JP6929529B2 - Screening methods for fluorescent proteins, DNA, vectors, transformants, and methods for monitoring intracellular redox status, as well as anticancer agents. - Google Patents

Description

The present invention relates to fluorescent proteins, DNAs, vectors, transformants, and methods for monitoring redox status in cells. The present invention also relates to a method for screening an anticancer drug using the fluorescent protein.

Various events related to the redox reaction occur in the cell. For example, in the electron transport chain located at the final stage of the metabolic system of cellular respiration, the electrons generated by the oxidation of NADH are finally transferred to oxygen to generate water. ATP is produced by oxidative phosphorylation that occurs in conjunction with the electron transport chain.

Redox reactions also occur in plant photosynthesis. In the thylakoid membrane of chloroplasts, chlorophyll (photosynthetic pigment) uses light energy to decompose water, producing protons, oxygen molecules and electrons. ^{NADP +} is reduced by the electrons generated at this time, and NADPH is generated. Furthermore, ATP is produced by ATP synthase using the proton concentration gradient inside and outside the thylakoid membrane.

In addition, active oxygen species generated in the process of aerobic organisms consuming oxygen have a very strong oxidative effect, and when present in excess, many cells are composed of intracellular proteins, enzymes, cell membranes, DNA, etc. It can oxidatively denature molecules and cause cell dysfunction.

In order to understand the details of such redox events in cells, it is desired to develop a molecular tool capable of monitoring the intracellular redox state.

As a molecular tool capable of monitoring the intracellular redox state, roGFP (Redox-sensitive green fluorescent protein) in which S147 and Q204 of green fluorescent protein (GFP), which is a fluorescent protein, is replaced with a cysteine residue is known. (Non-Patent Documents 1 and 2). roGFP exists in either an oxidized form in which the thiol group in the above two cysteine residues is linked by a disulfide bond, or a reduced form in which the thiol group in the cysteine residue is in a free state. The abundance ratio of the oxidized form to the reduced form changes depending on the redox state of the surrounding environment of roGFP. The change is detected as a shape change in the excitation spectrum of roGFP. There are two excitation maximum wavelengths (400 nm, 490 nm) in the excitation spectrum of roGFP, and by measuring the ratio of the fluorescence intensities at the excitation wavelength, it is possible to monitor the redox state of the ambient environment of roGFP. ..

In addition, Oba-Q (oxidation balance sensed quenching protein), which is a redox-responsive fluorescent protein whose fluorescence intensity changes with redox, has been reported (Patent Document 1, Non-Patent Document 3). Oba-Q is suitable for intracellular multicolor observation because it can observe changes in the redox state at a single excitation wavelength and fluorescence wavelength.

Japanese Unexamined Patent Publication No. 2015-91226

Hanson G.T.et al., Journal of Biological Chemistry, 2004, 279: 13044-13053. Lohman J.R.et.al., Biochemistry, 2008, 47, 8678-8688 Sugiura K. et.al., Biochemical and Biophysical Research Communications, 2015, 457, 242-248

When roGFP is used, in order to monitor the redox state in cells, it is necessary to measure the fluorescence intensities at two excitation wavelengths, which is troublesome. In addition, there is a problem that the change in the redox state cannot be directly evaluated by observation with a fluorescence microscope. Furthermore, roGFP has a problem that it is highly pH sensitive and difficult to use in cells.

Further, since Oba-Q detects a change in the redox state as a change in fluorescence intensity, the expression level of Oba-Q in the cell affects the observation result. In particular, when the expression level of Oba-Q is low, there is a problem that it is difficult to accurately quantify the redox state in cells.

Therefore, an object of the present invention is to provide a molecular tool capable of evaluating a change in a redox state in a cell by fluorescence at a single excitation wavelength and evaluating a change in the redox state regardless of the intracellular expression level. To provide.

As a result of diligent studies in view of the above problems, the present inventors have surprisingly found that the fluorescence of a fluorescent protein in which a specific amino acid mutation is applied to GFP changes color in response to a change in the redox state. We have found that by using the fluorescent protein, it is possible to measure the change in the redox state in the cell based on the discoloration of the fluorescence without depending on the expression level of the protein, and reached the present invention. bottom.

Therefore, the present invention has the following aspects.
[1]
In the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence having 90% or more sequence identity with respect to SEQ ID NO: 1.
The 149th amino acid residue was replaced with cysteine (C),
The 150th amino acid residue was replaced with aspartic acid (D),
The 202nd amino acid residue is replaced with cysteine (C),
A fluorescent protein consisting of an amino acid sequence in which the 203rd amino acid residue is substituted with valine (V) and the 205th amino acid residue is substituted with valine (V).
[2]
Insertion of valine (V) between the 1st and 2nd amino acid residues;
Substitution of the 26th amino acid residue with arginine (R);
Substitution of the 30th amino acid residue with arginine (R);
Substitution of the 39th amino acid residue with asparagine (N);
Substitution of amino acid residue 105 with threonine (T);
Substitution of amino acid residue at position 171 with valine (V);
The 206th amino acid residue is replaced with lysine (K);
The 224th amino acid residue is replaced with arginine (R); and the 230th amino acid residue is replaced with leucine (L);
The fluorescent protein according to [1], further comprising at least one mutation selected from the group consisting of.
[3]
Valine (V) is inserted between the 1st and 2nd amino acid residues,
The 26th amino acid residue is replaced with arginine (R),
The 30th amino acid residue is replaced with arginine (R),
The 39th amino acid residue was replaced with asparagine (N),
The 105th amino acid residue was replaced with threonine (T),
The 171st amino acid residue was replaced with valine (V),
The 206th amino acid residue is replaced with lysine (K),
The fluorescent protein according to [1] or [2], wherein the amino acid residue at position 224 is replaced with arginine (R) and the amino acid residue at position 230 is replaced with leucine (L).
[4]
A fusion protein obtained by fusing the fluorescent protein according to any one of [1] to [3] with a second protein.
[5]
A DNA encoding the fluorescent protein according to any one of [1] to [3] or the fusion protein according to [4].
[6]
A recombinant vector having the DNA according to [5].
[7]
A host cell transformed with the recombinant vector according to [6].
[8]
It is a method of monitoring the intracellular redox state.
The fluorescent protein according to any one of [1] to [3] or the fusion protein according to [4] is allowed to exist in the cell.
Detecting the fluorescence of the fluorescent protein or fusion protein,
A method comprising monitoring the intracellular redox state based on the detected fluorescence discoloration.
[9]
It is a screening method for anticancer drugs.
The fluorescent protein according to any one of [1] to [3] or the fusion protein according to [4] is allowed to be present in the cancer cell.
Fluorescence of the fluorescent protein or fusion protein before the addition of the candidate drug was detected.
Candidate drugs are added to the cancer cells and
The fluorescence of the fluorescent protein or fusion protein after the addition of the candidate drug is detected, and the fluorescence is detected.
The method comprising selecting a drug that oxidizes the cells as compared to before the addition of the candidate drug, based on the discoloration of the fluorescence before and after the addition of the candidate drug.

According to the fluorescent protein of the present invention, changes in the oxidation-reduction state in cells can be easily monitored by observation with a fluorescence microscope or the like based on the discoloration of the fluorescence of the fluorescent protein. The fluorescent protein of the present invention can monitor changes in the redox state in cells using a single excitation wavelength. In addition, changes in the redox state can be monitored regardless of the intracellular expression level of the fluorescent protein.

It is a fluorescence spectrum in each of the oxidized form and the reduced form of FOROG / B (Fluorescent protein with RedOx-dependent change in color between Green and Blue) (SEQ ID NO: 2). The fluorescence spectrum was measured with the excitation light wavelength set to 400 nm. It is a figure which shows the observation result of the reduced type and the oxidized type fluorescent color of FROG / B. The reduced form emits blue fluorescence, and the oxidized form emits green fluorescence. It is a figure which shows the oxidation-reduction potential change of FROG / B. It is a figure which shows the pH sensitivity of FROG / B. It is a figure which shows the time-dependent change of the fluorescence intensity at the time of adding an oxidizing agent (diamide) and a reducing agent (DTT) to HeLa cells expressing FROG / B. It is a figure which shows the result of the time-lapse observation by the confocal microscope when the oxidizing agent (diamide) and the reducing agent (DTT) were added to the HeLa cell which expressed FROG / B. It is a figure which shows the time-dependent change of the fluorescence intensity at the time of adding _{an oxidizing agent (H 2} O ₂ ) to the cell which expressed the All 1541-FROG / B fusion protein (SEQ ID NO: 3). It is a figure which shows the mechanism which the fluorescence discolors in response to the change of the redox state of FROG / B.

In the present specification, amino acids are represented by one-letter notation unless otherwise specified. In the notation of the amino acid sequence, for example, asparagine, which is the 149th amino acid residue from the N-terminal of the amino acid sequence of SEQ ID NO: 1, is expressed as N149 unless otherwise specified. The same applies to other amino acid residues. Further, the notation of substitution in the amino acid sequence is expressed as N149C unless otherwise specified, for example, when N149 of SEQ ID NO: 1 is substituted with C. The same applies to other amino acid substitutions. Further, the notation of insertion in the amino acid sequence is expressed as M1_S2insV unless otherwise specified, for example, when valine (V) is inserted between M1 and S2 of SEQ ID NO: 1. The same applies to the insertion of other amino acids. In addition, a mutant in which a plurality of amino acid residues are simultaneously substituted and / or inserted into the amino acid sequence (GFP) shown in SEQ ID NO: 1 is described as "GFP" at the beginning, and each substitution and / or insertion is hyphenated. It shall be connected by (-). For example, M1_S2insV is inserted into the amino acid sequence (GFP) shown in SEQ ID NO: 1, and amino acids of K26R, S30R, Y39N, N105T, N149C, V150D, I171V, S202C, T203V, S205V, A206K, V224R and H230L are substituted. The amino acid sequence is represented as "GFP-M1_S2insV_K26R_S30R_Y39N_N105T_N149C_V150D_I171V_S202C_T203V_S205V_A206K_V224R and H230L". Other variants for SEQ ID NO: 1 are similarly described.

As used herein, "sequence identity" refers to sequence identity between two DNAs or two proteins. The "sequence identity" is determined by comparing two optimally aligned sequences over the region of the sequence to be compared. Here, the DNA or protein to be compared may have additions or deletions (eg, gaps, etc.) in the optimal alignment of the two sequences. Such sequence identity can be calculated, for example, by creating an alignment using the Clustal W algorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)) using Vector NTI. Can be done. Sequence identity is measured using sequence analysis software, specifically Vector NTI, GENETYX, and analysis tools provided in public databases. The public database is generally available at, for example, the home page address http://www.ddbj.nig.ac.jp. In the present specification, the numbering of the amino acid sequence in the variant of the amino acid sequence of GFP (SEQ ID NO: 1) is performed based on the amino acid sequence of SEQ ID NO: 1, and the variant of the nucleotide sequence of GFP (SEQ ID NO: 22) is used as a reference. The base sequence numbering in the above is performed with reference to the base sequence of SEQ ID NO: 22.

In the present specification, the "excitation spectrum" is a spectrum obtained by fixing the wavelength of fluorescence to be detected, continuously changing the wavelength of excitation light, and plotting the obtained fluorescence intensity for each wavelength. In the present specification, the "excitation maximum wavelength" is a wavelength that gives the strongest fluorescence intensity in the excitation spectrum. The excitation spectrum is measured by a spectrofluorometer.

In the present specification, the "fluorescence spectrum" is a plot of the obtained fluorescence intensity for each wavelength with the wavelength of the excitation light fixed. The shape of the fluorescence spectrum does not change due to the difference in excitation wavelength. Normally, the excitation wavelength to be fixed is the maximum excitation wavelength.

The fluorescence intensity of the fluorescence emitted by the oxidized and reduced fluorescent proteins excited by the excitation light can be measured with a spectrofluorescent photometer, a fluorescence microscope, or the like.

One aspect of the present invention is the amino acid sequence of SEQ ID NO: 1, 90% or more, preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96 with respect to the amino acid sequence of SEQ ID NO: 1. In an amino acid sequence having a sequence identity of% or more, 97% or more, 98% or more, or 99% or more, the 149th amino acid residue is replaced with cysteine (C), and the 150th amino acid residue is aspartic acid (D). ), The 202nd amino acid residue is replaced with cysteine (C), the 203rd amino acid residue is replaced with valine (V), and the 205th amino acid residue is replaced with valine (V). It is a fluorescent protein consisting of an amino acid sequence. The fluorescent protein is also hereinafter referred to as "the fluorescent protein of the present invention".

The fluorescent protein of the present invention is either an oxidized form in which the thiol group in the two introduced cysteine residues is linked by a disulfide bond, or a reduced form in which the thiol group in the cysteine residue is in a free state. It exists in form. The half-reaction formula is as follows.

In the cell, there are electron acceptors (oxidizers) such as active oxygen containing hydrogen peroxide, and electron donors (reducing agents) such as glutathione and thioredoxin. These amounts vary depending on the environment in which the cells are present. Here, in the present invention, "monitoring the redox state in a cell" means monitoring the fluctuation of the amount of the oxidizing agent and the reducing agent in the cell. When the fluorescent protein of the present invention is present in the cell, the equilibrium of the half-reaction equation is biased toward the oxidized form when the amount of the oxidizing agent present in the cell increases. On the contrary, when the abundance of the reducing agent in the cell increases, the equilibrium of the half-reaction formula is biased to the reduced form. Then, the change in the redox state in the cell is observed as the discoloration of the fluorescence of the fluorescent protein of the present invention.

When the fluorescence is measured with the excitation light wavelength fixed, the oxidized fluorescence color of the fluorescent protein of the present invention is different from the reduced fluorescence color. Therefore, based on the fluorescent color, it becomes possible to distinguish whether the fluorescent protein exists in the oxidized form or the reduced form, and the redox state in the surrounding environment in which the fluorescent protein exists is monitored. can do.

"Fluorescence discoloration" is fluorescence derived from a peak on the long wavelength side (hereinafter, also referred to as "first fluorescence spectrum peak") among two fluorescence spectrum peaks existing in the fluorescence spectrum of the fluorescence protein of the present invention (hereinafter, also referred to as "first fluorescence spectrum peak"). It can be quantified as a change in the ratio (F1 / F2) of the fluorescence (F2) derived from the peak on the short wavelength side (hereinafter, also referred to as “second fluorescence spectrum peak”). Here, the larger the value of (F1 / F2) is, the more oxidative state is shown, and the smaller the value of (F1 / F2) is, the more reducing state is shown.

The ratio of F1 and F2 is not particularly limited, but may be, for example, the ratio of the peak area of the first fluorescence spectrum peak to the peak area of the second fluorescence spectrum peak. For example, the peak area of the first fluorescence spectrum peak is calculated as the area of the fluorescence spectrum in the region of 490 nm to 600 nm, and the peak area of the peak area of the second fluorescence spectrum peak is the peak area of the fluorescence spectrum in the region of 410 nm to 490 nm. It may be calculated as an area. Here, 490 nm is the boundary between the first fluorescence spectrum peak and the second fluorescence spectrum peak.

Further, for example, the ratio of F1 and F2 may be the ratio of the fluorescence intensity at the maximum fluorescence wavelength of the first fluorescence spectrum peak to the fluorescence intensity at the maximum fluorescence wavelength of the second fluorescence spectrum peak.

In order to further increase the fluorescence intensity of the fluorescent protein of the present invention, the fluorescent protein of the present invention is used.
Insertion of valine (V) between the 1st and 2nd amino acid residues;
Substitution of the 26th amino acid residue with arginine (R);
Substitution of the 30th amino acid residue with arginine (R);
Substitution of the 39th amino acid residue with asparagine (N);
Substitution of amino acid residue 105 with threonine (T);
Substitution of amino acid residue at position 171 with valine (V);
The 206th amino acid residue is replaced with lysine (K);
The 224th amino acid residue may have at least one mutation selected from the group consisting of a substitution for arginine (R); and a 230th amino acid residue for a leucine (L).

A preferred embodiment of the fluorescent protein of the present invention is the amino acid sequence of SEQ ID NO: 1 or 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more with respect to SEQ ID NO: 1. , 96% or more, 97% or more, 98% or more or 99% or more of amino acid sequences, the 149th amino acid residue is replaced with cysteine (C), and the 150th amino acid residue is aspartic acid. Substituted with (D), the 202nd amino acid residue is replaced with cysteine (C), the 203rd amino acid residue is replaced with valine (V), and the 205th amino acid residue is valine (V). Replaced and further
Insertion of valine (V) between the 1st and 2nd amino acid residues;
Substitution of the 26th amino acid residue with arginine (R);
Substitution of the 30th amino acid residue with arginine (R);
Substitution of the 39th amino acid residue with asparagine (N);
Substitution of amino acid residue 105 with threonine (T);
Substitution of amino acid residue at position 171 with valine (V);
The 206th amino acid residue is replaced with lysine (K);
A fluorescent protein having at least one mutation selected from the group consisting of the 224th amino acid residue being a substitution for arginine (R); and the 230th amino acid residue being a substitution for leucine (L).

A more preferred embodiment of the fluorescent protein of the present invention is the amino acid sequence of SEQ ID NO: 1 or 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% with respect to SEQ ID NO: 1. In the amino acid sequence having 96% or more, 97% or more, 98% or more, or 99% or more sequence identity, valine (V) is inserted between the first and second amino acid residues, and the 26th position. The amino acid residue of is replaced with arginine (R), the 30th amino acid residue is replaced with arginine (R), the 39th amino acid residue is replaced with asparagine (N), and the 105th amino acid residue is. Substituted with threonine (T), the 149th amino acid residue is replaced with cysteine (C), the 150th amino acid residue is replaced with aspartic acid (D), and the 171st amino acid residue is valine (V). The 202nd amino acid residue is replaced with cysteine (C), the 203rd amino acid residue is replaced with valine (V), the 205th amino acid residue is replaced with valine (V), 206. It consists of an amino acid sequence in which the second amino acid residue is replaced with lysine (K), the 224th amino acid residue is replaced with arginine (R), and the 230th amino acid residue is replaced with leucine (L). It is a fluorescent protein.

A plurality of redox-responsive fluorescent proteins having different fluorescent colors (that is, having significantly different maximum fluorescence wavelengths) can be distinguished even when used simultaneously in the same cell due to the difference in color. In addition, the combination of oxidation-reduction-responsive fluorescent proteins with significantly different maximum excitation wavelengths makes it possible to observe the fluorescence of only the other fluorescent protein by irradiating with excitation light at a wavelength at which one fluorescent protein can hardly react. do.

In the fluorescent protein of the present invention, for example, the fluorescence is measured by fixing the excitation light wavelength to around 380 to 410 nm, preferably around 400 nm.

The fluorescence intensity of wild-type GFP varies depending on the pH change of the surrounding environment. On the other hand, the fluorescent protein of the present invention has low pH sensitivity of fluorescent characteristics in the range of pH 7.0 to 8.0. Here, "low pH sensitivity" refers to fluorescence (F1) derived from a peak on the long wavelength side of the two fluorescence spectrum peaks existing in the fluorescence spectrum of the fluorescent protein of the present invention by changing the pH. The fluctuation rate of the ratio (F1 / F2) with the fluorescence (F2) derived from the peak on the short wavelength side is usually 40% or less, preferably 30% or less.

The fluorescent protein of the present invention can be prepared by introducing an amino acid mutation into the parent protein (GFP). The method of introducing the mutation will be described in detail below. Further, the fluorescent protein of the present invention is an organic chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method), or a commercially available suitable peptide synthesis. It can also be manufactured using a machine.

The fluorescent protein of the present invention is not limited to its own form, and can be used in an embodiment with arbitrary modifications, modifications, and the like. For example, fusion with a second protein, addition of peptide tags (eg, His tag, HQ tag, HN tag, HAT tag) to facilitate protein purification, various chemical modifications to proteins, polymers such as polyethylene glycol. Processing by various methods known to those skilled in the art, such as binding to, binding to an insoluble carrier, and encapsulation in a liposome, can be considered.

One aspect of the present invention is a fusion protein obtained by fusing the fluorescent protein of the present invention with a second protein. The fluorescent protein of the present invention and the second protein are linked via a linker having an amino acid sequence composed of a predetermined number of amino acid residues. The amino acid sequence constituting the linker and the number of amino acid residues thereof can be arbitrarily selected as long as the function of the fluorescent protein of the present invention is not impaired. For example, a GS linker containing one or more glycines and serines can be mentioned. For example, it may be _{(GGSGG) n.} Here, n = 1-10. The second protein is not particularly limited, and includes, for example, peroxiredoxin, peroxidase, and glutaredoxin. In particular, a protein in which the fluorescent protein of the present invention is fused with peroxiredoxin, peroxidase or glutaredoxin can improve redox sensitivity as compared with the fluorescent protein of the present invention before fusion.

In the fusion protein of the present invention, the DNA encoding the fluorescent protein of the present invention and the DNA encoding the second protein are inserted into a recombinant vector in the form of being linked to the DNA encoding the linker, and the vector is used as a host. It can be produced by transforming cells. It is also possible to prepare the fluorescent protein of the present invention and the second protein separately and connect them to each other. The timing of linking the fluorescent protein of the present invention and the second protein is not particularly limited, and they may be linked individually after preparation, or one of the proteins may be prepared and used for a specific purpose. After that, it may be linked to the other protein.

One aspect of the present invention is a DNA encoding the fluorescent protein of the present invention or the fusion protein of the present invention. In one embodiment, the DNA encoding the fluorescent protein of the present invention is 70% or more, 80% or more, 90% or more, 91% or more, based on the nucleotide sequence of SEQ ID NO: 22 encoding the parent protein (GFP). Amino acid mutations that characterize the fluorescent proteins of the invention in nucleotide sequences having sequence identity of 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater. It consists of a base sequence in which the codon corresponding to is substituted or inserted.

The method for producing the DNA encoding the fluorescent protein of the present invention is not particularly limited, but for example, 70% or more, 80% or more, 90% or more with respect to the base sequence of SEQ ID NO: 22 encoding the parent protein (GFP). , 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more based on DNA consisting of a base sequence having sequence identity. , PCR, site-specific mutagenesis and other common genetic engineering techniques. Genetic engineering techniques such as site-specific mutations are also widely used by those skilled in the art, such as Maniatis T. et al. (Molecular Cloning, a Laboratory Manual, Cold Spring harbor Laboratory, New York, 1982). It is described in the experimental operation manual.

One aspect of the invention is a recombinant vector comprising a DNA encoding the fluorescent protein of the invention or the fusion protein of the invention. The recombinant vector of the present invention may be in any form such as cyclic or linear, and has another base sequence if necessary in addition to the DNA encoding the fluorescent protein or fusion protein of the present invention. You may. Other base sequences include enhancer sequences, promoter sequences, ribosome binding sequences, base sequences used for the purpose of amplifying the number of copies, base sequences encoding signal peptides, polyA addition sequences, splicing sequences, replication initiation sites, and the like. Examples thereof include the base sequence of a gene that serves as a selectable marker.

At the time of gene recombination, a translation start codon or a translation stop codon is added to the DNA encoding the fluorescent protein of the present invention or the fusion protein of the present invention using an appropriate synthetic DNA adapter, or an appropriate restriction enzyme is added to the base sequence. It is also possible to generate a new truncated sequence or to eliminate it. These are within the range of work normally performed by those skilled in the art, and those skilled in the art can arbitrarily and easily process the DNA encoding the fluorescent protein of the present invention or the fusion protein of the present invention.

Further, as the vector carrying the DNA encoding the fluorescent protein of the present invention or the fusion protein of the present invention, an appropriate vector may be selected according to the host to be used. The vector may be an autonomous replication vector, that is, a vector that can autonomously replicate independently of the chromosomal replication of the host cell. It may also be integrated into the genome of the host cell and replicated with the integrated chromosome. For example, in addition to plasmids, various viruses such as bacteriophage, baculovirus, retrovirus, and vaccinia virus can be used.

Available commercially available expression vectors include pcDM8 (manufactured by Funakoshi), pcDNAI (manufactured by Funakoshi), pcDNAI / AmP (manufactured by Invitrogen), EGFP-C1 (manufactured by Clontech), pREP4 (manufactured by Invitrogen), and pGBT. -9 (manufactured by Clontech), pet23a (manufactured by Novagen) and the like can be exemplified. The expression of the fluorescent protein of the present invention or the fusion protein of the present invention can be expressed under the control of a promoter sequence unique to the gene encoding the protein. Alternatively, another appropriate expression promoter may be linked and used upstream of the base sequence encoding the fluorescent protein of the present invention or the fusion protein of the present invention. Such an expression promoter may be appropriately selected depending on the host and the purpose of expression. For example, when the host is Escherichia coli, the T7 promoter, the lac promoter, the trp promoter, the λPL promoter and the like are used, and when the host is yeast. The PHO5 promoter, GAP promoter, ADH promoter, etc. are used in the SV40-derived promoter, retrovirus promoter, cytomegalovirus (human CMV) IE (immediate early) gene promoter, metallothioneine promoter, etc. when the host is an animal cell. Heat shock promoter, SRα promoter and the like can be mentioned. Operations such as linking the DNA encoding the fluorescent protein or fusion protein of the present invention to the promoter exemplified above or incorporating it into an expression vector can also be performed by Maniatis T. et al. (Molecular Cloning, a Laboratory Manual, Cold Spring). It can be performed based on the description in the harbor Laboratory, New York, 1982) and other experimental operation manuals.

One aspect of the invention is a host cell transformed with the recombinant vector of the invention. Examples of host cells include Escherichia bacteria, Corynebacterium bacteria, Brevibacterium bacteria, Bacillus bacteria, Serratia bacteria, Pseudomonas. Bacteria, Arthrobacter, Erwinia, Methylobacterium, Rhodobacter, Streptomyces, Zymomonas , Bacteria such as Saccharomyces yeast, Insect cells such as silkworm, HEK293 cells, MEF cells, Vero cells, Hela cells, CHO cells, WI38 cells, BHK cells, COS-7 cells, MDCK cells, C127 cells, HKG Bacteria such as cells and human renal cell lines can be mentioned.

A method for introducing a recombinant vector into a host cell is described in an experimental operation manual such as Maniatis T. et al. (Molecular Cloning, a Laboratory Manual, Cold Spring harbor Laboratory, New York, 1982). For example, the electroporation method, the protoplast method, the alkali metal method, the calcium phosphate precipitation method, the DEAE dextran method, the microinjection method, the particle gun method and the like can be used. The use of insect cells such as Sf9 and Sf21 is described in Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), and BIO / TECHNOLOGY vol. 6, 1988, page 47, etc. ..

The fluorescent protein of the present invention or the fusion protein of the present invention can be obtained by expressing the recombinant vector of the present invention in the host cell, recovering the target protein from the host cell or medium, and purifying the protein. As a method for purifying a protein, an appropriate method can be appropriately selected from the methods usually used for purifying a protein. That is, various affinity chromatography such as salinization method, ultrafiltration method, isoelectric point precipitation method, gel filtration method, electrophoresis method, ion exchange chromatography, hydrophobic chromatography and antibody chromatography, chromatographic focusing method, adsorption. An appropriate method may be appropriately selected from commonly used methods such as chromatography and reverse phase chromatography, and if necessary, purification may be performed in an appropriate order using an HPLC system or the like.

One aspect of the present invention is a method of monitoring an oxidative-reduction state in a cell, in which the fluorescent protein or fusion protein of the present invention is present in the cell, and the fluorescence of the fluorescent protein or fusion protein is detected. A method comprising monitoring intracellular oxidation-reduction status based on the detected fluorescence discoloration. Hereinafter, it is also referred to as "a method for monitoring the redox state of the present invention". In the method, in order for the fluorescent protein or fusion protein of the present invention to be present in the cell, a recombinant vector having a DNA encoding the fluorescent protein or fusion protein of the present invention is transformed into the cell, and the cell is intracellular. The fluorescent protein or fusion protein of the present invention is expressed in, or the fluorescent protein or fusion protein of the present invention is directly introduced into the cell. The method for directly introducing the fluorescent protein or fusion protein of the present invention into the cell is not particularly limited, but for example, it is carried out using a protein introduction reagent based on a cationic lipid. It is also performed by the electroporation method or the microinjection method.

The detection of fluorescence of a fluorescent protein or fusion protein in the above is not particularly limited, but is carried out using, for example, a fluorescence microscope or a spectrofluorometer. For example, the intracellular redox state can be monitored based on the fluorescent discoloration of the fluorescent protein or fusion protein of the present invention while observing with a fluorescence microscope. Further, "monitoring the intracellular oxidation-reduction state based on the detected fluorescence discoloration" is a peak on the long wavelength side of the two fluorescence spectrum peaks existing in the fluorescence spectrum of the fluorescent protein of the present invention ( The ratio of fluorescence (F1) derived from the "first fluorescence spectrum peak" (hereinafter also referred to as "first fluorescence spectrum peak") to fluorescence (F2) derived from the peak on the short wavelength side (hereinafter also referred to as "second fluorescence spectrum peak"). It may be possible to monitor the intracellular oxidation-reduction state based on the change of (F1 / F2).

One aspect of the present invention is a method for screening an anticancer agent, wherein the fluorescent protein of the present invention or the fusion protein of the present invention is present in cancer cells, and the fluorescent protein or the fusion protein is fluorescent before the addition of the candidate drug. Detect, add the candidate drug to the cancer cells, detect the fluorescence of the fluorescent protein or fusion protein after the addition of the candidate drug, and compare with before the addition of the candidate drug based on the discoloration of the fluorescence before and after the addition of the candidate drug. The present invention relates to the above-mentioned method, which comprises selecting a drug that oxidizes the inside of cells. Hereinafter, it is also referred to as "the screening method of the present invention".

In general, the inside of cancer cells is kept in a reducing state as compared with the inside of normal cells. Drugs that can induce a more oxidative state in cancer cells can be candidates for anticancer drugs. In the conventional screening method for cancer cells, a drug candidate is added to the cancer cells, and whether or not the cancer cells die or a change in the shape of the cancer cells is confirmed. However, since it takes a certain amount of time for the cancer cells to die, it takes time to confirm the effect of the drug. In addition, cell image processing is required to confirm the shape change of cancer cells. By using the screening method of the present invention, a candidate drug can be screened for the redox state in cancer cells by changing the fluorescence, so that it can be performed quickly without waiting for the death of cancer cells. Moreover, it is not necessary to use an image processing means.

In the screening method of the present invention, the step of allowing the fluorescent protein of the present invention or the fusion protein of the present invention to exist in cancer cells and the step of detecting the fluorescence of the fluorescent protein or the fusion protein are described in the above-mentioned oxidation-reduction state of the present invention. It can be done in the same way as the monitoring method.

Examples will be shown below and the present invention will be described in more detail. However, the present invention is not limited to the following examples, and can be carried out with appropriate modifications.

In the following examples, the production of variants from the parent protein (GFP) was performed by a site-specific mutagenesis method. The sequence of primers used is shown in Table 1 below.

Here, the primer name in Table 1 is expressed as "Primer_primer number (F / R) -mutation to be introduced". "F" indicates a forward primer and "R" indicates a reverse primer. For example, Primer_1 (F) -M1_S2insV (SEQ ID NO: 4) indicates that it is a forward primer and a primer for introducing C147_S148insD. Other primers are described in the same manner.

Example 1:
FROG / B (GFP-M1_S2insV_K26R_S30R_Y39N_N105T_N149C_V150D_I171V_S202C_T203V_S205V_A206K_V224R and H230L) (SEQ ID NO: 2) Preparation of GFP (SEQ ID NO: 2) DNA (SEQ ID NO: 22) M1_S2insV, K26R, S30R, Y39N, N105T, N149C, V150D, I171V, S202C, T203V, S205V, A206K, V224R and A mutation in the nucleotide sequence corresponding to H230L was introduced into SEQ ID NO: 1. As primers for introducing mutations, combinations of SEQ ID NOs: 4 and 5, combinations of SEQ ID NOs: 6 and 7, combinations of SEQ ID NOs: 8 and 9, combinations of SEQ ID NOs: 10 and 11, combinations of SEQ ID NOs: 12 and 13, sequences. Combinations of numbers 14 and 15, combinations of SEQ ID NOs: 16 and 17, combinations of SEQ ID NOs: 18 and 19, and combinations of SEQ ID NOs: 20 and 21 were used.

PCR (50 μl reaction system) was performed using pet23a / GFP 10 pg as a template and the above primers and PrimeSTAR® Max Premix. PCR was carried out for 30 cycles, including DNA denaturation at 98 ° C. for 10 seconds, annealing reaction at 55 ° C. for 15 seconds, and extension / ligation reaction at 72 ° C. for 20 seconds.

The prepared mutant was transformed into Escherichia coli BL21 (DE3), cultured overnight in LB + 50 μM ampicillin plate medium, inoculated into 20 ml of 2 × YT medium, and pre-cultured at 37 ° C. for about 3 hours. This was inoculated into 1 L of 2 × YT medium and main-cultured at 37 ° C., and when the absorbance was 0.4 to 0.6, IPTG with a final concentration of 1 mM was added, and the culture temperature was lowered to 25 ° C. It was cultured in the evening. After culturing, the cells are collected by centrifugation, suspended in 20 mM Tris-HCl (pH 8.0), crushed using a French press, and centrifuged at 20000 × G for 30 minutes to perform FROG / B (Fluorescent protein with RedOx). A supernatant containing -dependent change in color between Green and Blue) was obtained.

The above supernatant was heated at 70 ° C. for 30 minutes to denature the E. coli-derived protein. Then, it was centrifuged at 20000 × G for 15 minutes to remove the denatured protein. The supernatant after centrifugation was applied to a TOYOPEARL Butyl-650 column (manufactured by Tosoh Corporation), and the ammonium sulfate concentration in the eluate was continuously reduced between 20% and 0% to separate fractions. The low-contamination portion of the fractionated fraction was dialyzed overnight in 20 mM Tris-HCl (pH 8.0) solution and then concentrated using Amicon Ultra 10K (Merck Millipore) to give FROG / B. rice field. The obtained protein was frozen in liquid nitrogen with glycerol added to a final concentration of 20% and stored. The DNA encoding the obtained protein is designated as SEQ ID NO: 23.

Example 2:
Measurement of Fluorescence Spectrum of FROG / B Using a 50 mM Tris-HCl (pH 7.0) buffer solution, prepare a 1 μM solution of FROG / B prepared in Example 1 and prepare a spectrofluorometer FP-8500 (JASCO Corporation). Fluorescence of FROG / B was measured using (Co., Ltd.). When the reduced fluorescence of FROG / B was measured, the fluorescent protein was kept warm at room temperature for 15 minutes in the presence of 10 mM dithiothreitol (DTT) before the fluorescence intensity was measured to obtain the reduced fluorescence in advance. When measuring the oxidation type fluorescence of FROG / B, the oxidation type was prepared in advance by air oxidation before measuring the fluorescence intensity. The fluorescence of the fluorescent protein at the maximum excitation wavelength (around 400 nm) was recorded.

The fluorescence spectra of the oxidized and reduced FROG / B are shown in FIG. There were two fluorescence spectrum peaks in both the oxidized and reduced fluorescence spectra. Of these, a peak having a maximum fluorescence wavelength near 510 nm is called a "first fluorescence spectrum peak", and a peak having a maximum fluorescence wavelength near 450 nm is called a "second fluorescence spectrum peak". From the reduced form to the oxidized form, the fluorescence intensity of the first fluorescence spectrum peak increases, while the fluorescence intensity of the second fluorescence spectrum peak decreases. Peak area of the first fluorescence spectrum region of 490nm~600nm (F _G) and calculating the ratio (F _G / F _B) of the peak area of the second of the fluorescence spectrum peak of 410nm~490nm region (F _B) By doing so, it is possible to quantitatively monitor the intracellular redox state without depending on the expression level of FROG / B in the cell.

Example 3: Observation of Fluorescent Color of FROG / B Using 50 mM Tris-HCl (pH 7.0) buffer, a 10 μM solution of FROG / B prepared in Example 1 was prepared and used as a sample. When observing the reduced fluorescence of FROG / B, the fluorescent protein was kept warm at room temperature for 15 minutes in the presence of 10 mM dithiothreitol (DTT) before the fluorescence intensity measurement, and the fluorescent protein was preliminarily reduced. When observing the oxidized type fluorescence of FROG / B, the oxidized type was prepared in advance by air oxidation before measuring the fluorescence intensity. The light from the high-pressure xenon lamp LambdaLS light source was separated using a dichroic mirror DM410 (OLYMPUS) and a bandpass filter BP340-390 (OLYMPUS) to obtain excitation light. The fluorescence emitted from the sample was separated using a long-pass filter BA420IF (OLYMPUS) and then detected using a color CCD camera ARTCAM-500MIWOM (ARTRAY). The results are shown in FIG. In FROG / B, the oxidized form fluoresces green and the reduced form fluoresces blue.

It is considered that the ESPT phenomenon contributes to the mechanism by which the fluorescence of FROG / B is discolored. FIG. 8 shows the estimation mechanism. FROG / B utilizes a phenomenon called ESPT (Excited-state proton transfer) in which a non-ionizing GFP chromophore, which originally emits blue fluorescence, emits green fluorescence by releasing protons at the time of excitation. There is. In the ESPT pathway inherent in GFP, protons released from the non-ionizing chromophore are transported via water molecules in the order of S205 (or T203) and E222. In FROG / B, the ESPT pathway was blocked by substituting S205 and T203 with valine (V), and instead H148, D150 (valine (V) at the 150th amino acid of GFP was replaced with aspartic acid (D)). A new ESPT pathway consisting of was formed. The positions of H148 and 150 that form this new pathway are altered by the formation of disulfide bonds of cysteine (C) introduced at positions 149 and 202. As a result, it is estimated that the probability of ESPT occurrence changes, and as a result, the fluorescence wavelength changes between blue and green.

横軸を酸化還元電位、縦軸を還元率としてプロットし、ネルンストの式でフィッティングし、ＦＲＯＧ／Ｂの中間酸化還元電位−２９５ｍＶを得た。結果を図３に示す。 Example 4:
Measurement of redox potentials Oxidized DTT with a final concentration of 20 mM in 50 mM Tris-HCl (pH 7.0) buffer and reduced DTT with a final concentration of 8.0 mM, 4.0 mM, 2.0 mM, 0.80 mM, 0, respectively. To 12 kinds of reaction solutions to which .40 mM, 0.20 mM, 80 μM, 40 μM, 20 μM, 8.0 μM, 4.0 μM and 2.0 μM were added, the FROG / B prepared in Example 1 was added to a final concentration of 1 μM. In addition, the sample was prepared by keeping it warm at room temperature for 2 hours. At this time, the redox potentials of each sample are 318 mM, -309 mV, -300 mV, -289 mV, -280 mV, -271 mV, -259 mV, -250 mV, -241 mV, -229 mV, -221 mV and -212 mV, respectively, according to the Nernst equation. Is calculated. The fluorescence spectrum of each sample was measured using a spectrofluorometer FP-8500 (JASCO Corporation), and the ratio of reduced FROG / B was determined as the reduction rate (R) using the following formula. Here, F _GO , F _BO , F _GR and F _BR are the peak area of the first fluorescence spectrum in the region of 490 nm to 600 nm and the peak area of the second fluorescence spectrum peak in the region of 410 nm to 490 nm at the time of complete oxidation, respectively. It represents the peak area of the first fluorescence spectrum in the region of 490 nm to 600 nm and the peak area of the second fluorescence spectrum peak in the region of 410 nm to 490 nm at the time of complete reduction.

The horizontal axis was plotted as the redox potential and the vertical axis was plotted as the reduction rate, and fitting was performed using the Nernst equation to obtain an intermediate redox potential of FROG / B of -295 mV. The results are shown in FIG.

Example 5:
PH Sensitivity of FROG / B The FROG prepared in Example 1 was prepared using a Tris-HCl buffer whose pH was adjusted to 7.0, 7.2, 7.4, 7.6, 7.8 and 8.0. / B was adjusted to a final concentration of 1 μM and used as a sample. When observing the reduced fluorescence of FROG / B, the fluorescent protein was kept warm at room temperature for 15 minutes in the presence of 10 mM dithiothreitol (DTT) before the fluorescence intensity measurement, and the fluorescent protein was preliminarily reduced. When observing the oxidized type fluorescence of FROG / B, the oxidized type was prepared in advance by air oxidation before measuring the fluorescence intensity. In Case pH, the fluorescence spectrum of the oxidation and reduction were measured using a fluorescence spectrophotometer FP-8500 (JASCO Corporation), was calculated value of F _G / F _B.

The pH sensitivity of FROG / B at pH 7.0 to 8.0 is shown in FIG. Rate of change in the value of F _G / F _B was less than 30%. This indicates that the pH sensitivity of FROG / B is low.

Example 6: Measurement of change in fluorescence intensity of FROG / B with time by addition of oxidizing agent (diamide) and reducing agent (DTT) FROG / B is expressed in HeLa cells and observed with a confocal microscope (LSM780 Zess). bottom. It was excited by a diode laser of 400 nm, and the fluorescence was separated into blue light of 490 nm or less and green light of 491 nm or more and detected by different detectors. After 5 minutes from the start of measurement, 500 μm of 1,1-azobis- (N, N-dimethylformamide) (diamide) was added, and after 15 minutes, 10 mM DTT was added. From the obtained fluorescence image (FIG. 6), fluorescence brightness information of a region surrounded by ROI of 490 nm or less and 491 nm or more was acquired, and the result of FIG. 5 was obtained.

After diamide addition as an oxidant, F _G / F _B was increased. On the other hand, after the addition of the reducing agent DTT, F _G / F _B was reduced (Fig. 5). The change in fluorescent color is shown in FIG. After the addition of the oxidizing agent diamide, the green fluorescence became stronger, and after the addition of the reducing agent DTT, the green fluorescence became weaker. Further, after the addition of the oxidizing agent diamide, the blue fluorescence became weaker, and after the addition of the reducing agent DTT, the blue fluorescence became stronger.

Example 7:
Preparation of fusion protein (FROG / B-All1541) (SEQ ID NO: 3) of FROG / B and All1541 For a vector (referred to as FROG / B / pet23a) in which the nucleotide sequence corresponding to FROG / B is inserted into pet23a. , The nucleotide sequence corresponding to All1541 which is a peroxyderedoxine was inserted. _{Further, a nucleotide sequence corresponding to the mobile linker (GGSGG) 6} was inserted between the nucleotide sequence corresponding to FROG / B and the nucleotide sequence corresponding to All1541. The obtained vector is referred to as FROG / B-All1541 / pet23a.

FROG / B-All1541 / pet23a was transformed into Escherichia coli BL21 (DE3), cultured overnight in LB + 50 μM ampicillin plate medium, inoculated into 20 ml of 2 × YT medium, and pre-cultured at 37 ° C. for about 3 hours. .. This was inoculated into 1 L of 2 × YT medium and main-cultured at 37 ° C., and when the absorbance was 0.4 to 0.6, IPTG with a final concentration of 1 mM was added, and the culture temperature was lowered to 25 ° C. It was cultured in the evening. After culturing, the cells were collected by centrifugation, suspended in 20 mM Tris-HCl (pH 8.0), crushed using a French press, and centrifuged at 20000 × G for 30 minutes to produce a supernatant containing FROG / B-All1541. Got

The obtained supernatant is applied to a Ni-NTA column (manufactured by QIAGEN) and washed with a washing buffer containing 20 mM imidazole-20 mM Tris-HCl (pH 8.0), and then 100 mM imidazole and 20 mM Tris-HCl (pH 8.0). ) Was used to elute. The low-contamination portion of the fractionated fraction was dialyzed overnight in 20 mM Tris-HCl (pH 8.0) solution, then concentrated using Amicon Ultra 10K (Merck Millipore) and FROG / B-All1541. Got The obtained fusion protein was frozen in liquid nitrogen with glycerol added to a final concentration of 20% and stored. The DNA encoding the obtained protein is designated as SEQ ID NO: 24.

Example 8:
Measurement of changes in the fluorescence intensity of FROG / B-All1541 with time due to the addition of H ₂ O ₂ A microdialysis membrane Minii using a anaerobic chamber to previously reduce the fluorescence intensity of FROG / B-All1541 with 10 mM DTT in the absence of oxygen. After dialysis using Dynalysis Kit 8 kDa cut-off, 250 μl (GE Healthcare) to remove DTT, the final concentration of protein was adjusted to 1 μM using 50 mM Tris-HCl (pH 8.0) buffer. Used as a sample. The fluorescence intensity of the sample was measured using a spectrofluorometer FP-8500 (JASCO Corporation). The sensitivity of the spectral fluorometer was set to medium, the excitation wavelength was set to 400 nm, the excitation and fluorescence bandwidths were both set to 5 nm, and the fluorescence spectrum was observed. A predetermined concentration of H ₂ O ₂ was added, and the fluorescence spectrum after 3 minutes was observed (FIG. 7).

FROG / B-All1541 was observed to change to the oxidized form 3 minutes after the addition of _{H 2} O _2. By fusing FROG / B with All1541, the redox sensitivity could be further improved.

By using the fluorescent protein of the present invention, changes in the oxidation-reduction state in cells can be easily monitored by fluorescence microscope observation or the like based on the discoloration of the fluorescence of the fluorescent protein. In addition, by using the fluorescent protein of the present invention, quick and simple screening of anticancer agents becomes possible.

Claims (8)

In the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence having 90% or more sequence identity with respect to SEQ ID NO: 1.
The 148th amino acid residue is histidine (His),
The 149th amino acid residue was replaced with cysteine (C),
The 150th amino acid residue was replaced with aspartic acid (D),
The 202nd amino acid residue is replaced with cysteine (C),
Fluorescence for measuring changes in intracellular oxidation-reduction state, consisting of an amino acid sequence in which the 203rd amino acid residue is replaced with valine (V) and the 205th amino acid residue is replaced with valine (V). The above-mentioned fluorescent protein, which is a protein and has a function of discoloring fluorescence between an oxidized form and a reduced form when fluorescence is measured at a single excitation wavelength. Valine (V) is inserted between the 1st and 2nd amino acid residues,
The 26th amino acid residue is replaced with arginine (R),
The 30th amino acid residue is replaced with arginine (R),
The 39th amino acid residue was replaced with asparagine (N),
The 105th amino acid residue was replaced with threonine (T),
The 171st amino acid residue was replaced with valine (V),
The 206th amino acid residue is replaced with lysine (K),
The fluorescent protein according to claim 1, wherein the amino acid residue at position 224 is replaced with arginine (R) and the amino acid residue at position 230 is replaced with leucine (L). A fusion protein obtained by fusing the fluorescent protein according to claim 1 or 2 with a second protein. A DNA encoding the fluorescent protein according to claim 1 or 2 or the fusion protein according to claim 3. A recombinant vector having the DNA according to claim 4. A host cell transformed with the recombinant vector according to claim 5. It is a method of monitoring the intracellular redox state.
The fluorescent protein according to claim 1 or 2 or the fusion protein according to claim 3 is allowed to exist in the cell.
Detecting the fluorescence of the fluorescent protein or fusion protein,
A method comprising monitoring the intracellular redox state based on the detected fluorescence discoloration. It is a screening method for anticancer drugs.
The fluorescent protein according to claim 1 or 2 or the fusion protein according to claim 3 is allowed to exist in cancer cells.
Fluorescence of the fluorescent protein or fusion protein before the addition of the candidate drug was detected.
Candidate drugs are added to the cancer cells and
The fluorescence of the fluorescent protein or fusion protein after the addition of the candidate drug is detected, and the fluorescence is detected.
The method comprising selecting a drug that oxidizes the cells as compared to before the addition of the candidate drug, based on the discoloration of the fluorescence before and after the addition of the candidate drug.

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