,
As a forward primer, corresponding to the base sequence of the T7 promoter site,
T7 primer: GTAATACGACTCACTATAGGGC
As a reverse primer, complementary to the base sequence of the T3 promoter site
TTAATTGGGAGTGATTTCCC
T3 primer: AATTAACCCTCACTAAAGGG
Was used as a sequence primer and was prepared from the region containing cDNA inserted in a plasmid vector. Table 5 shows the temperature conditions of the used DNA chain extension reaction and the composition of the reaction solution.

The prepared sample for base sequence analysis was purified by the following procedure.

From each reaction tube, the prepared sample solution was transferred to another 0.5 mL tube. Separately, a solution prepared by mixing a 3M sodium acetate aqueous solution 0.5 μL and 95% ethanol 12.5 μL per 5 μL of the sample solution was separately prepared in a 1.5 mL tube. The sample solution collected in advance was put into the 1.5 mL tube. After mixing uniformly, the mixture was allowed to stand for 10 minutes under ice-cooling to precipitate the contained DNA fragment in ethanol. Centrifugation (14,000 rpm) was performed for 20 minutes to deposit the precipitated DNA fragment, and the supernatant was removed. Subsequently, 125 μL of 70% ethanol was added to rinse the precipitated DNA fragment. Again, the mixture was centrifuged (14,000 rpm) for 5 minutes to deposit the precipitated DNA fragments, and the supernatant was removed by suction. The remaining pellet of the precipitated DNA fragment was dried.

The purified analytical sample DAN fragment was redispersed in Template suppressor Reagent (TSR). After vortexing and mixing, the solution was collected by centrifugation. The mixture was heated at 95 ° C. for 2 minutes, separated into single-stranded DNA, and cooled on ice. Once vortexed, it was centrifuged to deposit the template plasmid. After separation of the template plasmid, the template was stored at -20 ° C. Thereafter, the sample DAN fragment for analysis was subjected to a base sequence analysis by applying it to a commercially available sequence device: ABI PRISM 3100 Genetic Analyzer.

The sequence analysis results from the 5 ′ end side and the sequence analysis results from the 3 ′ end side were synthesized, and the base sequence of the cDNA fragment prepared from mRNA encoding the fluorescent protein derived from Red Copepoda was determined. .

(Recombinant expression of fluorescent protein derived from Red Copepoda)
Insertion of Red Copepoda-derived fluorescent protein gene into plasmid pET101 / D-TOPO First, based on the above base sequence, as a PCR forward primer and reverse primer,
Forward primer: pET-UP1 (28mer)
5-CACCATGACAACCTTCAAAATCGAGTCC
Reverse primer: SalI-LP1 (35mer); the corresponding base sequence is added to introduce a restriction enzyme SalI cleavage site at the 3 ′ end.
5-CTCGTCGACCTACATGTCTCTTGGGGCGCTGTTGA
A PCR amplification product was obtained under the following conditions using the vector recovered from the isolated clone as a template. Table 6 shows the temperature conditions of the PCR reaction used and the composition of the reaction solution.

The PCR amplification product thus prepared was purified by the following procedure.

  After performing PCR reaction with a reaction volume of 25 μL each, combine a total of 3 reaction liquids, collect 2 μL of the reaction liquid, and run on a 1% agarose gel to confirm a PCR amplification product with a target molecular weight of 673 bp did.

  Next, the product DNA was concentrated from the reaction solution by the MinElute method. 5 volumes of PB buffer was added per volume (73 μL) of the reaction solution, subjected to vortexing, and transferred to a MinElute column. The solution was centrifuged for 30 seconds to deposit the precipitated DNA, and the supernatant was removed. The precipitated DNA was washed with 0.7 mL of PE buffer and centrifuged (15,000 rpm) for 1 minute. Further, 10 μL of EB buffer is added, and the mixture is allowed to stand at room temperature for 1 minute. Thereafter, the mixture was centrifuged (15,000 rpm) for 1 minute, and the supernatant was collected.

  After adding 2 μL of 10 × loading dye solution to the recovered DNA solution, 12 μL of each lane DNA solution was run on a 1.0% TAE agarose gel. The desired 688 bp band was excised from the gel. The gel slice was placed in a 1.5 mL eppen tube, and the DNA was collected.

As shown in FIG. 2, the purified double-stranded DNA was inserted into a commercially available plasmid pET101 / D-TOPO (manufactured by Invitrogen) to prepare an expression vector for the fluorescent protein derived from the Red Capepoda: pET101-NFP. .

In addition, the fluorescent protein derived from Red Copepoda is linked to the C-terminal of the fusion partner glutathione S-transferase (GST) via a linker sequence having a cleavage site by endopeptidase Factor Xa. An expression vector for GST-tagged fluorescent protein: pGEX6P1-NFP was also prepared. That is, as a PCR forward primer and reverse primer,
Forward primer: GST-UP1 (43mer); a partial sequence of ATCGAAGGCCGC encoding the cleaved amino acid sequence of the protease: Facter Xa with respect to the pET-UP1 (28mer), and a restriction enzyme Eco RI cleavage site at the 5 ′ end The corresponding base sequence GAATTC has been added to introduce
5-CGAATTCATCGAAGGCCGCATGACAACCTTCAAAATCGAGTCC

5-CACC ATGACAACCTTCAAAATCGAGTCC
Reverse primer: SalI-LP1 (35mer); the corresponding base sequence is added to introduce a restriction enzyme SalI cleavage site at the 3 ′ end.
5-CTCGTCGACCTACATGTCTCTTGGGGCGCTGTTGA
A PCR amplification product was obtained using a vector recovered from the isolated clone as a template. Table 7 shows the temperature conditions of the PCR reaction used and the composition of the reaction solution.

The prepared PCR amplification product was purified according to the procedure described above.

  First, after carrying out PCR reaction with a reaction liquid volume of 25 μL each, a total of 3 reaction liquids were combined, 2 μL reaction liquid was collected and run on a 1% agarose gel, and a target molecular weight of 688 bp (19 + 660 + 9) was obtained. PCR amplification products were confirmed. Subsequent separation, purification procedures, and conditions were the same.

The purified double-stranded DNA was once incorporated into pCR4 Blunt-TOPO (manufactured by Invitrogen), and clone selection was performed using a selection marker. Each selected clone was cultured to increase the clone plasmid pCR4Blunt-NFP. After culturing, the contained plasmid was purified, and its molecular weight size and the base sequence of the inserted DNA fragment were confirmed. Next, as shown in FIG. 3, the Eco RI / SalI fragment of the 688 bp inserted DNA containing the ORF (translation frame) following the site encoding the cleavage sequence of Factor Xa was expressed in a commercially available GST-tagged fusion protein. A plasmid vector for use: pGEX-6P-1 (manufactured by Amersham Biosciences) was inserted to prepare a GST-tagged fluorescent protein expression vector: pGEX6P1-NFP.

(Fluorescence characteristics of a recombinant expression product of Red Copepoda-derived fluorescent protein NFP)
First, Escherichia coli was transformed with the fluorescent protein NFP expression vector: pET101-NFP shown in FIG. The resulting transformed Escherichia coli was subjected to clone selection using the ampicillin resistance gene as a selection marker.

  For the selected clone, expression of the inserted gene was induced from the promoter derived from the vector pET101 / D-TOPO using IPTG, and after 2 hours and 4 hours after induction, the recombinant protein was expressed recombinantly. The presence or absence was confirmed. In addition, after induction of IPTG expression, the fact that the recombinantly expressed fluorescent protein is a mature protein is confirmed by showing fluorescence of the colony of the transformed strain under ultraviolet irradiation.

  With respect to the cultured transformant, the cultured cells were collected when 4 hours had elapsed after the induction of IPTG expression. After disrupting the cells, SDS-PAGE analysis was performed on the proteins contained in the soluble fraction (cytoplasmic component) and insoluble fraction (membrane component) separated by centrifugation (15,000 rpm; 18,800 × g). . As a result, a new band having a molecular weight of 25 kDa was found in the soluble fraction (cytoplasmic component) of transformed E. coli. That is, the molecular weight of the fluorescent protein derived from Red Copepoda is estimated to be 24.7 kDa from the above-mentioned deduced amino acid sequence, and the new band with a molecular weight of 25 kDa shown in the SDS-PAGE analysis result of FIG. 4 corresponds. .

  The Red Copepoda-derived fluorescent protein NFP expression vector: The transformed E. coli harboring pET101-NFP was cultured in large quantities, and separation and purification of the fluorescent protein recombinant produced by inducing IPTG expression was attempted.

  As a result of estimating (calculating) the isoelectric point (pI) of the fluorescent protein recombinant based on the deduced amino acid sequence in advance, it was calculated that pI = 6.50. Referring to the results, the soluble fraction (cytoplasmic component) was applied to an anion exchange column: HiTrap DEAE FF (manufactured by Amersham Biosciences), elution conditions: A buffer 20 mM Tris-HCl pH 7.6 B buffer 1M NaCl in A buffer, The fluorescent protein recombinant was recovered in a fraction of 4.8-8.8% B buffer in a linear gradient 0-20% B buffer (0-200 mM NaCl concentration).

  Next, the collected fraction was concentrated in advance under the conditions of VIVASPIN20 MW 10,000 cut. This concentrated sample was subjected to gel filtration: HiLoad 16/60 Superdex 200 pg (manufactured by Amersham Biosciences), elution conditions: A buffer 20 mM Tris-HCl pH 7.6, and the fluorescent protein as a fluorescent fraction having a molecular weight of 100 kDa or less. The recombinant was purified and recovered. As a result of SDS-PAGE analysis at this stage, the target fluorescent protein recombinant was almost purified as shown in FIG.

  As shown in FIG. 6, it is confirmed that the purified protein solution sample at this stage emits yellow-green fluorescence when irradiated with Dark Reader light (wavelength range: 420 nm to 500 nm). Of course, no fluorescence was observed in the control sample in which the soluble fraction (cytoplasmic component) of the host E. coli was subjected to the same purification treatment. Actually, the fluorescence spectrum and the excitation spectrum were measured using the purified protein solution sample at this stage. As shown in FIG. 7, a maximum peak was found at a wavelength of 507 nm in the excitation spectrum measured while monitoring the fluorescence intensity at a wavelength of 500 nm. On the other hand, in the fluorescence spectrum measured while being excited at a wavelength of 508 nm, fluorescence having a maximum peak at a wavelength of 518 nm and covering the yellow region (wavelength region of 570 nm to 590 nm) has been confirmed.

Furthermore, the protein solution sample after gel filtration was applied to a MonoQ HR 5/5 column (manufactured by Amersham Biosciences), and elution conditions: A buffer 20 mM Tris-HCl pH 7.6 B buffer 1 M NaCl in A buffer, linear gradient 10-20% In B buffer (100-200 mM NaCl concentration), the purified fluorescent protein recombinant was recovered in a fraction of 12.0-14.0% B buffer. FIG. 10 shows the results of SDS-PAGE analysis of each protein solution sample in the purification process of the fluorescent protein recombinant expression product derived from Red Copepoda described above.

Moreover, Escherichia coli was transformed using the GST-tagged fluorescent protein expression vector: pGEX6P1-NFP shown in FIG. The obtained transformed Escherichia coli, host Escherichia coli (negative control), and cloning vector: isolated clone (positive control) in which the cDNA encoding the fluorescent protein derived from Red Copypoda is inserted in pBluescript II SK Each was cultured on a medium. As shown in FIG. 8, each colony was observed under irradiation with Dark Reader light (wavelength range: 420 nm to 500 nm). As a result, the obtained colonies of transformed Escherichia coli emitted fluorescence, and the GST-tagged fluorescent protein It was confirmed that expression was made. In addition, as a fusion protein linked to another protein through an appropriate linker sequence, cyclization of an internal tripeptide site that forms a fluorophore from a translated peptide chain even when recombinantly expressed, Through subsequent oxidation, it is confirmed that a mature fluorescent protein having fluorescent properties is obtained.

(Temperature Dependence of Fluorescence Properties of Recombinant Expressed Fluorescent Protein NFP from Red Copepoda)
The purified fluorescent protein NFP recombinant prepared by the above method is subjected to incubation treatment at various temperatures according to the following evaluation method, and the dependence of the fluorescence characteristics on the treatment temperature is evaluated.

  10 μL each of the solution containing the fluorescent protein NFP recombinant purified at a protein concentration of 0.577 mg / mL in 20 mM Tris-HCl pH 8.5 buffer was added at a temperature of 4, 20, 30, 37, 45, 50. , 55, 60, 65, 70, 75, and 80 ° C. for 10 minutes. After the treatment, the solution containing the NFP recombinant is ice-cooled (0 ° C.). To 10 μL of each ice-cooled (0 ° C.) solution, 192.5 μL of 20 mM Tris-HCl pH 8.5 buffer is added and diluted to obtain a diluted solution having a final protein concentration of 20 μg / mL. Using this diluted solution, a fluorescence spectrum of the fluorescent protein NFP recombinant is measured using a spectrofluorometer manufactured by Hitachi while being excited at a wavelength of 507 nm.

  The fluorescence spectrum measured in a diluted solution containing the fluorescent protein NFP recombinant that has not undergone incubation treatment has a maximum peak at a wavelength of 518 nm. Measured in a diluted solution containing an NFP recombinant that has been incubated at the various temperatures, based on the fluorescence spectrum of the fluorescent protein NFP recombinant, measured when the incubation treatment is not performed. Measure the fluorescence spectrum. In the measured fluorescence spectrum, the fluorescence intensity at a wavelength of 517 nm was plotted against the treatment temperature during the incubation treatment on the horizontal axis to obtain a plot shown in FIG.

In the plot shown in FIG. 11, no deterioration in the fluorescence characteristics of the fluorescent protein NFP recombinant has been found in the range of the treatment temperature of 70 ° C. or lower when ice-cooled (0 ° C.) after the incubation treatment. On the other hand, when the treatment temperature is 75 ° C. or higher, a decrease in the fluorescence characteristics of the fluorescent protein NFP recombinant is observed even after ice treatment (0 ° C.) after the incubation treatment. Therefore, when the fluorescent protein NFP recombinant is in the range of a treatment temperature of 70 ° C. or less, when it is ice-cooled (0 ° C.) after the incubation treatment, there is no deterioration in the fluorescence characteristics due to the structural change caused by thermal denaturation In the said temperature range, it is evaluated that high temperature stability is shown.

(Drug resistance of fluorescent properties of recombinant expression product of fluorescent protein NFP derived from Red Copepoda)
The purified fluorescent protein NFP recombinant prepared by the above method is subjected to incubation treatment in the presence of various reagents (drugs) according to the following evaluation method to stabilize the fluorescence characteristics against the action of various drugs. Assess sex.

  Various reagent (drug) solutions 192 listed below are added to 10 μL each of the solution containing the fluorescent protein NFP recombinant purified at a protein concentration of 0.577 mg / mL in 20 mM Tris-HCl pH 8.5 buffer. Add 5 μL to a drug-containing solution with a final protein concentration of 20 μg / mL. This drug-containing solution is incubated for 10 minutes under ice-cooling (0 ° C.). After the treatment, the fluorescence spectrum of the fluorescent protein NFP recombinant is measured using the drug-containing solution while being excited at a wavelength of 507 nm using a Hitachi spectrofluorometer.

FIG. 12 shows a fluorescent protein recombinant expressed product derived from Red Copepoda according to the present invention after incubation with a recombinant expressed product of fluorescent protein in the presence of various reagents (drugs). The characteristic (fluorescence intensity measured at a wavelength of 517 nm) is displayed as a relative value based on the fluorescence characteristic when a buffer solution for dilution (20 mM Tris-HCl pH 8.5 buffer) is added.

  When an organic solvent is added and the solvated water molecules covering the fluorescent protein recombinant expressed product are removed, the fluorescence characteristics exhibited by the fluorescent protein recombinant expressed product are significantly reduced. On the other hand, when guanidine hydrochloride and urea, which show the function of a protein denaturing agent, are allowed to act, the fluorescence characteristics of the fluorescent protein recombinant expression product undergo a considerable decrease. However, the action of SDS (sodium dodecyl sulfate) of about 1% has no influence on the fluorescence characteristics exhibited by the fluorescent protein recombinant expression product.

Even if 2-mercaptoethanol or DTT (dithiothreitol), which shows the action of reducing the Cys-Cys bond in the protein, is allowed to act,
It only receives a slight decline.

(PH dependence of fluorescence characteristics of a recombinant expression product of the fluorescent protein NFP derived from Red Copepoda)
The purified fluorescent protein NFP recombinant prepared by the above method is subjected to an incubation treatment in a solution to which various pH buffer solutions are added according to the following evaluation method, and the dependence of fluorescence characteristics on pH is determined. To evaluate.

In 20 mM Tris-HCl pH 8.5 buffer, each buffer solution 192.5 μL listed below is added to 10 μL each of the solution containing the fluorescent protein NFP recombinant purified at a protein concentration of 0.577 mg / mL. In addition, the pH is adjusted with the buffer solution to obtain a final protein concentration of 20 μg / mL. This solution having various pH values is incubated for 10 minutes under ice-cooling (0 ° C.). After the treatment, the fluorescence spectrum of the fluorescent protein NFP recombinant is measured using the solutions exhibiting various pHs while being excited at a wavelength of 507 nm using a Hitachi spectrofluorometer.
Buffer solution for pH adjustment:
pH3.0 Glycine-HCl
pH3.5 Glycine-HCl
pH4.0 Acetate
pH4.5 Acetate
pH5.0 Acetate
pH5.5 Acetate
pH6.0 Phosphate
pH7.0 HEPES
pH8.0 Tris-HCl
pH8.5 Tris-HCl
pH8.9 Tris-HCl
pH10.0 Carbonate
pH10.5 Carbonate
pH11.0 Carbonate
pH11.5 Phosphate-NaOH
pH12.0 Phosphate-NaOH
pH12.5 Phosphate-NaOH
pH13.0 Phosphate-NaOH
pH13.6 NaOH

FIG. 13 shows the fluorescence exhibited by the fluorescent protein recombinant expression product after subjecting the Red Copepoda-derived fluorescent protein recombinant expression product according to the present invention to incubation treatment at pH adjusted by various buffer solutions. The results of plotting the characteristics (fluorescence intensity measured at a wavelength of 517 nm) against pH values adjusted by various buffer solutions are shown. At least in the range of pH 6.0 to 11.0, it is confirmed that the fluorescent property exhibited by the fluorescent protein recombinant expression product exhibits high stability.

In FIG. 14, the measurement result in pH3.0-pH11.0 is shown collectively about the fluorescence spectrum of this fluorescent protein NFP recombinant measured in the solution of various pH in said evaluation. At least in the range of pH 3.5 to pH 11.0, the fluorescence intensity of the maximum peak measured at a wavelength of 517 nm varies, but the relative shape of the fluorescence spectrum of the fluorescent protein NFP recombinant is generally retained. It is confirmed that

(Resistance to fluorescent light of the recombinant expression product of Red Copepoda-derived fluorescent protein NFP with respect to ultraviolet light irradiation)
The purified fluorescent protein NFP recombinant prepared by the above method is subjected to a treatment for irradiation with ultraviolet light having a wavelength of 302 nm for a long time according to the following evaluation method, and the ultraviolet light having a fluorescent property for a long time. Evaluate stability against irradiation.

A solution containing the fluorescent protein NFP recombinant purified at a protein concentration of 0.577 mg / mL in 20 mM Tris-HCl pH 8.5 buffer was irradiated with ultraviolet light at a wavelength of 302 nm at an irradiation light amount of 7300 μW / cm ^2. A process of continuous irradiation for minutes is performed. Meanwhile, 10 μL each before the start of irradiation (irradiation time 0 minutes), at the time when 0, 1, 5, 10, 15, 30, 45 minutes have elapsed after the start of irradiation and at the end of irradiation (irradiation time 60 minutes). Take a sample of. To 10 μL of each collected sample solution, 192.5 μL of 20 mM Tris-HCl pH 8.5 buffer is added and diluted to obtain a diluted solution having a final protein concentration of 20 μg / mL. Using this diluted solution, a fluorescence spectrum of the fluorescent protein NFP recombinant is measured using a spectrofluorometer manufactured by Hitachi while being excited at a wavelength of 507 nm.

FIG. 17 shows the fluorescent properties of the fluorescent protein recombinant expression product obtained by subjecting the red protein recombinant expression product derived from Red Copepoda according to the present invention to continuous irradiation with ultraviolet light having a wavelength of 302 nm. The fluorescence intensity measured at a wavelength of 517 nm is displayed as a relative value based on the fluorescence characteristics before irradiation. From the results shown in FIG. 17, it is confirmed that the fluorescent protein NFP recombinant is not substantially deteriorated in fluorescence characteristics within a range where the continuous irradiation time with ultraviolet light having a wavelength of 302 nm is within 60 minutes. Is done.

(Recombinant expression in human cells of fluorescent protein derived from Red Copepoda)
Insertion of Red Copepoda-derived Fluorescent Protein Gene into Plasmid pcDNA3.2 / V5-GW / D-TOPO According to the above-mentioned red Copepoda-derived fluorescent protein expression vector: Red Copepoda Purified double-stranded DNA prepared by PCR based on the coding gene of the fluorescent protein NFP derived from the cloning site of the commercially available plasmid pcDNA3.2 / V5-GW / D-TOPO (manufactured by Invitrogen) The expression vector for expression in human cells of the fluorescent protein derived from Red Copepoda was prepared. FIG. 15 shows the construction of the expression vector for expression in human cells of the produced Red Copepoda-derived fluorescent protein NFP. Note that the double-stranded DNA fragment inserted into such an expression vector has the same base sequence as that of the coding protein of the fluorescent protein NFP derived from Red Copepoda, and is subjected to codon conversion suitable for codon selection in humans. It has not been. That is, it is the same as the double-stranded DNA fragment inserted into the expression vector: pET101-NFP.

The prepared plasmid was collected and purified using a commercially available plasmid purification kit; QIAGEN Plasmid Maxi Kit (manufactured by QIAGEN).

Introduction of an expression vector for expression of a fluorescent protein derived from Red Copepoda into a human cell into HeLa cells The purified expression vector for expression in human cells of the fluorescent protein NFP derived from Red Copepoda was introduced into a HeLa cell using PolyFect. Introduced by applying the Transfection method. As the host HeLa cells, those cultured in a serum-added medium (DMEM + 10% FBS + 100 μg / ml Kanmycin) on a 100 mm dish to 70% confluent are used. At that time, 6.0 μL of an expression vector / plasmid solution (DNA content: 5 ng / mL) was added to 50.0 μL of a commercially available reagent solution for PolyFect Transfection; PolyFect Transfection Reagent (manufactured by QIAGEN), and the mixture was stirred for 10 minutes. To do. After the PolyFect Transfection treatment, the HeLa cells are maintained at 37 ° C. in an atmosphere of 5% CO ₂ and subjected to an incubation treatment for 24-48 hours to recover cell damage associated with the treatment and culture.

  When the recombinant expression of the fluorescent protein NFP derived from Red Copepoda in HeLa cells introduced with this expression vector was induced, as shown in an example in FIG. In addition, fluorescence derived from a recombinant fluorescent protein NFP was observed. That is, even in HeLa cells derived from humans, translation into a recombinant fluorescent protein NFP is performed based on the coding gene of fluorescent protein NFP derived from Red Copepoda, and into mature fluorescent protein NFP. It clearly shows that post-translational processing in the cell has progressed.

Therefore, the coding gene for the fluorescent protein NFP derived from Red Copepoda can be used in an in vitro culture system that uses various human-derived cell systems without sufficient codon conversion to match codon selection in human cells. It is verified that it can be used as a coding gene for an in vivo fluorescent marker protein that can be expressed in a host cell.

Furthermore, it was verified as described below that the fluorescent protein NFP derived from Red Copepoda can actually be used as an in vivo fluorescent marker protein that can be expressed in a host cell.

  Similar to the widely used fluorescent marker protein EGFP, a fusion protein obtained by linking the fluorescent protein NFP derived from Red Copperpoda to the N-terminus of the target protein can be expressed in the host cell. It is verified that the fusion protein retains the fluorescence characteristics derived from the fluorescent protein NFP at the N-terminal part, as well as the function of the target protein at the C-terminal part.

  Commercially available plasmid vector for expressing fusion protein of fluorescent marker protein EGFP and human cytoplasmic β-actin, which are widely used; pGFP-Actin (5820 bp: manufactured by BD Biosciences Clontech), EGFP and human α-tubulin A commercially available plasmid vector for expression of a fusion protein with pGFP-Tub (6045 bp: manufactured by BD Biosciences Clontech), and the coding sequence portion of the fluorescent marker protein EGFP is converted to the fluorescent protein NFP derived from the Red Copepoda NFP-Human cytoplasmic β-Actin (1128 bp, 375aa) fusion protein, and NFP-Human α-tu ulin (in frame with RedGFP 1356bp, 451aa) a recombinant expression vector for fusion protein, to build the following procedure.

  To the coding gene of the fluorescent protein NFP derived from Red Copepoda, a kozak sequence (GCCACC) is added immediately before the start codon, and a restriction enzyme NheI recognition sequence (GCTAGC) is provided in the upstream untranslated region, while terminating. In order to add a base sequence (TCCGAC TCA GAT) that constitutes a part of the coding base sequence of the linker sequence, excluding codons, and to provide a restriction enzyme SalI recognition sequence (GTCGAC) downstream of it, the following forward primer : NFP NheI / Kozak-UP1 and reverse primer: NFP SalI-LP1 is used for PCR amplification.

NFP NheI / Kozak-UP1 48mer
5'-CCA GCT AGC GCT ACG GTC GCC
ACC ATG ACA ACC TTC AAA ATC GAG TCC-3 '
NFP SalI-LP1 45mer
5'-AAA GTC GAC ATC TGA
GTC CGG ACA TGT CTC TTG GGG CGC TGT TGA-3 '
In the commercially available plasmid vectors pGFP-Actin and pGFP-Tub, the coding sequence part of the fluorescent marker protein EGFP is digested with restriction enzymes NheI and XhoI and excised. On the other hand, the obtained PCR product (703 bp) is digested with the newly introduced restriction enzymes NheI and SalI sites. The resulting DNA fragment is ligated with a vector fragment of a plasmid vector digested with a restriction enzyme to construct a recombinant protein recombinant expression vector. The prepared plasmid was purified using a commercially available purification kit; Endo Free Plasmid Maxi Kit (Qiagen).

The prepared two types of expression vectors were analyzed for their base sequences, the DNA fragment derived from the inserted PCR product and the base sequence of the ligation part were confirmed, and the fluorescent protein NFP derived from the Red Copepoda and the C-terminal It was confirmed that the coding sequence with the target protein on the side was linked without a frame shift via the linker portion. In particular, the amino sequence of the linker portion provided between the fluorescent protein NFP derived from Red Copepoda and the target protein on the C-terminal side is the base sequence (T CCG GAC included in the reversal primer: NFP SalI-LP1). TCA GAT) and a cleavage site in the recognition sequence (GTCGAC) of the restriction enzyme SalI downstream thereof are ligated to the vector fragment, whereby the following base sequence:
TCC GGA CTC AGA TGT CGA GCT
AGG CCT GAG TCT A CA GCT CGA
It was confirmed that the partial amino acid sequence encoded by is SGLRCRA 7a.a.

  In accordance with the transfection procedure described above, human intracellular expression vectors for each fusion protein are introduced into HeLa cells by applying the PolyFect Transfection method. When the cells were cultured for 16 hours after gene recombination, helium cells that were cultivated were found to emit fluorescence. When observed with a fluorescence microscope, HeLa cells into which the above-described expression vector for the fluorescent protein NFP alone is introduced, uniform fluorescence is observed from the entire cell, but the fusion protein expression vector with the fluorescent protein NFP is observed. In the HeLa cells into which the fluorescence was introduced, the distribution of fluorescence intensity in the cells showed localization.

That is, it can be recombinantly expressed as a fusion protein in human cells, and after translation, the fluorescent protein NFP derived from the Red Copepoda at its N-terminal part exhibits fluorescent properties through proper holding, Along with the localization of β-actin or α-tubulin at the C-terminal part in the cell, it is observed as a localized fluorescence intensity distribution. FIG. 18 shows the fluorescence intensity in a cell caused by an NFP-Human cytoplasmic β-Actin (1128 bp, 375aa) fusion protein expressed in HeLa cells into which a fusion protein expression vector with the fluorescent protein NFP has been introduced. The result of having observed the localization of distribution by the fluorescence microscope is shown. As a result, the coding gene of the fluorescent protein NFP derived from Red Copepoda is actually an in vivo fluorescent marker that can be expressed in the host cell in an in vitro culture system using various human-derived cell systems. It is a visa that it can be used as a protein coding gene.

  The fluorescent protein of the present invention can be used as an in vivo fluorescent marker protein that can be expressed in a host cell in an in vitro culture system using mammalian cells.

Claims (8)

Taxonomically, a fluorescent protein derived from a rodent that is classified as Chiridius poppei,
The full-length amino acid sequence of the fluorescent protein is
MTTFKIESRI HGNLNGEKFE LVGGGVGEEG RLEIEMKTKD KPLAFSPFLL SHCMGYGFYH 60
FASFPKGTKN IYLHAATNGG YTNTRKEIYE DGGILEVNFR YTYEFNKIIG DVECIGHGFP 120
SQSPIFKDTI VKSCPTVDLM LPMSGNIIAS SYARAFQLKD GSFYTAEVKN NIDFKNPIHE 180
SFSKSGPMFT HRRVEETHTK ENLAMVEYQQ VFNSAPRDM 219
(The amino acid sequence described in SEQ ID NO: 1)
The fluorescent protein can be recombinantly expressed in a mammalian cell in vitro culture system and in Escherichia coli as a fluorescent protein exhibiting fluorescence resulting from a fluorophore formed after translation. Fluorescent protein. A gene that encodes a fluorescent protein derived from a rodent class classified as Chiridius poppei,
A gene comprising DNA encoding the amino acid sequence set forth in SEQ ID NO: 1. The base sequence of DNA encoding the amino acid sequence set forth in SEQ ID NO: 1 is
ATG ACA ACC TTC AAA ATC GAG TCC CGG ATC CAT GGC AAC CTC AAC GGG 48
GAG AAG TTC GAG TTG GTT GGA GGT GGA GTA GGT GAG GAG GGT CGC CTC 96
GAG ATT GAG ATG AAG ACT AAA GAT AAA CCA CTG GCA TTC TCT CCC TTC 144
CTG CTG TCC CAC TGC ATG GGT TAC GGG TTC TAC CAC TTC GCC AGC TTC 192
CCA AAG GGG ACT AAG AAC ATC TAT CTT CAT GCT GCA ACA AAC GGA GGT 240
TAC ACC AAC ACC AGG AAG GAG ATC TAT GAA GAC GGC GGC ATC TTG GAG 288
GTC AAC TTC CGT TAC ACT TAC GAG TTC AAC AAG ATC ATC GGT GAC GTC 336
GAG TGC ATT GGA CAT GGA TTC CCA AGT CAG AGT CCG ATC TTC AAG GAC 384
ACG ATC GTG AAG TCG TGT CCC ACG GTG GAC CTG ATG TTG CCG ATG TCC 432
GGG AAC ATC ATC GCC AGC TCC TAC GCT AGA GCC TTC CAA CTG AAG GAC 480
GGC TCT TTC TAC ACG GCA GAA GTC AAG AAC AAC ATA GAC TTC AAG AAT 528
CCA ATC CAC GAG TCC TTC TCG AAG TCG GGG CCC ATG TTC ACC CAC AGA 576
CGT GTC GAG GAG ACT CAC ACC AAG GAG AAC CTT GCC ATG GTG GAG TAC 624
CAG CAG GTT TTC AAC AGC GCC CCA AGA GAC ATG TAG 660
The gene according to claim 2, wherein the gene is (the base sequence described in SEQ ID NO: 2). A cDNA prepared from mRNA of a fluorescent protein derived from a rodent class classified as Chiridius poppei, which encodes the amino acid sequence set forth in SEQ ID NO: 1.
The cDNA base sequence is
AGAACACTCA GTGTATCCAG TTTTCCGTCC TACTACAAAC 40
ATG ACA ACC TTC AAA ATC GAG TCC CGG ATC CAT GGC AAC CTC AAC GGG 88
GAG AAG TTC GAG TTG GTT GGA GGT GGA GTA GGT GAG GAG GGT CGC CTC 136
GAG ATT GAG ATG AAG ACT AAA GAT AAA CCA CTG GCA TTC TCT CCC TTC 184
CTG CTG TCC CAC TGC ATG GGT TAC GGG TTC TAC CAC TTC GCC AGC TTC 232
CCA AAG GGG ACT AAG AAC ATC TAT CTT CAT GCT GCA ACA AAC GGA GGT 280
TAC ACC AAC ACC AGG AAG GAG ATC TAT GAA GAC GGC GGC ATC TTG GAG 328
GTC AAC TTC CGT TAC ACT TAC GAG TTC AAC AAG ATC ATC GGT GAC GTC 376
GAG TGC ATT GGA CAT GGA TTC CCA AGT CAG AGT CCG ATC TTC AAG GAC 424
ACG ATC GTG AAG TCG TGT CCC ACG GTG GAC CTG ATG TTG CCG ATG TCC 472
GGG AAC ATC ATC GCC AGC TCC TAC GCT AGA GCC TTC CAA CTG AAG GAC 520
GGC TCT TTC TAC ACG GCA GAA GTC AAG AAC AAC ATA GAC TTC AAG AAT 568
CCA ATC CAC GAG TCC TTC TCG AAG TCG GGG CCC ATG TTC ACC CAC AGA 616
CGT GTC GAG GAG ACT CAC ACC AAG GAG AAC CTT GCC ATG GTG GAG TAC 664
CAG CAG GTT TTC AAC AGC GCC CCA AGA GAC ATG TAG 700
AATGTGGAAC GAAACCTTTT TTTCTGATTA CTTTCTCTGT TGACTCCACA 750
TTCGGAACTT GTATAAATAA GTTCAGTTTA AA 782
The gene according to claim 2, wherein the gene is (the base sequence of SEQ ID NO: 3). A plasmid vector obtained by inserting a cDNA prepared from mRNA of a fluorescent protein derived from a rodent class classified as Chiridius poppei, which encodes the amino acid sequence represented by SEQ ID NO: 1,
The cDNA base sequence is
AGAACACTCA GTGTATCCAG TTTTCCGTCC TACTACAAAC 40
ATG ACA ACC TTC AAA ATC GAG TCC CGG ATC CAT GGC AAC CTC AAC GGG 88
GAG AAG TTC GAG TTG GTT GGA GGT GGA GTA GGT GAG GAG GGT CGC CTC 136
GAG ATT GAG ATG AAG ACT AAA GAT AAA CCA CTG GCA TTC TCT CCC TTC 184
CTG CTG TCC CAC TGC ATG GGT TAC GGG TTC TAC CAC TTC GCC AGC TTC 232
CCA AAG GGG ACT AAG AAC ATC TAT CTT CAT GCT GCA ACA AAC GGA GGT 280
TAC ACC AAC ACC AGG AAG GAG ATC TAT GAA GAC GGC GGC ATC TTG GAG 328
GTC AAC TTC CGT TAC ACT TAC GAG TTC AAC AAG ATC ATC GGT GAC GTC 376
GAG TGC ATT GGA CAT GGA TTC CCA AGT CAG AGT CCG ATC TTC AAG GAC 424
ACG ATC GTG AAG TCG TGT CCC ACG GTG GAC CTG ATG TTG CCG ATG TCC 472
GGG AAC ATC ATC GCC AGC TCC TAC GCT AGA GCC TTC CAA CTG AAG GAC 520
GGC TCT TTC TAC ACG GCA GAA GTC AAG AAC AAC ATA GAC TTC AAG AAT 568
CCA ATC CAC GAG TCC TTC TCG AAG TCG GGG CCC ATG TTC ACC CAC AGA 616
CGT GTC GAG GAG ACT CAC ACC AAG GAG AAC CTT GCC ATG GTG GAG TAC 664
CAG CAG GTT TTC AAC AGC GCC CCA AGA GAC ATG TAG 700
AATGTGGAAC GAAACCTTTT TTTCTGATTA CTTTCTCTGT TGACTCCACA 750
TTCGGAACTT GTATAAATAA GTTCAGTTTA AA 782
A plasmid vector pBluescript II SK-NFP (FERM BP-08681), characterized in that it is (the base sequence described in SEQ ID NO: 3).   Recombinant expression in a mammalian cell of a fluorescent protein derived from Chiropius poppei having the full-length amino acid sequence of SEQ ID NO: 1 in an in vitro culture system of mammalian cells The recombinant expression type fluorescent protein that exhibits fluorescence resulting from the fluorophore formed after translation is used as an in vivo fluorescent marker protein and is described in SEQ ID NO: 1. Use of DNA having the base sequence set forth in SEQ ID NO: 2 as a base sequence encoding a peptide chain having the amino acid sequence of Use of DNA according to claim 6, characterized in that the mammalian cells are human derived cell lines which can be cultured in vitro. 8. Use of DNA according to claim 6 or 7, characterized in that the mammalian cells are human-derived HeLa cells.