RU2402566C2 - Mutant photoprotein (versions) for determining intracellular calcium simultaneously in different cell organelles - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention represents mutant photoprotein for determining intracellular calcium simultaneously in different cell organelles, which possesses light0emitting function and is characterised by amino acid sequence of wild type calcium-regulated photoprotein, in which natural residues of phenylalanine, related to conservative regions of said sequence and corresponding to residue 119 of photoprotein obelin Obelia longissima, as well as to residue 88 of obelin Obelia longissima, are substituted with other residues, different from phenylalanine, substitution of which results in the reduction of sensitivity of calcium-regulated photoprotein to calcium ions in comparison with wild type photoprotein and change of emitted light wavelength in comparison with wild type photoprotein. Invention also relates to one more mutant protein, which has substitution in position 144 of photoprotein obelin Obelia longissima. Invention also relates to DNA, which codes said proteins, as well as to vectors for expression of proteins.

EFFECT: invention allows to obtain novel forms of photoprotein with changed sensitivity to calcium and changes wavelength of emitted light.

13 cl, 5 dwg, 4 ex

Description

The invention relates to the field of genetic and protein engineering, in particular to new proteins, mutant Ca ²⁺ -regulated photoproteins that exhibit distinctive properties compared to wild-type photoprotein, cDNA encoding these proteins, and can be used in in vivo studies, in in particular, to determine the flow of calcium in cells. The main use of Ca ²⁺ -regulated photoproteins is due to the very nature of the reaction - light is emitted in response to the appearance of calcium ions, therefore, the greatest use of Ca ²⁺ -regulated photoproteins was found as bioluminescent indicators of intracellular calcium in various types of cells. Bioluminescence is initiated by calcium ions and arises as a result of oxidative decarboxylation of a protein-bound substrate, celenterazine. The result is a reaction product, celenteramide, in an excited state and CO ₂ . The transition of celenteramide from the excited state to the ground state is accompanied by the emission of light. Wild type obelin generates “blue” bioluminescence. To monitor the content of intracellular calcium immediately in different parts of the cell, it would be useful to change the wavelength of emission so that it is possible to separate the signals coming from different organelles. Wild type photoproteins make it possible to measure the concentration of Ca ²⁺ in a concentration range of 10 ^-7 -10 ^-4 M. Different contents of free calcium in different organelles (in the EPR, which is Ca ²⁺ depot, and in mitochondria, the maximum concentration of free calcium is very high and reaches 0.1-1 mm, and in the cytosol, on the contrary, low 10 ^-8 -10 ^-7 M) necessitates the use of photoproteins with reduced and increased sensitivity to calcium.

Known mutants of aquorin and obelin with a shifted emission spectrum [US 7345160, C12Q 1/66, publ. March 18, 2008], where the shift was also achieved by using modified celenterazine analogues as a substrate, which led to radiation in the longer wavelength part of the spectrum.

Known for a number of mutants of aquorin with reduced [Kendall J.M. et al, Biochem. Biophys. Res. Commun., 1992] and increased sensitivity to calcium [Tricoire L. et al, PNAS, 2006, (prototype)].

The disadvantage of the mutants of aquorin and wild-type aquorin is the effect of magnesium ions on sensitivity to calcium ions. This is significant, since magnesium ions are able to bind at calcium binding sites and change the sensitivity to calcium, and in cells the magnesium content is quite high (1-3 mm).

The technical result of the invention is to obtain, on the basis of photoprotein, an intracellular calcium reporter having both reduced sensitivity to calcium and “blue bioluminescence”, and a reporter with increased sensitivity to calcium and “green” bioluminescence.

The technical result is achieved in that in a mutant photoprotein for determining intracellular calcium simultaneously in different cell organelles having a light-emitting function and characterized by the amino acid sequence of a wild-type calcium-regulated photoprotein, in which the natural phenylalanine residues belong to the conserved regions of the named sequence and correspond to 119 photoprotein residue Obelia obelia longissima (or the remainder of the 120 photoprotein of aquorin Aequorea victoria, or the remainder of the 122 photoprotein clitin of Clytia gregaria), as well as residue 88 of Obelin longissima obelin (or residue 89 of Aequorea victoria aquorin photoprotein, or residue 91 of Clytia gregaria clitin photoprotein), replaced by other residues other than phenylalanine, the substitutions of which lead to a change in the sensitivity of calcium to calcium ions decreases compared to the wild-type photoprotein and the wavelength of the emitted light changes in comparison with the wild-type photoprotein. The amino acid residue corresponding to residue 119 in Obelia longissima obelin is tryptophan, and the amino acid residue corresponding to Obelia longissima obelin residue 88 is tyrosine.

The technical result is also achieved by the fact that a mutant photoprotein for determining intracellular calcium simultaneously in different cell organelles, having a light-emitting function and characterized by the amino acid sequence of a wild-type calcium-regulated photoprotein, in which the natural isoleucine residue, which is a conservative amino acid and corresponding to the residue 144 of Obelia longissima obelin photoprotein (or residue 145 of Aequorea victoria aquorin photoprotein, or residue 147 of clitin Clytia gregaria photoprotein), replacement n other amino acid residue, other than isoleucine, in such a way that the sensitivity of the calcium-regulated photoprotein to calcium ions increases and the radiation wavelength changes in comparison with the wild-type photoprotein. In the mutant photoprotein, the hydrophobic amino acid residue at position 144 of the Obelia longissima obelin photoprotein is replaced by a hydrophilic amino acid residue. The hydrophilic amino acid, which provides an increase in the sensitivity of calcium-regulated photoprotein to calcium ions and a change in the wavelength of the emitted light, is histidine.

The technical result is also provided by DNA, in which the codons corresponding to the amino acid residues at positions 119 and 88 are replaced by codons defining amino acids other than phenylalanine, so that the sensitivity of the calcium-regulated photoprotein to calcium ions decreases and the wavelength of the emitted light changes in comparison with wild type photoprotein. DNA has the nucleotide sequence described in Seq. No. 1, where the nucleotides at positions 262-264 form a codon encoding an amino acid that provides a shift of the maximum radiation in the shorter wavelength region of the spectrum, and the nucleotides at positions 355-357 form a codon encoding an amino acid that provides a decrease in the sensitivity of photoprotein to calcium ions. DNA in which the nucleotides at positions 262-264 encode tyrosine and the nucleotides at positions 355-357 encode tryptophan.

The technical result is also provided by DNA in which the codon corresponding to the amino acid residue at position 144 is replaced by a codon defining an amino acid other than isoleucine, so that the sensitivity of the calcium-regulated photoprotein to calcium ions increases and the wavelength of the emitted light changes in comparison with the photoprotein wild type. DNA has the nucleotide sequence described in Seq. No. 3, where the nucleotides at positions 430-432 form a codon encoding an amino acid that provides a shift in the radiation maximum to the longer wavelength region of the spectrum and an increase in the sensitivity of the photoprotein to calcium ions. DNA in which the nucleotides at positions 430-432 encode histidine.

The vector for expression of the protein, in mammalian cells, is the plasmid pCMV / myc / mito (Invitrogene), into which the DNA characterized by the Seq sequence is ligated. No. 1, the expression product of which will be localized in the mitochondria and emit “blue” light.

A vector for the expression of protein in mammalian cells, which is the pcDNA3.1 plasmid into which Seq-characterized DNA is ligated. No. 3, the expression product of which will be localized in the cytoplasm of cells and, when calcium ions appear (with a stimulus), emit “green” light. Plasmids containing the mutant photoprotein genes in such forms that are capable of expressing the proteins of the invention constitutively when introduced into the host cell.

The invention provides mammalian CHO cell lines capable of expressing the mutant photoproteins of the invention, as well as an assay method in which the intracellular calcium content is measured simultaneously in two parts of the cell.

In the claimed invention, through site-directed mutagenesis, proteins are obtained, one molecule of which simultaneously contains substitutions, leading to a change in two properties of the photoprotein: sensitivity to calcium ions and the bioluminescence spectrum. It does not require additional use of analogs of the substrate. And, unlike the prototype, in addition to the altered sensitivity to calcium, the mutant photoproteins of the present invention have altered radiation spectral characteristics so that the bioluminescence maxima of two mutant photoproteins are spaced apart from each other on a spectral wavelength scale of 50 nm. Also, unlike the prototype, the photoproteins of the invention have calcium sensitivity, which is independent of the presence of magnesium ions.

The new properties acquired by mutant photoproteins make it possible to effectively use them as reporter agents in the study of calcium fluxes in cells, since having different sensitivity to Ca ^{2+ they} make it possible to measure the concentration of ions in cell compartments with different calcium contents, and different radiation maxima allow measurements to be carried out simultaneously.

The above distinguishing features from the prototype signs allow us to conclude that the claimed technical solutions meet the criterion of "novelty."

The features distinguishing the claimed technical solutions from the prototype are not identified in other technical solutions and, therefore, provide the claimed solution with the criterion of "inventive step".

Description of drawings

The figure 1 presents the sequence of oligonucleotides used as primers for mutations by site-directed mutagenesis.

Figure 2 is a graph illustrating the bioluminescence spectra of wild-type WT obelin and obelin mutants with a double substitution: phenylalanine at position 88 at tyrosine and at position 119 at tryptophan (F88Y & F119W) and with isoleucine at position 144 of the obelin amino acid sequence replaced by histidine (I144H).

The figure 3 shows a graph of the dependence of the luminescence intensity on the concentration of Ca ²⁺ (A) mutant obelin OL I144H, (B) mutant obelin OL F88Y & F119W. Designations: Δ - luminescence of mutants, ○ - luminescence of obelin WT. Filled symbols - calcium concentration was set using Ca ²⁺ - EGTA buffers. Open symbols - calcium concentration was set by diluting a CaCl ₂ solution. The light signal intensity (L) is expressed in units of L / L _int , where L _int is the total number of quanta emitted during the reaction, determined using kinetic measurements under the same conditions in the same protein sample. Measurement temperature 20 ° C. The lower right corner of the graphs shows the bioluminescence spectra of the corresponding mutant proteins, the luminescence intensity is expressed in relative units obtained by normalizing the spectra to L _max . Next to the curve, the numbers indicate the wavelength at which the maximum bioluminescence (λ _max ) is observed.

The figure 4 presents graphs of the dependence of the luminescence intensity of the mutant obelin OL F88Y (A) and aquorin Aeq N33D (B) on the concentration of Ca ²⁺ . Designations: ○ - luminescence of mutants in the absence of Mg ²⁺ ; □ - luminescence of mutants in the presence of 1 mm Mg ²⁺ . The remaining designations are similar to FIG.

Figure 5 presents a table that shows the main characteristics of the bioluminescence of obelin WT and its mutant variants: OL I144H and OL F88Y & F119W. The characteristics of bioluminescence in solution at pH 7.0 are presented. Bioluminescent activity is expressed as a percentage relative to the specific specific activity of wild-type obelin (recombinant). The parameter L _min / Int denotes the intensity of Ca ²⁺ -independent luminescence, normalized to the total light output. Ca ²⁺ -independent luminescence (spontaneous emission) is an indicator of the stability of photoproteins. k _decay - characterizes the decay rate of a bioluminescent signal.

The sequences presented at the end of the description describe the DNA and amino acid sequences as follows:

Sequence No. 1: represents the DNA sequence encoding the mutant photoprotein of the present invention, in which wild-type codons Obelia longissima at positions 262-264 and 355-357 underwent mutations, for tyrosine, base 263 as a result of the mutation is A, for tryptophan bases 356 and 357 b The result of the mutation is T.

Sequence No. 2: represents the amino acid sequence of the photoprotein of the present invention, in which the amino acid phenylalanine at positions 88 and 119 of Obelia longissima are replaced by tyrosine and tryptophan, respectively.

Sequence No. 3: represents the DNA sequence encoding another mutant photoprotein of the present invention, in which the wild-type codon Obelia longissima at position 430-432 is mutated so that the bases at positions 430 and 431 are represented by C and A.

Sequence No. 4: represents the amino acid sequence of another photoprotein of the present invention, in which the isoleipine amino acid is replaced by histidine.

Example 1

Obtaining mutant photoproteins with altered sensitivity to calcium and radiation spectra.

All genetic engineering manipulations, unless otherwise noted, were performed using methods well known to those skilled in the art.

Site-directed mutagenesis to obtain mutant obelin forms was performed by PCR using the QuikChange® Site-Directed Mutagenesis Kit (Stratagene, United States) site-directed mutagenesis kit, following the instructions for the kit. A plasmid containing the wild-type obelin gene pET19-OL8 was used as a matrix. Primers for introducing single amino acid substitutions were synthesized at Syntol LLC (Russia). The sequence of oligonucleotide primers and their complementary partners are presented in figure 1. All obtained mutations were confirmed by sequencing of the obtained DNA. The nucleotide sequence of the DNA was determined on an Amerlight sequencing system (Amersham, UK) using Themo Sequenase ™ Cy ™ 5 Dye Terminator Kit (Amersham, UK). For each set of 4 reactions (one sample), the reaction mixture was prepared by a combination of the following components in one tube: 1 μg DNA template (0.5 μg / μl), 4 pmol of sequencing primer (20 μM), reaction buffer, thermosequenase (10 U / μ1), 2 μl of A, G, C or T-mixture. The amplification program included 31 cycles with the following parameters: 95 ° C for 30 sec, the next 30 cycles (95 ° C for 30 sec, 56 ° C for 30 sec, 72 ° C for 1 min 20 sec). The resulting samples were reprecipitated with ethanol with ammonium acetate and glycerol, the precipitate was obtained by centrifugation for 15 min at 16,000 g, dried in air and dissolved in 8 μl of a stop solution each. Then, the DNA was denatured by heating for 3 minutes at 72 ° C and placed in ice until applied to the gel. As an electrophoresis buffer, TBE × 0.5 was used. 1/2 of the reaction volume was applied to the gel. Separation was carried out according to the standard ALFWin Control program (Amerlight, UK) under the following conditions: 1500 V, 25 mA, 750 min. Data was processed using the ALF Express program.

Proteins were expressed in E. coli cells and obtained in highly purified form according to the method developed for wild-type obelin (WT). The cell paste was suspended in a five-fold volume of 0.02 M Tris-HCl, pH 7.0, and destroyed by sonication using a UD-20 ultrasonic disintegrator (Techpan, Poland) 5 times for 20 seconds under ice-cooling. The resulting mixture was centrifuged (14000 g, 2.9 min). The precipitate, consisting mainly of inclusion bodies, was washed by resuspending successively with solutions of 0.02 M Tris-HCl, pH 7.0, containing 0.9% NaCl, 0.1% Triton X-100, 5 mM CaCl ₂ . Inclusion bodies were dissolved in a 2.5-fold volume of 6 M urea containing 20 mM Tris-HCl and 5 mM CaCl ₂ (40 ° C. overnight), then centrifuged again. The supernatant was chromatographed on a column filled with DEAE-Sepharose FF (2 × 3.5 cm) (FPLC chromatographic system, Pharmacia, Sweden). The substances from the column were eluted with a buffer containing 6 M urea, 5 mM CaCl ₂ 0.02 M Tris-HCl pH 7.0 in a gradient of NaOAc concentrations from 0 to 0.5 M. The elution rate was 2 ml / min.

The purity of the final preparations of recombinant apoproteins obtained after isolation was evaluated by electrophoresis in 12.5% SDS page under denaturing conditions (in the presence of 0.1% SDS) according to the Laemmli method.

Active photoproteins were obtained by incubation of apoproteins with a 10-fold molar excess of the substrate, celenterazine, in a buffer containing 20 mM Tris-HCl, pH 7.0, 5 mM EDTA, 5 mM dithiothreitol at 4 ° C for at least 4 hours.

Activated proteins were separated from alobella by ion exchange chromatography on a Mono Q 5/5 column using the FPLC chromatographic system, Pharmacia, Sweden. Protein elution from the column was performed in 20 mM Tris-HCl, pH 7, 5 mM EDTA with a NaCl gradient (0-0.35 M).

Example 2

Determination of the sensitivity of photoproteins to calcium ions

The determination of the sensitivity of photoproteins to Ca ^{2+ is} based on the fact that low Ca ²⁺ concentrations are set using calcium buffer solutions (Ca ²⁺ -EGTA buffers), and high ones are determined by simple dilution of a CaCl ₂ solution. Calcium buffer solutions were prepared from two stock solutions by reciprocal dilution: one of them contained an equimolar amount of Ca ²⁺ and a chelator - EGTA, the other exactly the same amount of chelator, but without Ca ²⁺ . To determine the concentration of calcium in the finished solutions, the equilibrium chelator dissociation constant was used, which depends on the pH, ionic strength, and temperature of the solution. All solutions and utensils used require careful handling of traces of Ca ²⁺ and EGTA ^ions . To this end, all the solutions used were passed through a column with a Chelex-100 sorbent that binds trace amounts of calcium ions (Sigma). For the preparation of solutions, deionized water (18.2 MΩ) was used. During the measurements, the temperature of both the solutions and the measuring cuvette block was maintained at a constant level (20 ° C).

Protein solutions were also purified from traces of Ca ²⁺ and EGTA ions by gel filtration on a column: 1.5 × 6.5 cm D-Salt Dextran Desalting column (Pierce) in a buffer containing 150 mM KCl, 5 mM PIPES, pH 7.0. Fractions containing protein were identified by bioluminescent activity, and in order to avoid EGTA contamination, only the very first protein fractions leaving the column were collected to determine the dependence of bioluminescent activity on Ca ²⁺ concentration. The light signal was measured by injecting 10 μl of the thus prepared solution of photoprotein in 1 ml of a Ca ²⁺ solution. The light signal intensity (L) was expressed in units of L / L _int , where L _int is the total number of quanta emitted during the reaction, determined using kinetic measurements under the same conditions in the same protein sample. The luminometer had three filters of different optical density, attenuating the radiation, which made it possible to record signals differing in magnitude by 8 orders of magnitude.

To determine the sensitivity of photoproteins to Ca ²⁺ in the presence of 1 mM Mg ²⁺ , the desalted protein sample was kept for 1 hour with 1 mM MgCl ₂ . All Ca ²⁺ solutions also contained 1 mM Mg ²⁺ .

As a result of studying the dependence of the luminescence intensity on the concentration of Ca ²⁺ , the following indicators were determined:

a) the range of sensitivity to Ca ²⁺ ;

b) L _min / L _max - the ratio of the lower and upper boundaries of the range of the luminescent response, characterizing the width of the range of the luminescent response;

c) L _min / Int is the intensity of Ca ²⁺ -independent luminescence, normalized to the total light output.

The dependence of the luminescence intensity of mutant photoproteins on the concentration of Ca ²⁺ is presented as a function of the logarithm of normalized luminescence (log (L / L _int )) on the logarithm of the concentration of Ca ²⁺ (log [Ca ²⁺ ]). The sensitivity range for Ca ²⁺ in the mutant I144H photoprotein is in the range of -7.6 - -4.7 and is shifted towards lower Ca ²⁺ concentrations compared to the wild-type photoprotein, which means an increase in the sensitivity of mutants to Ca ²⁺ . In contrast, substitutions of F88Y & F119W lead to a shift in the sensitivity range to Ca ²⁺ towards higher ion concentrations, and the range is -6.5 - -3.6 (an order of magnitude smaller compared to the wild-type obelin). These mutant photoproteins were selected as the most promising of a number of planned and investigated point substitutions in the obelin sequence, including: C51A, F72V, V118F, E41G, D133E & I132V, F119W, L160D, F88W, F88Y, F88H, F88R, W92F, W92H, W92H, W92H, W92H W92R, W92F & H22E, H22E, H22N, M25F, M25Y, M25H, M25K, M25R, I144W, I144H, I144Q, Y138F, Y138W. For the mutant photoprotein F88Y & F119W, the value of L _min / L _max is almost 10 ^-7 , for the mutant I144H - 10 ^-6 . The narrowing of the width of the range of the luminescent response in the case of the latter is explained by an increase in the luminescence intensity in the absence of Ca ²⁺ (an increase in the lower boundary of the range) L _min / Int.

Magnesium ions (Mg ²⁺ ) have less affinity for the calcium-binding sites of photoproteins than calcium ions (Ca ²⁺ ). But their presence may affect sensitivity to Ca ²⁺ . The presence of Mg ²⁺ at physiological concentration (1 mM) does not affect the sensitivity of the mutant photoproteins of the present invention to Ca ²⁺ . This is expressed in the fact that the Ca ²⁺ curves in the absence of magnesium ions and in the presence of 1 mM Mg ^{2+ are} identical. The absence of the effect of magnesium ions on sensitivity to calcium ions is also characteristic of the initial protein, wild-type obelin (WT). According to this feature, obelin differs from another photoprotein - aquorin, despite the fact that both proteins are closely related and structural differences between them are minimal. We constructed 2 aquorin mutants: the first with the replacement of the 33rd asparagine with aspartic acid in the 1st Ca ²⁺ -binding site of the aquorin (N33D), the second with the replacement of phenylalanine at the 156th position with serine located in the hydrophobic region of the protein globule (F156S). The sensitivity of the obtained aquorin mutants to calcium was determined. It turned out that both photoproteins have an increased sensitivity to Ca ²⁺ compared to wild-type aquorin, which is characterized by a shift in the sensitivity range to Ca ²⁺ towards lower Ca ²⁺ concentrations. The addition of 1 mM Mg ²⁺ to all the solutions used in the experiment radically shifts the Ca ²⁺ curves toward higher ion concentrations. The figure 4 presents examples of the influence of the presence of magnesium ions on the sensitivity to calcium of an obelin mutant and an aquorin mutant.

Example 3

Basic bioluminescent properties of photoproteins

The relative specific bioluminescent activity (relative to wild-type obelin) of the mutants was determined using a Luminoskan vl.30 plate luminometer (Labsystems, Finland). For this, 50 μl of a solution of photoprotein in 20 mM Tris-HCl buffer, pH 7.0, containing 5 mM EDTA and 0.2 M NaCl was added to the wells of an opaque plate. The reaction was started by injecting 50 μl of 100 mM Tris-HCl buffer, pH 8.8, containing 100 mM CaCl ₂ into each well of the plate. The signal scanning time is from 5 sec to 1 min, depending on the speed of the luminescent reaction of each of the proteins.

The bioluminescent activity of the F88Y & F119W mutant photoprotein is identical to the wild-type (WT) photoprotein activity, the activity of the mutant I144H photoprotein is 40% lower (Fig. 5).

Calcium-independent luminescence was measured in protein samples (0.5-2.0 mg / ml) containing 2 mM EDTA placed in a luminometer cuvette.

The level of calcium-independent luminescence, which characterizes the stability of the photoprotein as a protein-substrate complex, in the "double" mutant is the same as in the wild-type obelin, and in the "single" 10 times higher, but when using this protein for in vivo studies , this is not significant, since the protein-substrate complex is formed upon the penetration of a hydrophobic substrate into the cell and does not require its long-term storage.

Bioluminescence spectra were measured on an AMINCO spectrofluorimeter (Thermo Spectronic, USA). All emission spectra were corrected for the sensitivity of the PMT to various wavelengths using the instrument software. Bioluminescence spectra were measured in protein solutions containing 1 mM EDTA, 50 mM Bis-Tris propane, pH 7.0, by injection of a CaCl ₂ solution in the same buffer. The concentration of free calcium in the reaction mixture was about 0.5 μM (the concentration was calculated using the Maxichelator computer program) in order to ensure a constant level of luminescence during recording of the radiation spectrum. Measurements were performed at room temperature.

The wild-type obelin generates blue bioluminescence with a maximum of 482 nm and a shoulder in the region of 400 nm. The maximum bioluminescence of the mutant F88Y & F119W photoprotein is located in the “blue” part of the spectrum and is equal to 455 nm. The maximum bioluminescence of the mutant I144H photoprotein is located in the "green" part of the spectrum and is equal to 505 nm. The spectra of both photoproteins of the invention are monomodal and do not contain other peaks.

The decay constant of the bioluminescent signal was calculated from the decay curve, which was recorded when the saturating concentration of Ca ²⁺ was injected into the sample in the luminometer cuvette, by the _decay formula k = ln (I ₁ / I ₂ ) / Δt (sec).

The kinetics of the bioluminescent reaction of the mutants is characterized by a slower decrease in intensity compared to the wild-type obelin. The decay constant of the bioluminescent signal for F88Y & F119W is 1.6 sec ^-1 , which is 4 times less than that of Obelin WT, for I144H it is 6.1, which is also less than the value of the wild-type obelin constant. Slow luminescence is another advantage of photoprotein as an intracellular reporter protein, because it simplifies the registration of light flux.

Example 4

Obtaining plasmids containing the DNA of the present invention

Plasmids pCMV / myc / mito and pcDNA3.1 (+) were obtained from hwtrogene Corporation, USA. Oligonucleotide primers were synthesized by Biosan CJSC, Russia.

The primer containing the restriction site for KpnI nuclease for constructing a vector containing the mutant photoprotein gene OL I144H, based on pcDNA3.1 (+) using PCR is characterized by the sequence:

olig No. 1 - 5'-TAACTTTAAGAAGGAGA GGTACC ATGGCT-3 '.

The primer containing the restriction site for PstI nuclease, for constructing a vector containing the mutant photoprotein gene OL F88Y & F119W, based on pCMV / myc / mito using PCR, has the following sequence:

olig No. 2 - 5'-CTTTAAGAAGGAGATAT CTGCAG GCTTCAAAATACGCA-3 '.

The primer for the synthesis of the restriction site for XhoI nuclease, complementary to the end of the obelin sequence is:

olig No. 3 - 5'-TACTCGAGATTAGGGAACTCCGTTGC-3 '.

To obtain pCMV-OL88 / 119, an insert containing the coding sequence of the mutant photoprotein OL F88Y & F119W (Seq. 1) with terminal restriction sites PstI and XhoI was obtained on the basis of pET19b-OL88 / 119 by PCR with olig primers No. 2 and olig No. 3. The fragment and vector restriction was performed using PstI and XhoI (SibEnzyme, Russia), after which they were preparatively purified by agarose gel electrophoresis and ligated.

To obtain pcDNA3.1 (+) - OL144 an insert containing the coding sequence of the mutant photoprotein OL I144H (Seq. 3) with restriction end sites KpnI and XhoI was obtained on the basis of pET19b-OL144 by PCR with primers olig No. 1 and olig No. 3. The restriction of the fragment and the vector was performed using KpnI and XhoI (SibEnzyme, Russia), after which they were preparatively purified by agarose gel electrophoresis and ligated.

The resulting constructs were verified by DNA sequencing. Transformation of plasmids into CHO cells (Chinese hamster ovary cells) was performed using the FreeStyle MAX transfection agent (Invitrogene). Selective selection of clones containing the plasmid was carried out on a medium with neomycin.

New mutant forms of the obelin photoprotein are obtained, characterized by altered sensitivity to calcium and at the same time altered by the wavelength of the emitted light. New photoproteins differ from the original variant by point substitutions in the amino acid sequence. Two reporters were obtained that can be used as intracellular calcium indicators. The blue and green color of bioluminescence, as well as high and low sensitivity to calcium, allows one to study the change in calcium concentration simultaneously (using two-wave detection) in two cell compartments with different Ca ²⁺ contents, for example, in the cytosol, where [Ca ²⁺ ] is in a state rest is 0.05-0.1 μM, increasing to 10 μM with various stimuli, and mitochondria, where [Ca ²⁺ ] reaches hundreds of μM. This method of determining the content of intracellular calcium is applicable for studying processes in the cell mediated by the action of calcium ions, for determining the function of receptors, ligands, ion channels, for studying the effects on cells of various substances: drugs, toxins, etc.

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Claims (13)

1. A mutant photoprotein for determining intracellular calcium simultaneously in different cell organelles, having a light-emitting function, and characterized by the amino acid sequence of a wild-type calcium-regulated photoprotein, in which the natural residues of phenylalanine belong to the conserved regions of this sequence and correspond to the residue 119 of Obelia longima obelin photoprotein (Obelia longima or residue 120 of Aequorea victoria aquorin photoprotein, or residue Clytia gregaria photoprotein 122), as well as 88 obelin residue Obelia longissima (or residue 89 of the aquorin Aequorea victoria photoprotein, or residue 91 of the clitin Clytia gregaria photoprotein) has been replaced by other residues other than phenylalanine, the substitutions of which lead to a decrease in the sensitivity of the calcium-regulated photoprotein to calcium ions compared to the type protoprotein wild the wavelength of the emitted light changes in comparison with the wild-type photoprotein. 2. The mutant photoprotein according to claim 1, characterized by the amino acid sequence described in Seq. No. 2, characterized in that the amino acid residue corresponding to residue 119 in Obelia longissima obelin is tryptophan, and the amino acid residue corresponding to Obelia longissima obelin residue 88 is tyrosine. 3. A mutant photoprotein for determining intracellular calcium simultaneously in different cell organelles, having a light-emitting function and characterized by the amino acid sequence of a wild-type calcium-regulated photoprotein, in which the natural isoleucine residue, which is a conserved amino acid and corresponding to residue 144 of Obelia longissima obelin photoprotein (or 145 aquorin Aequorea victoria, or residue 147 of the clitin photoprotein Clytia gregaria) is replaced by a different amino acid residue other than iso leucine, thus increasing the sensitivity of the calcium-regulated photoprotein to calcium ions and changing the radiation wavelength compared to wild-type photoprotein. 4. The mutant photoprotein according to claim 3, characterized in that the hydrophobic amino acid residue at position 144 of the Obelia longissima obelin photoprotein is replaced by a hydrophilic amino acid residue. 5. The mutant photoprotein according to claim 4, characterized by the amino acid sequence described in Seq. No. 4, where the hydrophilic amino acid, providing an increase in the sensitivity of calcium-regulated photoprotein to calcium ions and a change in the wavelength of the emitted light, is histidine. 6. The DNA encoding the mutant photoprotein according to claim 2, characterized in that its polynucleotide sequence is a wild-type obelin photoprotein sequence in which the codons corresponding to amino acid residues at positions 119 and 88 are replaced by codons defining amino acids other than phenylalanine, Thus, the sensitivity of the calcium-regulated photoprotein to calcium ions decreases and the wavelength of the emitted light changes in comparison with the wild-type photoprotein. 7. DNA according to claim 6, characterized in that it has the nucleotide sequence described in Seq. No. 1, where the nucleotides at positions 262-264 form a codon encoding an amino acid that provides a shift of the maximum radiation in the shorter wavelength region of the spectrum, and the nucleotides at positions 355-357 form a codon encoding an amino acid that provides a decrease in the sensitivity of photoprotein to calcium ions. 8. The DNA according to claim 7, where the nucleotides at positions 262-264 encode tyrosine and the nucleotides at positions 355-357 encode tryptophan. 9. The DNA encoding the mutant photoprotein according to claim 5, characterized in that its polynucleotide sequence is a wild-type obelin photoprotein sequence in which the codon corresponding to the amino acid residue at position 144 is replaced by a codon defining an amino acid other than isoleucine, thus that the sensitivity of the calcium-regulated photoprotein to calcium ions increases and the wavelength of the emitted light changes in comparison with the wild-type photoprotein. 10. The DNA according to claim 9, characterized in that it has the nucleotide sequence described in Seq. No. 3, where the nucleotides at positions 430-432 form a codon encoding an amino acid that provides a shift in the radiation maximum to the longer wavelength region of the spectrum and an increase in the sensitivity of the photoprotein to calcium ions. 11. The DNA of claim 10, where the nucleotides at positions 430-432 encode histidine. 12. A vector for the expression of the protein characterized in any of claims 1 and 2 in mammalian cells, which is a plasmid pCMV / myc / mito into which the DNA characterized in any of claims 6-8 is ligated. 13. A vector for the expression of a protein characterized in any one of claims 3 to 5 in mammalian cells, which is a pcDNA3.1 plasmid into which the DNA characterized in any of claims 9 to 11 is ligated.

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