JP2014060946A - Red fluorescent protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a red fluorescent protein being highly useful.SOLUTION: Provided is a red fluorescent protein having a specified amino acid sequence derived from disc coral (Discosoma sp.). Also provided is a red fluorescent protein whose amino acid sequence has 80% or more homology to the above amino acid sequence, and it fluoresces after the expressing cell is cultured at least 16 hours at 37°C.

Description

  The present invention relates to a red fluorescent protein.

  Clontech's red fluorescent protein (Red Fluorescent Protein, RFP), cloned from the coral insect Discosoma, is widely used as a fluorescent marker in cytology and molecular biology. RFP is used as a reporter for imaging changes in intracellular localization and expression level by fusing with other proteins. The wavelength used for the excitation of RFP has less light scattering and lower phototoxicity when used in cells and tissues than the excitation wavelength of GFP. Moreover, since red fluorescence has high tissue permeability, it is excellent for observation of cells and living tissues. Furthermore, RFP is suitable for multicolor imaging by using in combination with fluorescent proteins having other fluorescent properties such as GFP, and is widely used in analysis for visualizing a plurality of life phenomena simultaneously.

  Although the need for RFP is high due to the effectiveness of its fluorescence characteristics, DsRed, which is representative of RFP, has a plurality of problems. Wild-type DsRed has a slow formation of a fluorescent chromophore, forms a stable tetramer, and tends to aggregate. For this reason, when it is going to detect fluorescence using DsRed, it will be necessary to wait until fluorescence of sufficient intensity | strength arises.

  In order to overcome such problems, various improvements of DsRed have been made as described in the prior art documents. Among them, DsRed mutants widely used are DsRed Express (Clontech) and DsRed T4 disclosed in Patent Literature 1 and Non-Patent Literature 1. These are tetramer-type mutants that are produced by randomly introducing mutations into DsRed and that mature quickly and have a lower degree of aggregation than the wild-type. The amino acid mutation that most affects the promotion of maturation rate is considered to be N42Q (mutation in which the 42nd asparagine is replaced with glutamine) which DsRed Express and DsRed T4 have in common. Is about 17 times as mature as wild-type DsRed.

  In addition, although monomerized DsRed has been prepared, a large number of mutations are required for obtaining such mutants. Even the brightest currently available monomer type DsRed has a fluorescence intensity about half that of the wild type.

JP2011-92198A

Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Bevis BJ, Glick BS et al., Nat. Biotechnol., 20 (1), 83-87 (2002) A monomeric red fluorescent protein. Campbell RE, Tour O et al., Proc. Natl Acad. Sci. USA, 99, 7877-7882 (2002) Improved monomeric red, orange and yellow foluorescent proteins derived form Discosoma sp.red fluorescent protein Shaner NC, Campbell RE et al., Nat. Biotechnol., 22 (12), 1567-1572 (2004)

  An object of the present invention is to provide a red fluorescent protein having high utility.

  The red fluorescent protein according to the present invention has the amino acid sequence set forth in SEQ ID NO: 1.

  The red fluorescent protein according to the present invention has an amino acid sequence having 80% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and produces fluorescence when cultured at 37 ° C. for at least 16 hours.

  The red fluorescent protein according to the present invention has the amino acid sequence set forth in SEQ ID NO: 2, 3 or 4.

  The red fluorescent protein according to the present invention has an amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 2, 3 or 4, and is fluorescent when cultured at 37 ° C. for at least 16 hours. Produce.

  The red fluorescent protein according to the present invention is derived from a disc coral (Discosoma sp.) Produced in Ishigaki Island, Okinawa Prefecture, and produces fluorescence when cultured at 37 ° C. for at least 16 hours.

  The nucleic acid according to the present invention encodes the red fluorescent protein according to the present invention.

  The nucleic acid according to the present invention includes a gene encoding the red fluorescent protein represented by SEQ ID NO: 5.

  The nucleic acid according to the present invention includes a gene encoding the red fluorescent protein described in SEQ ID NO: 6, 7 or 8.

  According to the present invention, a red fluorescent protein having high utility is provided.

It is a figure which shows the absorption spectrum of the red fluorescent protein which concerns on embodiment. It is a figure which shows the fluorescence spectrum of the red fluorescent protein which concerns on embodiment. It is a figure which shows the excitation spectrum of the red fluorescent protein which concerns on embodiment. It is a figure which shows the mode of fluorescence when colon_bacillus | E._coli which expresses the red fluorescent protein which concerns on embodiment is culture | cultivated at 37 degreeC for about 16 hours, and when cultured at 37 degreeC for about 23 hours.

  An example of the red fluorescent protein according to the embodiment is a red fluorescent protein having the amino acid sequence set forth in SEQ ID NO: 1.

  The red fluorescent protein means a protein that is excited by receiving excitation light and emits red light. The generation of this luminescence does not require a substrate.

  The red fluorescent protein having the amino acid sequence set forth in SEQ ID NO: 1 is derived from Ishigaki Island, Okinawa Prefecture Disc Coral (Discosoma sp.). This organism is an organism whose species has not yet been identified.

  The red fluorescent protein having the amino acid sequence shown in SEQ ID NO: 1 consists of 228 amino acids, exhibits a fluorescence peak at 593 nm, and an absorption peak at 568 nm. The homology between this amino acid sequence and the amino acid sequence of conventional DsRED (AET75774) exhibiting red fluorescence is about 89%. Therefore, a red fluorescent protein having an amino acid sequence showing 90% or more homology with SEQ ID NO: 1, preferably a red fluorescent protein having an amino acid sequence showing 95% or more homology with SEQ ID NO: 1. In addition to the protein, more preferably having such an amino acid sequence exhibiting high homology, the red fluorescent protein produced in Japan (particularly from Okinawa) has the amino acid sequence set forth in SEQ ID NO: 1. Properties similar to those of red fluorescent protein can be expected and are included in the present invention.

The amino acid sequence of this red fluorescent protein is shown below:
MSCSKNVIKEFMRFKVHMEGTVNGHEFEIRGEGEGRPYEGQNTVKLKVIKGGPLPFAWDILSPQFQYGSKVYVKHPPDIPDYKKLSFPEGFKWERVMNFEDGGVVTVTQDSSLIDGCFVYEVKDIFIGVNFPSDGPVMDIYKKDGWLVEFQ

  As with other RFPs such as DsRed, the red fluorescent protein having the amino acid sequence set forth in SEQ ID NO: 1 itself promotes the formation of a fluorescent chromophore only at low temperatures, has a low maturation rate, and has a low fluorescence intensity. However, unlike other RFPs such as DsRed, this red fluorescent protein introduces a slight mutation that promotes the formation of a fluorescent chromophore even at low temperatures, has a high maturation rate, and has a strong fluorescence intensity. A type of red fluorescent protein can be obtained. Such mutant red fluorescent proteins are mutants 1, 2, and 3 as described below.

  In view of the above, the red fluorescent protein having the amino acid sequence shown in SEQ ID NO: 1 is a mutant red fluorescent protein that promotes the formation of a fluorescent chromophore even at low temperatures, has a high maturation rate, and has a strong fluorescence intensity. Resulting in a useful red fluorescent protein.

  Another example of the red fluorescent protein according to the embodiment has an amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 1, and is fluorescent when cultured at 37 ° C. for at least 16 hours. Is a red fluorescent protein.

  This red fluorescent protein is a red fluorescent protein in which mutation has been introduced into the red fluorescent protein having the amino acid sequence described in SEQ ID NO: 1, and as a result, maturation has been accelerated. “Maturation is accelerated” means that the time until red fluorescence can be measured after red fluorescent protein is expressed in the cell is shortened. The reason why fluorescence is emitted early includes, for example, that the formation of the fluorescent chromophore is accelerated. Note that “producing fluorescence” includes generating sufficient fluorescence that can be measured by a general measuring device such as a fluorescence microscope or a luminometer.

  The homology is preferably 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

  Another example of the red fluorescent protein according to the embodiment is a red fluorescent protein having the amino acid sequence set forth in SEQ ID NO: 2, 3 or 4.

  These red fluorescent proteins introduce mutations into the red fluorescent protein obtained from Ishigaki-jima disc coral (Discosoma sp.) Having the amino acid sequence set forth in SEQ ID NO: 1 at 37 ° C. for at least 16 hours. It is a red fluorescent protein that has acquired the property of generating fluorescence when cultured.

  The amino acid sequence described in SEQ ID NO: 2 is obtained by substituting the 118th phenylalanine from the N-terminus with leucine (F118L) in the amino acid sequence described in SEQ ID NO: 1. Throughout this application, the red fluorescent protein having the amino acid sequence set forth in SEQ ID NO: 2 will be referred to as “mutant 1”, “mutant1” or “mut1” for convenience. In addition, a red fluorescent protein obtained from Ishigaki-jima disc coral (Discosoma sp.) Having the amino acid sequence set forth in SEQ ID NO: 1 is referred to as “wild type” or “WT” for convenience.

  Mutant 1 exhibits a fluorescence peak at 592 nm and an absorption peak at 568 nm. The fluorescence intensity at the fluorescence peak of variant 1 is 1.4 times the fluorescence intensity at the wild-type fluorescence peak.

The amino acid sequence of variant 1 is shown below:
MSCSKNVIKEFMRFKVHMEGTVNGHEFEIRGEGEGRPYEGQNTVKLKVIKGGPLPFAWDILSPQFQYGSKVYVKHPPDIPDYKKLSFPEGFKWERVMNFEDGGVVTVTQDSSLIDGCLVYEVKFIGVNFPSDGPVMDIYYKNGGWEPSTERLYVERGYPV

  The amino acid sequence shown in SEQ ID NO: 3 is the amino acid sequence shown in SEQ ID NO: 1 in which the 41st glutamine from the N-terminus is substituted with histidine (Q41H) and the 118th phenylalanine is substituted with leucine (F118L). is there. Throughout this application, the red fluorescent protein having the amino acid sequence set forth in SEQ ID NO: 3 will be referred to as “mutant 2”, “mutant 2” or “mut 2” for convenience.

  Mutant 2 shows a fluorescence peak at 592 nm and an absorption peak at 568 nm. The fluorescence intensity at the fluorescence peak of variant 2 is 1.5 times the fluorescence intensity at the wild-type fluorescence peak.

The amino acid sequence of variant 2 is shown below:
MSCSKNVIKEFMRFKVHMEGTVNGHEFEIRGEGEGRPYEGHNTVKLKVIKGGPLPFAWDILSPQFQYGSKVYVKHPPDIPDYKKLSFPEGFKWERVMNFEDGGVVTVTQDSSLIDGCLVYEVKFIGVNFPSDGPVMDIQYTMGWEPSTERLYVERGYPV

  The amino acid sequence shown in SEQ ID NO: 4 is the amino acid sequence shown in SEQ ID NO: 1 in which the 71st valine from the N-terminus is substituted with alanine (V71A) and the 226th proline is substituted with glutamine (P226Q). is there. Throughout this application, the red fluorescent protein having the amino acid sequence set forth in SEQ ID NO: 4 will be referred to as “mutant 3”, “mutant 3” or “mut 3” for convenience.

  Mutant 3 shows a fluorescence peak at 593 nm and an absorption peak at 567 nm. The fluorescence intensity at the fluorescence peak of variant 1 is 1.7 times the fluorescence intensity at the wild-type fluorescence peak.

The amino acid sequence of variant 3 is shown below:
MSCSKNVIKEFMRFKVHMEGTVNGHEFEIRGEGEGRPYEGQNTVKLKVIKGGPLPFAWDILSPQFQYGSKAYVKHPPDIPDYKKLSFPEGFKWERVMNFEDGGVVTVTQDSSLIDGCFVYEVKFIGVNFPSDGPVMQYTMTM

  Another example of the red fluorescent protein according to the embodiment has an amino acid sequence having 80% or more homology with the amino acid sequence shown in SEQ ID NO: 2, 3 or 4, and is cultured at 37 ° C. for at least 16 hours It is a red fluorescent protein that produces fluorescence at the time when it is applied.

  In other words, this red fluorescent protein is one in which a mutation in the amino acid sequence is further introduced into mutants 1, 2, or 3, but the characteristic that mutants 1, 2, or 3 mature quickly is maintained. is there.

  The homology is preferably 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

  Another example of the red fluorescent protein according to the embodiment is a red fluorescent protein that is derived from a disc coral (Discosoma sp.) From Ishigaki Island, Okinawa Prefecture, and generates fluorescence when cultured at 37 ° C. for at least 16 hours.

  One example of a red fluorescent protein derived from a disc coral (Discosoma sp.) From Ishigaki Island, Okinawa Prefecture is a red fluorescent protein having the amino acid sequence set forth in SEQ ID NO: 1, but the amino acid sequence set forth in SEQ ID NO: 1 On the other hand, for example, a protein in which a mutation is introduced in a natural state and a property of early maturation is acquired is also included in the red fluorescent protein according to the embodiment.

  The red fluorescent protein that produces fluorescence when cultured at 37 ° C. for at least 16 hours according to the embodiment matures faster than the conventional red fluorescent protein, and fluorescence can be measured quickly. Some conventional red fluorescent proteins require the cells expressing them to be kept at a low temperature for maturation, but the red fluorescent protein according to the embodiment does not need such, for example, 37 ° C. This makes it possible to detect fluorescence quickly. Therefore, the red fluorescent protein according to the embodiment is easy to handle experimentally and enables measurement in a state closer to the original state of the cell. In addition, the red fluorescent protein according to the embodiment exhibits strong fluorescence as compared with the conventional red fluorescent protein.

  The red fluorescent protein according to the embodiment may be provided in the form of a nucleic acid. That is, a nucleic acid containing a gene encoding the red fluorescent protein according to the embodiment is also provided.

  A specific example of such a nucleic acid is a nucleic acid comprising the base sequence set forth in SEQ ID NO: 5. This base sequence encodes a wild type gene.

The nucleotide sequence encoding the wild type gene is shown below:
(SEQ ID NO: 5).

  Further, specific examples of such nucleic acids are nucleic acids comprising the base sequences set forth in SEQ ID NOs: 6, 7 and 8. These base sequences encode the genes of mutants 1, 2 and 3, respectively.

The base sequence encoding the gene of variant 1 is shown below:
(SEQ ID NO: 6).

The base sequence encoding the gene of variant 2 is shown below:
(SEQ ID NO: 7).

The base sequence encoding the gene of variant 3 is shown below:
(SEQ ID NO: 8).

  The nucleic acid may further include a region to which a factor for transcription control such as a promoter binds. The nucleic acid may further include a gene encoding a protein different from the red fluorescent protein according to the embodiment. In this case, the genes are arranged on the nucleic acid so that the red fluorescent protein and the different protein are expressed in a fused state.

  The nucleic acid may also be in the form of a vector. Vectors may contain elements contained in common vectors such as antibiotic resistance genes and origins of replication.

  According to such a nucleic acid, the gene of the red fluorescent protein according to the embodiment can be stably stored and used in the form of a nucleic acid.

(1) Cloning of wild-type fluorescent protein gene from disc coral from Ishigaki Island in Okinawa Prefecture (Discosoma sp.) Obtaining a full-length cDNA library from disc coral from Ishigaki Island The gene was cloned.

  A full-length cDNA library was obtained as follows. 0.62g of disc coral from Ishigakijima, Okinawa, purchased from a pet dealer in Musashimurayama, Tokyo, was used to prepare cells using a glass homogenizer in 10mL of total RNA extraction reagent TRIzol Reagent (Invitrogen). After crushing, total RNA was extracted according to the instruction manual. Thereafter, mRNA was extracted from the extracted total RNA according to the instruction manual using Micro-FastTrack 2.0 kit (Invitrogen). The separated and purified mRNA was synthesized using a full-length cDNA synthesis reagent GeneRacer (Invitrogen) according to the instruction manual.

As a primer set for cloning, a candidate primer was prepared based on the amino acid sequence GTXNGH (SEQ ID NO: 9) which is well conserved in the following four types of cnidarian fluorescent proteins. X includes any amino acid of 20 amino acids.
Montipola efflorescens (ABB17950)
Galaxea fascicularis (ABB17967)
Discosoma stricta (AAU04445)
Discosoma sp. RC-2004 (AY679106)

The prepared primers are the following four types.
coral-universal-F1-ta, TGGAAGGcrmtGTaAACGGGCAC (SEQ ID NO: 10)
coral-universal-F1-tt, TGGAAGGcrmtGTtAACGGGCAC (SEQ ID NO: 11)
coral-universal-F1-ca, TGGAAGGcrmcGTaAACGGGCAC (SEQ ID NO: 12)
coral-universal-F1-ct, TGGAAGGcrmcGTtAACGGGCAC (SEQ ID NO: 13)

  Using these primer sets, 3 'Race PCR was performed using a full-length cDNA library as a template, using a full-length cDNA synthesis reagent GeneRacer (Invitrogen) according to the instruction manual.

Based on the sequence obtained from 3 'Race PCR, the following two primer sets are prepared, using a full-length cDNA synthesis reagent GeneRacer (Invitrogen), and using the full-length cDNA library as a template according to the instruction manual, 5' Race PCR was performed.
Discosoma (FP586) -R1, TTCTTTCATACTGCTCCAACGAGTGT (SEQ ID NO: 14)
Discosoma (FP586) -R2, TTCAACTAGTAATGACCACCGTCT (SEQ ID NO: 15)

Based on the sequence obtained from 5 'Race PCR, the following primers are prepared, and full length cDNA synthesis reagent GeneRacer (Invitrogen) is used to obtain the full length using the full length cDNA library as a template according to the instruction manual. 3 'Race PCR was performed.
Discosoma-Full-FP586-F1, ATTCCAGTTTCAGTCAGGTGACGGTCAGAGA (SEQ ID NO: 16)

A base sequence encoding the gene of red fluorescent protein derived from disc coral produced in Ishigaki Island, Okinawa Prefecture was obtained by full length acquisition 3 ′ Race PCR. The base sequence is shown below.
(SEQ ID NO: 17)

The amino acid sequence of the wild type fluorescent protein deduced from this base sequence is shown below.
MSCSKNVIKEFMRFKVHMEGTVNGHEFEIRGEGEGRPYEGQNTVKLKVIKGGPLPFAWDILSPQFQYGSKVYVKHPPDIPDYKKLSFPEGFKWERVMNFEDGGVVTVTQDSSLIDGCFVYEVKQFIGVNFPSDGPVYY

(2) Discosoma sp. Preparation of mutant of fluorescent protein gene Discosoma sp. Obtained in (1) above. Mutations were introduced into wild-type red fluorescent protein residues derived from the mutants to obtain mutants.

  In order to eliminate one EcoRI restriction enzyme site contained in the base sequence of the wild-type red fluorescent protein gene obtained in (1) above, a DNA sequence can be used without changing the amino acid sequence using a general kit. The process which modifies was performed. Then, pRSETB (Invitrogen) was inserted into the vector using a general kit. In pRSETB, between BamHI (5′-end side) and EcoRI (3′-end side), the Discosoma sp. The derived fluorescent protein gene was inserted.

  Discosoma sp. A mutant of the derived red fluorescent protein was produced using GeneMorph II Random Mutagenesis Kit (Agilent Technologies) as follows.

Discosoma sp. PRSET-B (Invitrogen) plasmid in which a wild-type fluorescent protein derived from is cloned as a template,
Primer1 (CGTTACGACGGATGACGATAAGGATCCAATG) (SEQ ID NO: 18), and Primer2 (GTTAGCAGCCGGATCAGCTTCGAATTCTTAC) (SEQ ID NO: 19)
PCR reaction was carried out using as a primer according to the manual attached to the kit. After the PCR reaction, electrophoresis was performed using 1% agarose gel, and the target PCR product was purified using Wizard SV Gel and PCR Clean-Up (Promega). After purification, PCR reaction was performed according to the manual, DpnI was added and treated at 37 degrees, followed by ethanol precipitation. After precipitation, DNA was dissolved in a small amount of distilled water and transformed into JM109 (DE3) by electroporation using MicroPulser (BioRad). The transformed Escherichia coli was spread on a 25 cm square LB (containing 50 μg / mL ampicillin) plate to form colonies at 37 ° C. and screened. After formation of the colony, observation was performed by irradiating with VISRAYS (ATTO) GREEN light, and a colony having bright fluorescence was selected and cultured, and the DNA sequence of the plasmid retained by the E. coli was determined. Thereby, variants 1 and 3 were obtained. Mutant 2 was obtained by performing the same operation using the plasmid of mutant 1 as a template and the same primers as described above.

  Escherichia coli carrying the wild-type and the three mutant plasmids was cultured at 28 ° C. for 3 nights, then lysed and purified by column chromatography using His Link (Promega).

(3) Discosoma sp. Spectra measurement of wild type and mutant of fluorescent protein Each spectrum of purified wild type red fluorescent protein and three mutants was measured.

  For the wild type and the three mutants purified as described in (2) above, the absorption spectrum was measured using a spectrophotometer U-3010 spectrophotometer (HITACHI) (FIG. 1), and the fluorometer F-2500 Fluorescence Spectrometer The fluorescence spectrum (FIG. 2) and the excitation spectrum (FIG. 3) were measured using (HITACHI). The fluorescence spectrum was excited at 568 nm, and the excitation spectrum was measured with the fluorescence wavelength set at 610 nm.

  As shown in FIGS. 1 to 3, the three mutants showed higher fluorescence intensity and absorbance than the wild type. The fluorescence intensity at the peak values of the mutants 1, 2, and 3 was 1.4 times, 1.5 times, and 1.7 times the fluorescence intensity of the wild type peak value, respectively.

(4) Fluorescence observation in 37 degreeC culture conditions in colon_bacillus | E._coli The state of fluorescence when wild type and three types of mutants were cultured at 37 degreeC was observed.

  Escherichia coli expressing the wild type and the mutant was cultured at 37 ° C., and the follow-up was performed. Excited with VISIARAYS (ATTO) GREEN light, a fluorescent image was acquired with DP70 (Olympus) using a red fluorescent filter (Olympus). The result is shown in FIG.

  FIG. 4 shows a fluorescent image (a) and a transmitted light image (c) about 16 hours after the start of culture, and a fluorescent image (b) and a transmitted light image (d) after about 23 hours. From FIG. 4 (a), it can be seen that, at the stage of about 16 hours, fluorescence cannot be detected with the wild type, but detectable fluorescence with the mutants 1 to 3. From FIG. 4 (b), it can be seen that at the stage of about 23 hours, the wild type also shows detectable fluorescence, while the mutants 1 to 3 show sufficiently intense fluorescence.

Claims (8)

  A red fluorescent protein having the amino acid sequence set forth in SEQ ID NO: 1.   A red fluorescent protein having an amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 1 and producing fluorescence when cultured at 37 ° C for at least 16 hours.   A red fluorescent protein having the amino acid sequence of SEQ ID NO: 2, 3 or 4.   A red fluorescent protein having an amino acid sequence having 80% or more homology with the amino acid sequence set forth in SEQ ID NO: 2, 3 or 4, and producing fluorescence when cultured at 37 ° C. for at least 16 hours.   A red fluorescent protein derived from disc coral (Discosoma sp.) From Ishigaki Island, Okinawa Prefecture, which produces fluorescence when cultured at 37 ° C. for at least 16 hours.   A nucleic acid comprising a gene encoding the red fluorescent protein according to any one of claims 1 to 5.   A nucleic acid comprising a gene encoding the red fluorescent protein represented by SEQ ID NO: 5.   A nucleic acid comprising a gene encoding the red fluorescent protein represented by SEQ ID NO: 6, 7 or 8.

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