RU2599443C2 - Modified genetically coded photosensitizer - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and can be used for recombinant production of mutants of phototoxic fluorescent protein. Mutants of KillerRed phototoxic fluorescent protein are obtained by recombinant production. KillerOrange mutant with SEQ ID NO: 02 comprises replacements Y68W, D115G, N147S, F179L, Y223H and E238Q, wherein KillerGreen with SEQ ID NO: 04 comprises replacements V46A, Y68W, A74T, D115G, N147S, Y223H and E238Q.

EFFECT: invention enables to obtain mutants of KillerRed phototoxic fluorescent protein having changed spectral characteristics.

6 cl, 3 dwg, 1 tbl, 5 ex

Description

The present invention relates mainly to the field of biology and chemistry, and is directed, in particular, to fluorescent proteins.

Fluorescent proteins, including green fluorescent protein (Green Fluorescent Protein, GFP), its mutants and homologs, are now widely known for their intensive use as in vivo fluorescent markers in biomedical research, as discussed in detail by Lippincott-Schwartz and Patterson in Science (2003) 300 (5616): 87-91.

Fluorescent proteins are proteins that are capable of fluorescence when irradiated with light of a suitable wavelength. The fluorescence properties of these proteins are due to the interaction of two or more amino acid residues that form the chromophore, and not the fluorescence of any one amino acid residue.

GFP hydromedusa Aequorea aequorea (synonym for A. victoria), has been described by Johnson et al. at J Cell Comp Physiol. (1962), 60: 85-104, as part of a jellyfish bioluminescent system, where GFP acts as a secondary emitter that converts blue light from an equorin photo protein to green cDNA light, encoding A. victoria GFP was cloned by Prasher et al. (Gene (1992), 111 (2): 229-33). It turned out that this gene can be heterologically expressed in almost any organism due to the unique ability of GFP to autocatalytically form a chromophore (Chalfie et al., Science 263 (1994), 802-805). This information has opened up broad prospects for the use of GFP in cell biology as a genetically encoded fluorescent label.

GFP has been used in a wide range of applications, including gene expression studies and protein localization (Chalfie et al., Science 263 (1994), 802-805, and Heim et al. In Proc. Nat. Acad. Sci. (1994), 91 : 12501-12504), as a tool for visualizing the intracellular distribution of organelles (Rizzuto et al., Curr. Biology (1995), 5: 635-642), for visualizing protein transport along the secretory pathway (Kaether and Gerdes, FEBS Letters (1995) 369: 267-271).

Numerous studies have been conducted to improve the properties of avGFP (Aequorea victoria GFP) and to obtain GFP variants that are suitable and optimized for various research purposes. The avGFP (codon usage) genetic code was optimized to increase expression in mammalian cells (“humanized” GFP, Haas, et al., Current Biology (1996), 6: 315-324; Yang, et al., Nucleic Acids Research (1996), 24: 4592-4593). Various GFP mutants have been prepared, including the Enhanced Green Fluorescent Protein (EGFP) having two amino acid substitutions: F64L and S65T (Heim et al., Nature 373 (1995), 663-664). Other mutants are blue, blue, and yellow-green spectral variants of avGFP and contain substitutions of amino acid residues that form the chromophore and / or residues that form the environment of the chromophore.

In 1999, GFP homologues were cloned from non-bioluminescent species of Anthozoa (Matz et al., Nature Biotechnol. (1999), 17: 969-973). This discovery has demonstrated that these proteins are not necessarily a component of the bioluminescent system. GFP-like proteins from Anthozoa had a large spectral diversity and included cyanic, green, yellow, red fluorescent proteins and violet-blue non-fluorescent chromoproteins (CPs) (Matz et al., Bioessays (2002), 24 (10): 953-959) . Subsequently, cDNAs of GFP-like proteins were cloned from a number of hydroid jellyfish and from copepods (Shagin et al., Mol Biol Evol. (2004), 21 (5): 841-850). Today, the family of GFP-like proteins includes hundreds of fluorescent and stained GFP homologs. The similarity of these proteins to GFP varies from 80-90% to less than 25% amino acid sequence identity.

The crystal structures of wild-type avGFP, GFP S65T mutant, and a number of GFP homologues were obtained (Ormo et al. Science (1996) 273: 1392-1395; Wall et al. Nat Struct Biol (2000), 7: 1133-1138; Yarbrough et al . Proc Natl Acad Sci USA (2001) 98: 462-467; Prescott et al. Structure (Camb) (2003), 11: 275-284; Petersen et al. J Biol Chem (2003), 278: 44626-44631; Wilmann et al. J Biol Chem (2005), 280: 2401-2404; Remington et al. Biochemistry (2005), 44, 202-212; Quillin et al. Biochemistry (2005), 44: 5774-5787). It was postulated that all members of the family have a common 3D structure, which is the so-called barrel of 11 beta layers, forming a compact antiparallel structure, inside which there is an alpha helix containing a chromophore. The chromophore is formed by oxidative cyclization of three conserved amino acid residues in the central region of the alpha helix (Cody et al., Biochemistry (1993) 32, 1212-1218). The positions of the amino acid residues forming the chromophore correspond to the Ser65-Tyr66-Gly67 region of avGFP. These amino acid residues can easily be identified in any GFP-like protein by aligning its sequence with the avGFP sequence.

The process of autocatalytic formation of a chromophore of proteins with different spectral properties is described in detail in several articles and includes several chemical reactions (Heim et al. Proc Natl Acad Sci USA. 1994; 91: 12501-12504; Ormo et al. Science. 1996; 273: 1392- 1395; Yang et al. Nat Biotechnol. 1996; 14: 1246-1251; Brejc et al. J. Proc Natl Acad Sci USA. 1997; 94: 2306-2311; Palm et al. Nat Struct Biol. 1997; 4: 361 -365; Gurskaya et al., BMC Biochem. 2001; 2: 6; Gross et al. Proc Natl Acad Sci USA. 2000; 97: 11990-11995; Wall et al. Nat Struct Biol. 2000; 7: 1133-1138 ; Yarbrough et al., J. Proc Natl Acad Sci USA. 2001; 98: 462-467; Pakhomov, AA and Martynov, VI Chem. Biol. 2008, 15, 755-764; Quillinet al. (2005) Biochemistry 44, 5774-5787; Yampolsky et al. (2005) Biochemistry 44, 5788-5793; Shu et al. (2006) Biochemistry 45, 9639-9647; Kikuch i et al. (2008) Biochemistry 47, 11573-11580; Yampolsky et al. Biochemistry, 2009, 48 (33), pp 8077-8082).

It was shown that the fluorescent protein KillerRed has phototoxic properties, which allows it to be used for targeted destruction of cells and biological molecules (proteins, DNA) directly in living organisms (Bulina et al., Nat Biotechnol. 2006 Jan; 24 (1): 95- 9; Bulina et al., Nat Protoc. 2006; 1 (2): 947-53). In the inactive state, KillerRed is a red fluorescent protein, low-toxic to cells and proteins. Under the influence of green light, KillerRed begins to produce reactive oxygen species. Activation of the phototoxicity of the protein is accompanied by structural rearrangement of the chromophore and loss of ability to fluorescence.

The spatial organization of the KillerRed monomer is a β-barrel, typical of fluorescent proteins, formed by 11 β-segments, with a central δ-helix containing a chromophore obtained by post-translational modification of the chromophore-forming sequence Gln65-Ty66-Gly67 (Pletnev et al. J Biol Chem. 2009; 284 (46): 32028-32039). In the KillerRed protein, the GFP-like domain (β-barrel) is composed of amino acid residues at positions 6 through 225 corresponding to positions 6 through 229 of the avGFP sequence.

The KillerRed chromophore in active fluorescent form is a planar bicyclic conjugated double bond system consisting of five-membered imidazolinone and phenolic rings. The Tuk66 phenolic cycle assumes a cis orientation with respect to the Ca-N bond (66), which corresponds to the active fluorescent state of the protein. This orientation is stabilized by two hydrogen bonds with Asn145 and through a water molecule with Thr201. In the process of the formation of the C ^α chromophore, the atom of the first Gln65 residue accepts sp ² hybridization, characterized by a planar trigonal arrangement of adjacent bonds. In this case, the partially formed double N-acylimine bond N = C ^α (Gln65) leads to the expansion of the conjugated π-electron system, causing a shift of the maxima of the excitation and emission wavelengths to the red region of the spectrum. The immediate environment of the chromophore is formed from the side chains of 17 residues, including catalytic Arg94 and Glu218. Most of these residues are involved in a developed system of hydrogen bonds, both directly and through water molecules.

A unique feature of the spatial structure of KillerRed is the presence of a channel filled with water, going from the end of the β-cylindrical protein architecture to the central region of the chromophore. This channel can facilitate the access of oxygen to the chromophore, facilitate the release of reactive oxygen species from the protein to the outside, and also serve as a conductor of protons or electrons upon photoexcitation of the chromophore. This feature, apparently, is one of the key structural factors of the observed phototoxic properties of the protein.

The KillerRed protein is currently the only GFP-like fluorescent protein with pronounced phototoxic properties. The creation of mutants of this protein with different spectral characteristics significantly expands the possibilities of targeted inactivation of cellular proteins (the method of chromophore-dependent light inactivation, CALI, from chromophore-assisted light inactivation), as it opens up the possibility, by varying the irradiation wavelength, more than one targeted inactivation of light cell protein, but several. The introduction of several phototoxic proteins with different spectral characteristics into the cell at once also allows one to increase the overall phototoxicity for cells when exposed to white light. In addition, the creation of mutants, the phototoxicity of which is activated by light with a wavelength of 450-470 nm, allows their use in two-photon microscopy.

The approach proposed in this application is aimed at creating such mutants and expanding the range of phototoxic GFP-like fluorescent proteins.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules encoding mutant forms of KillerRed having different spectral characteristics than the original protein.

In preferred embodiments, these proteins are characterized by an amino acid sequence that differs from the KillerRed protein sequence by at least replacing Y68W (tyrosine at position 68 with tryptophan; hereinafter, the amino acid residues are indicated in a single letter code, the numbering of amino acids corresponds to the numbering of amino acids in KillerRed, SEQ ID NO: 6).

In preferred embodiments, these proteins also contain several additional substitutions selected from the group V46A, A74T, D115G, N147S, F179L, Y223H, E238Q.

Examples of amino acid sequences of the functional proteins of the invention are shown in SEQ ID NO: 02 and SEQ ID NO: 04. Examples of their nucleotide sequences are shown in SEQ ID NO: 01 and SEQ ID NO: 03.

Nucleic acids encoding the proteins of the invention fused to the intracellular localization signal are also provided. Being expressed in cells, these proteins are sent to certain cellular compartments or organelles that are sensitive to oxidation by reactive oxygen species.

Nucleic acid molecules that differ from the presented nucleotide sequences due to the degeneracy of the genetic code are also included in the scope of the invention.

The proteins of the invention can be expressed in living cells and used for targeted destruction of target proteins, selected cells or cell populations, both in cell culture and in whole organisms.

In other embodiments, vectors comprising the nucleic acid of the invention are also provided. In addition, the invention provides expression cassettes comprising the nucleic acid of the invention and regulatory elements necessary for expression of the nucleic acid in a selected host cell. In addition, cells, stable cell lines, transgenic animals and transgenic plants, including nucleic acids, vectors or expression cassettes of the invention are also provided.

In other embodiments, the functional fluorescent proteins of the invention are provided that are encoded by the nucleic acids described above (for example, isolated proteins having the amino acid sequences of SEQ ID NO: 02 and SEQ ID NO: 04).

In addition, kits comprising nucleic acids or vectors or expression cassettes comprising said nucleic acids of the invention are provided.

A brief description of the figures

Figure 1 shows the alignment of the KillerRed protein (KillR, SEQ ID NO: 06) and other fluorescent proteins, the amn2CP chromoprotein (SEQ ID NO: 7) from the antomedusa, on the basis of which KillerRed, the red fluorescent protein DsRed (SEQ ID NO: 8) from Discosoma coral polyp, and avGFP green fluorescent protein (SEQ ID NO: 9) from Aequorea victoria jellyfish. The amino acid residues forming the chromophore are underlined. Amino acid residues whose side chains are embedded inside a protein globule are shown against a gray background.

Figure 2 presents the spectra of excitation (1) and emission (2) of fluorescence of the KillerOrange protein (SEQ ID NO: 1, 2).

Figure 3 presents the spectra of excitation (1) and emission (2) of fluorescence of the KillerGreen protein (SEQ ID NO: 3, 4).

Detailed Description of the Invention

The present invention is directed to nucleic acid molecules that encode mutant forms of KillerRed. Nucleic acids of the present invention obtained using recombinant technologies. In preferred embodiments, the nucleic acids of the invention encode proteins having different spectral characteristics than KillerRed. In some embodiments, the nucleic acids of the invention encode mutant forms of KillerRed operatively fused to intracellular localization signals.

Also provided are vectors and expression cassettes comprising the nucleic acid of the invention. In addition, provided are cells, stable cell lines, transgenic animals and transgenic plants, including nucleic acids, vectors or expression cassettes of the invention.

In other embodiments, the functional fluorescent proteins of the invention are provided, which are encoded by the nucleic acids indicated above.

These protein and nucleotide compositions are used in many different applications and methods, in particular, in applications of inactivation of target proteins and targeted destruction of cells by irradiation with light of a certain wavelength. Finally, kits are provided for their use in such methods and applications.

Definitions

Various terms related to the biological molecules of the invention are used above and also in the description and in the claims.

As used here, the term "fluorescent protein" means a protein belonging to the family of GFP-like proteins, which contains the GFP domain and is capable of fluorescence; for example, it may exhibit low, medium, or intense fluorescence when irradiated with light at a suitable wavelength for excitation. The fluorescent property of these proteins is such a property, which is the result of the work of a chromophore formed by autocatalytic cyclization of three or more amino acid residues in a polypeptide chain. As such, the fluorescent proteins of the invention do not include proteins that exhibit fluorescence due to individual fluorescent residues, such as tryptophan, tyrosine and phenylalanine.

As used here, the term "avGFP" refers to the green fluorescent protein from Aequorea victoria jellyfish, including avGFP variants known in the art, designed to provide greater fluorescence or fluorescence in other color areas. The wild-type avGFP sequence has been disclosed in Prasher et al. (1992, Gene 111: 229-33).

The term “humanized” refers to a change in the nucleotide sequence of a fluorescent protein made to optimize the genetic code of codons for expression in mammalian cells (Yang et al., 1996, Nucleic Acids Research 24: 4592-4593).

As used here, the term "isolated" means a molecule or cell that is in an environment other than the environment in which the molecule or cell is in vivo.

As used here, the term "mutant" or "derivative" refers to a protein disclosed in the present invention, in which one or more amino acids are added and / or substituted and / or deleted (deleted) and / or inserted (inserted) at the N-terminus and / or C-terminus and / or within the native amino acid sequences of the proteins of the present invention. As used here, the term "mutant" refers to a nucleic acid molecule that encodes a mutant protein. In addition, the term “mutant” refers to any variant that is shorter or longer than a protein or nucleic acid.

As used herein, “homology” is a term used to describe the relationship of nucleotide or amino acid sequences to other nucleotide or amino acid sequences, which is determined by the degree of identity and / or similarity between said compared sequences.

[001] As used here, the amino acid or nucleotide sequences are "substantially similar" or "essentially the same as the reference sequence if the amino acid or nucleotide sequences have at least 85% identity with the specified sequence within the region selected for comparison. thus, substantially similar sequences include those that have, for example, at least 85% identity, at least 90% identity, at least 95% identity, or at least 96%, 97%, 98% or 99% identity Two sequences that are identical to one another are also essentially similar.

For the purposes of the present invention, the length of the compared sequences of fluorescent proteins corresponds to the length of the GFP domain. The GFP domain can be identified by analyzing the crystal structure of the fluorescent protein or by aligning the amino acid sequence of the protein with avGFP. The GFP domain can also be identified using protein domain analysis programs such as the Conserved Domain Database (CDD) and SMART (a Simple Modular Architecture Research Tool). It was shown that the barrel-forming domain includes amino acid residues corresponding to positions 6 through 229 of the avGFP sequence, where the positions of the corresponding amino acid residues are determined by aligning the amino acid sequence of the protein with avGFP (Fig. 1). For the KillerRed protein, the domain contains amino acid residues corresponding to positions 6 to 225. It was shown that amino acid fragments that are not part of the GFP domain may contain deletions, insertions, and substitutions of amino acid residues; however, there is no significant change in the spectral properties of the fluorescent protein (Shimozono et al., Biochemistry 2006, 45, 6267-6271; Crameri et al., Nat. Biotechnol. 1996, 14: 315-319).

The percentage of sequence identity is determined based on the reference sequence. Algorithms for sequence analysis are known in the art, such as BLAST described in Altschul et al., J. Mol. Biol., 215, pp. 403-10 (1990). For the purposes of the present invention, the comparison of nucleotide and amino acid sequences performed using the Blast software package provided by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/blast) using alignment breaks with standard parameters can be used to determine the level of identity and similarity between nucleotide sequences and amino acid sequences.

As used here, the term "similar fluorescent proteins" or "essentially similar fluorescent proteins" refers to fluorescent proteins that have GFP domains that are at least 85% identical, typically 90% or more identical, most often identical. at least 95% or more (for example, 96% or more, 97% or more, 98% or more, 99% or more, 100%).

In some embodiments, the term “similar fluorescent proteins” or “substantially similar fluorescent proteins” refers to fluorescent proteins that have whole protein amino acid sequences that are at least 85% identical, typically 90% or more identical. most often identical, at least 95% or more (for example, 96% or more, 97% or more, 98% or more, 99% or more, 100%).

As used here, the term "functional" means that the nucleotide or amino acid sequence can function for the specified test or task. The term "functional", used to describe the KillerRed and chimeric protein of the present invention, means that the protein has phototoxic properties.

As used herein, “biochemical properties” refer to protein folding (folding) and maturation rate, half-life, aggregation ability, oligomerization ability, pH and temperature stability, and other similar properties.

As used herein, “fluorescence properties” or “spectral properties” refer to the molar extinction coefficient at a suitable wavelength, the quantum yield of fluorescence, the shape of the fluorescence excitation spectrum or emission spectrum, the wavelength corresponding to the maximum fluorescence excitation, and the wavelength corresponding to the maximum emission, the ratio of the amplitude of the excitation of fluorescence at two different wavelengths, the ratio of the amplitude of the emission at two different wavelengths, the lifetime of the excited oyaniya and optical anisotropy. The measured difference can be defined as the amount of any quantitative fluorescence property, for example, the fluorescence intensity at a certain wavelength, or the integral fluorescence over the entire emission spectrum.

As used here, the terms “phototoxic properties” or “phototoxicity” refer to the ability of a protein to damage nearby molecules, such as nearby proteins, nucleic acids, and / or lipids, which in turn can cause cell death, cell division arrest or impaired cell differentiation and / or proliferation. A bacterial system can be used to compare phototoxic properties. For example, proteins can be expressed in bacterial cells (e.g., E. coli) by transfection with suitable expression vectors encoding these proteins under the control of a promoter that provides protein expression in these bacterial cells. Colonies are grown overnight at 37 ° C and then the plates are incubated at 4 ° C until the proteins mature completely. A single colony was selected for each protein, which was suspended in 0.1 ml of PBS buffer, after which half of the volume was irradiated with activating light of a certain intensity for 30 minutes, and half was left in the dark as a control. The resulting cell suspensions were plated on Petri dishes in various dilutions and, after overnight growth at 37 ° C, the number of colonies grown was counted. The stronger the phototoxic properties of the protein, the fewer colonies grow after irradiation.

Reference to the nucleotide sequence of the "coding" polypeptide means that the polypeptide is produced from the nucleotide sequence during translation and transcription of mRNA. In this case, both a coding strand identical to mRNA and usually used in the list of sequences can be indicated, as well as a complementary strand that is used as a template for transcription. As is obvious to any person skilled in the art, the term also includes any degenerate nucleotide sequences encoding the same amino acid sequence. Nucleotide sequences encoding a polypeptide include sequences containing introns.

The term “operably linked” or the like when describing chimeric proteins refers to polypeptide sequences that are in physical and functional relationship with one another. In the most preferred embodiments, the functions of the polypeptide components of the chimeric molecule are not changed compared with the functional properties of the isolated polypeptide components. For example, the protein of the present invention may be crosslinked with a fusion partner of interest. In this case, the chimeric protein retains the phototoxic properties of the protein of the invention, and the polypeptide of interest (for example, a signal of a certain intracellular localization) retains its original biological activity. For example, when a protein of the invention is crosslinked with an intracellular localization signal of interest, the chimeric protein retains fluorescence and phototoxicity, but is localized in a particular cellular compartment. As is obvious to any person skilled in the art, nucleotide sequences encoding a chimeric protein including “operably linked” components (proteins, polypeptides, linker sequences, protein domains, etc.) consist of fragments encoding these components, where these the fragments are covalently linked in such a way that a full-sized chimeric protein is produced during translation and transcription of the nucleotide sequence. In other words, the fragments are connected in such a way that there are no “failures” in the reading frame and stop codons at their junctions.

Nucleic acid molecules

The present invention provides isolated nucleic acid molecules encoding a KillerRed protein mutant having altered spectral characteristics. In particular, the present invention provides isolated nucleic acid molecules encoding a protein having an amino acid sequence selected from SEQ ID NO: 02, 04 and the like. Examples of nucleotide sequences provided by the invention are shown in SEQ ID NO: 01 and SEQ ID NO: 03.

As used herein, a nucleic acid molecule is a DNA molecule, such as a genomic DNA or cDNA molecule, or an RNA molecule, such as an mRNA molecule. As used here, the term "cDNA" refers to nucleic acids that possess the arrangement of sequence elements found in native mature types of mRNA, where sequence elements are exons and 5 ′ and 3 ′ non-coding regions.

[002] A nucleic acid molecule encoding a protein according to this invention can be synthesized from suitable nucleoside triphosphates. The method is well described in protocols known in the art. For example, the availability of amino acid sequence information (e.g., SEQ ID NO: 02 or SEQ ID NO: 04) or nucleotide sequence information (e.g., SEQ ID NO: 01 or SEQ ID NO: 03) makes it possible to produce isolated nucleic acid molecules of the present invention using oligonucleotide synthesis. In the case of amino acid sequence information, several nucleic acids that differ from each other due to the degeneracy of the genetic code can be synthesized. Methods for selecting codon variants for a desired host are well known in the art.

Synthetic oligonucleotides can be prepared using the phosphoramidite method, and the resulting constructs can be purified using methods well known in the art, such as high performance liquid chromatography (HPLC) or other methods as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY, and according to the instructions described in, for example, United States Dept. of HHS, National Institute of Health (NIH) Guidelines for Recombinant DNA Research. The long double-stranded DNA molecules of the present invention can be synthesized in the following stages: several smaller fragments with the necessary complementarity, which contain suitable ends capable of cohesion with the neighboring fragment, can be. Neighboring enzymes can be crosslinked using a DNA ligase or PCR based method.

[003] In some embodiments, the nucleic acid molecule of the present invention is a DNA (or cDNA) molecule containing an open reading frame that encodes a chimeric protein of the present invention and is capable of under suitable conditions (for example, expression of a protein in a host cell. The present invention is as follows it encompasses nucleic acids that are homologous, substantially similar, identical, or derived from nucleic acids encoding the proteins of the present invention, said nucleic acids being in a medium other than where they are located in vivo, e.g., they are isolated, presented in an increased amount, or are expressed in in vitro systems, or in cells or organisms other than those in which they are located in vivo.

Changes or differences in the nucleotide sequence between highly similar nucleotide sequences may represent nucleotide substitutions in the sequence that occur during normal replication or duplication. Other substitutions can be specifically calculated and inserted into the sequence for specific purposes, such as changing the codons of certain amino acids or the nucleotide sequence of a regulatory region. Such special substitutions can be made in vitro using various mutagenesis technologies or obtained in host organisms under specific breeding conditions that induce or select these changes. Such specially prepared sequence variants may be called “mutants” or “derivatives” of the original sequence.

The nucleotide sequences of the invention encode KillerRed protein mutants having a GFP domain that is at least 85% identical (more often at least 90% identical, typically at least 95% identical) to the KillerRed protein GFP domain.

For example, the specified GFP domain may be at least 96%, 97%, 98%, 99%, or 100% identical to the Giller domain of the KillerRed protein. Mutant or derivative nucleic acids can be obtained on a matrix nucleic acid selected from the above nucleic acids, by modifying, deletion or addition of one or more nucleotides in the matrix sequence or a combination thereof, to obtain a variant of the matrix nucleic acid. Modifications, additions or deletions can be performed by any method known in the art (see, for example, Gustin et al., Biotechniques (1993) 14: 22; Barany, Gene (1985) 37: 111-123; and Colicelli et al., Mol. Gen. Genet. (1985) 199: 537-539, Sambrook et al., Molecular Cloning: A Laboratory Manual, (1989), CSH Press, pp. 15.3-15.108), including error prone PCR ), shuffling, oligonucleotide-directed mutagenesis, assembly PCR, paired GSH mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive multiple mutagenesis, exponential multiple mutagenesis, site-specific mutagenesis, random mutagenesis, gene re assembly (gene reassembly), gene site-saturating mutagenesis (GSSM), artificial ligation rearrangement (SLR), or combinations thereof. Modifications, additions or deletions can also be performed by a method including recombination, recursive recombination of sequences, phosphotioate-modified DNA mutagenesis, mutagenesis on a uracil-containing matrix, double-pass mutagenesis, mismatch spot recovery mutagenesis, strain mutagenesis, recovery-deficient mutagenesis, radioactive mutagenesis, deletion mutagenesis, restriction-selective mutagenesis, restriction mutagenesis with purification, synthesis of artificial g new, multiple mutagenesis, creation of multiple chimeric nucleic acids, and combinations thereof. In some embodiments, the fluorescent proteins encoded by mutant or derivative nucleic acids have the same fluorescent or biochemical properties as wild-type fluorescent protein. In other embodiments, mutant or derivative nucleic acids encode fluorescent proteins with altered properties.

In addition, degenerate nucleic acid variants that encode the proteins of the present invention are also provided. Degenerate nucleic acid variants include substitutions of nucleic acid codons with other codons encoding the same amino acids. In particular, degenerate nucleic acid variants are created to increase expression in the host cell. In this embodiment, nucleic acid codons that are not preferred or less preferred in the genes of the host cell are replaced by codons that are abundantly represented in the coding sequences of the genes in the host cell, where said substituted codons encode the same amino acid. Of particular interest are humanized versions of the nucleic acids of the present invention. As used herein, the term “humanized” refers to substitutions made in a nucleic acid sequence to optimize codons for protein expression in mammalian (human) cells (Yang et al., Nucleic Acids Research (1996) 24: 4592-4593). See also US patent No. 5795737, which describes the humanization of proteins, and the disclosure of which is incorporated herein by reference. Examples of degenerate variants of interest are described in more detail in the experimental part below.

Nucleic acids encoding shortened and elongated variants of the proteins of the invention are also included in the scope of the invention. As used here, these protein variants contain amino acid sequences with altered C-, N-, or both ends. In extended embodiments, the C- or N-terminus of the protein may contain additional amino acid residues. In shortened versions, one or more (usually up to 11, more often up to 7 and mostly up to 5) amino acid residues can be removed from the sequence or replaced with any other amino acid residues. Such modifications do not essentially change the properties of the proteins, but can facilitate protein folding in the host cell, reduce the ability to aggregate, or modulate other biochemical properties of the proteins, for example, half-life. In some embodiments, these modifications do not alter the biochemical properties of the protein. All types of modifications and mutations mentioned above are carried out at the nucleic acid level.

Also provided are nucleic acids encoding chimeric proteins consisting of the protein of the present invention and a signal of a specific intracellular localization. Such chimeric proteins can be obtained by operatively combining a nucleic acid of the present invention encoding a KillerRed mutant and a nucleic acid encoding an intracellular localization signal. Methods for producing such nucleic acids are well known to those skilled in the art.

The claimed nucleic acids can be isolated and obtained essentially in purified form. Essentially purified form means that the nucleic acids are at least 50% pure, usually at least 90% pure and usually are "recombinant", that is, flanked by one or more nucleotides - with which it usually not bound on the chromosome found naturally in its natural host organism.

A vector and other nucleic acid constructs containing the claimed nucleic acids are also provided. Suitable vectors include viral and non-viral vectors, plasmids, cosmids, phages, etc., preferably plasmids, and are used for cloning, amplification, expression, transfer, etc., the nucleic acid sequence of the invention in a suitable host. The selection of a suitable vector is understandable to a person skilled in the art, and many such vectors are commercially available. To prepare the construct, a full-length nucleic acid or part thereof is usually inserted into the vector by attaching a DNA ligase to a restriction enzyme cleaved site in a vector. Alternatively, the desired nucleotide sequence can be inserted by in vivo homologous recombination, usually by attaching homologous regions to a vector on the flanks of the desired nucleotide sequence. Homologous regions are added by ligation of oligonucleotides or by polymerase chain reaction using primers including, for example, both homologous regions and part of the desired nucleotide sequence.

Also provided are expression cassettes or systems used inter alia to produce the claimed fluorescent proteins or chimeric proteins based on them or to replicate the claimed nucleic acid molecules. The expression cassette may exist as an extrachromosomal element or may be incorporated into the genome of a cell as a result of introducing said expression cassette into the cell. For expression, the gene product encoded by the nucleic acid of the invention is expressed in any convenient expression system, including, for example, bacterial systems, yeast, insects, amphibians, or mammalian cells. In an expression vector, said nucleic acid is operably linked to a regulatory sequence that may include promoters, enhancers, terminators, operators, repressors and inducers. Methods for making expression cassettes or systems for expressing a desired product are known to those skilled in the art.

Cell lines that stably express the proteins of the present invention can be selected by methods known in the art (e.g., co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin, which makes it possible to identify and isolate transfected cells that contain the gene included in the genome).

The above expression systems can be used in prokaryotic or eukaryotic hosts. To obtain the protein, host cells, such as E. coli, B. subtilis, S. cerevisiae, insect cells in combination with baculovirus vectors, or cells of a higher organism such as vertebrates, for example, COS 7 cells, HEK 293, CHO, can be used Xenopus oocytes, etc.

If any of the aforementioned host cells or other suitable host cells or organisms are used to replicate and / or express the nucleic acids of the invention, the resulting replicated nucleic acid, expressed protein or polypeptide is within the scope of the invention as a product of the host cell or organism. The product may be isolated by a suitable method known in the art.

The nucleic acid molecules of the invention can be used to produce protein (gene expression) in a host cell. A method in which cells are examined for the presence of specific nucleotide sequences, such as genomic DNA or RNA, is well established in the art. Briefly, DNA or mRNA is isolated from a cell sample. mRNA can be amplified by RT-GSR, using reverse transcriptase to form a complementary DNA strand, followed by amplification by polymerase chain reaction using primers specific for the declared DNA sequences. Alternatively, the mRNA sample is separated by gel electrophoresis, transferred to a suitable carrier, for example, nitrocellulose, nylon, etc., and then tested with a fragment of the claimed DNA as a sample. Other methods may also be used, such as oligonucleotide crosslinking assays, in situ hybridization, and hybridization with DNA probes immobilized on a solid chip. Detection of mRNA hybridizing with the claimed sequence indicates gene expression in the sample.

Squirrels

The nucleic acids of the invention encode KillerRed protein mutants. In preferred embodiments, said proteins comprise a Y68W substitution. The specified amino acid is in the position that corresponds to Y66 in avGFP and is part of the chromophore. In preferred embodiments, these proteins also contain several additional substitutions selected from the group V46A, A74T, D115G, N147S, F179L, Y223H, E238Q. For example, in some embodiments, said proteins comprise said combinations of amino acid substitutions V46A + Y68W + A74T + D115G + N147S + Y223H + E238Q and Y68W + D115G + N147S + F179L + Y223H + E238Q. Examples of amino acid sequences of the functional proteins of the invention are given in SEQ ID NO: 02 and SEQ ID NO: 04. The proteins of the invention are GFP-like proteins substantially similar to the KillerRed protein and having a GFP domain whose amino acid sequence has at least 85% identity (e.g., at least 90% identity, at least 95% identity, or more, e.g. 96%, 97%, 98% or 100%) with the Giller domain of the KillerRed protein.

Prior to the transition to an activated state, the proteins of this invention have low toxicity in host cells.

Prior to the transition to the activated state, the proteins of this invention are capable of detectable fluorescence. In some embodiments, an unactivated (prior to activation of phototoxic properties) protein has a maximum fluorescence excitation in the range from about 400 nm to 550 nm, usually from about 420 nm to 500 nm. In some embodiments, the protein of this invention has an emission maximum in the range of from about 450 to 600 nm, usually from about 460 nm to 590 nm, and most often from about 470 to 560 nm.

The claimed protein usually has a maximum extinction coefficient in the range of from about 30,000 to 150,000 and usually from about 60,000 to 120,000.

The fluorescence of the protein of this invention can be detected by conventional methods (e.g., visual screening, spectrophotometry, spectrofluorimetry, fluorescence microscopy, FACS devices, etc.)

The protein of this invention goes into an activated state under the influence of light of a certain wavelength (activating light). In some embodiments, the wavelength of the activating light is in the range of about 350 to 550 nm, usually about 400 to 500 nm, and most often about 430 to 480 nm, for example, at 440-470 nm.

In the activated state, the protein of this invention has pronounced toxicity to cells. In some embodiments, in the activated state, the protein of this invention is capable of producing reactive oxygen species. In some embodiments, in the activated state, the protein of this invention changes spectral characteristics, for example, loses its fluorescence ability or changes the spectrum of excitation and / or fluorescence emission.

In some embodiments, the protein of this invention matures rapidly after expression in the host cell. By rapid maturation, it is understood that the protein reaches its tertiary structure, which provides its spectral and phototoxic properties, in a short period of time. In these embodiments, the protein is laid over a period of time that generally does not exceed about 60 hours, usually does not exceed about 48 hours, and more often does not exceed 24 hours (for example, a half-period of laying may be 4-6 hours). Specific proteins of interest include the KillerOrange (Seq ID NO: 1, 2) and KillerGreen (SEQ ID No: 03 and 04) proteins, described in detail in the Examples section below, and their functional mutants.

Mutants may retain the properties of the parent protein or may have biological properties different from the forms of the parent protein. The term "biological properties" of the proteins of the present invention includes, but is not limited to, spectral properties such as maximum fluorescence excitation, maximum emission, maximum extinction coefficient, brightness (for example, compared with reference proteins), photo stability and the like; biochemical properties such as in vivo and / or in vitro stability (for example, half-life); maturation rate, a tendency to aggregation and a tendency to oligomerization, and other similar properties (compared to a reference protein). Mutations include single amino acid substitutions, deletions and insertions of one or more amino acids, shortening or lengthening of the N-terminus, shortening or lengthening of the C-terminus, and the like.

Mutants can be obtained using standard methods of molecular biology, as described in detail in the section "nucleic acid molecules" above. The examples provide general techniques and the use of standard methods, so that those skilled in the art can easily obtain a large number of additional mutants and check whether the biological (e.g. biochemical, spectral, etc.) property has been changed. For example, fluorescence intensity can be measured using a spectrofluorimeter at various excitation wavelengths. Also provided are proteins that are substantially similar to the above specific proteins, where substantially similar means that these proteins have an amino acid sequence identical to the sequence of the original protein, at least 85% identity, usually at least 90% and more often, at least 95% (for example, 95% and higher; 96% and higher, 97% and higher; 98% and higher: 99% and higher or 100% sequence identity).

The proteins of the invention are present in an environment different from their natural environment; for example, they are recombinant. The proteins of the invention may be in an isolated state, which means that the proteins are substantially free of other proteins and other biological molecules present in the natural environment, such as oligosaccharides, nucleic acids and fragments thereof and the like, where the term "essentially free "in this case means that less than 70%, usually less than 60% and more often less than 50% of the composition containing the isolated protein, are some other biological molecules than those found in nature. In some embodiments, the proteins are present in a substantially purified form, where “substantially purified form” means purified at least 95%, usually at least 97%, and more often at least 99%.

The claimed proteins can be obtained artificially, for example, by expression of a recombinant nucleic acid encoding a sequence of a protein of interest in an appropriate host, as described above. For protein purification, any conventional methodology may be employed where suitable protein purification methods are described in Guide to Protein Purification, (Deuthser ed., Academic Press, 1990). For example, a lysate can be prepared from a source and purified using HPLC, size exclusion chromatography, gel electrophoresis, affinity chromatography, and the like.

Also provided are fusion proteins, including the proteins of the present invention, fused to a sequence of intracellular localization (for example, with a localization signal in the nucleus, in peroxisomes, Golgi apparatus, mitochondria, etc.). A polypeptide providing a certain intracellular localization can be operatively attached to the N-terminus and / or C-terminus of the biosensor. Signals of intracellular localization are well known to specialists in this field and are described, for example, by Nakai K. (Advances in Protein Chemistry, Vol. 54, 2000, p. 277-344).

Transformants

Nucleic acids of the present invention can be used to obtain transformants, including transgenic organisms or site-specific gene changes in cell lines. The transgenic cells of the invention comprise one or more nucleic acids of the invention as a transgene. For the purposes of the invention, any suitable host cell can be used, including prokaryotic (e.g., Escherichia coli, Streptomyces sp., Bacillus subtilis, Lactobacillus acidophilus, etc.) or eukaryotic host cells. A transgenic organism of the invention may be a prokaryotic or eukaryotic organism, including bacteria, cyanobacteria, fungi, plants, and animals in which one or more cells of the body contain a heterogeneous nucleic acid of the invention, introduced by human intervention, by methods such as technologies transgenosis, which are known in the art. The isolated nucleic acid of the invention can be introduced into the host by methods known in the art, for example, infection, transfection, transformation or trans-conjugation. Methods for transferring a nucleic acid molecule (i.e., DNA) into such organisms are widely known and provided in references such as Sambrook et al. (Molecular Cloning: A Laboratory Manual, 3nd Ed., (2001) Cold Spring Harbor Press, Cold Spring Harbor, NY).

In one embodiment, the transgenic organism may be a prokaryotic organism. Methods for transforming prokaryotic hosts are well described in the art (e.g., see Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edition (1989) Cold Spring Harbor Laboratory Press and Ausubel et al., Current Protocols in Molecular Biology (1995) John Wiley & Sons, Inc).

In another embodiment, the transgenic organisms may be fungi, for example, yeast. Yeast is widely used as a carrier for heterogeneous gene expression (e.g. see Goodey et al Yeast biotechnology, DR Berry et al, eds, (1987) Allen and Unwin, London, pp 401-429) and King et al Molecular and Cell Biology of Yeasts EF Walton and GT Yarronton, eds, Blackie, Glasgow (1989) pp 107-133). Several types of yeast vectors are available, including integrative vectors that require recombination with the host genome to support them, and autonomously replicating plasmid vectors.

Another host organism is an animal. Transgenic animals can be obtained by transgenosis methods known in the art and are provided in references such as Pinkert, Transgenic Animal Technology: a Laboratory Handbook, 2nd edition (2203) San Diego: Academic Press; Gersenstein and Vintersten, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed, (2002) Nagy A. (Ed), Cold Spring Harbor Laboratory; Blau et al., Laboratory Animal Medicine, 2nd Ed., (2002) Fox J.G., Anderson L.C., Loew F.M., Quimby F.W. (Eds), American Medical Association, American Psychological Association; Gene Targeting: A Practical Approach by Alexandra L. Joyner (Ed.) Oxford University Press; 2nd edition (2000). For example, transgenic animals can be obtained by homologous recombination, where the endogenous locus changes. Alternatively, the nucleic acid construct is randomly incorporated into the genome. Vectors for sustainable incorporation include plasmids, retroviruses and other animal viruses, YAC, and the like.

Nucleic acids can be introduced into a cell directly or indirectly, by introducing into a cell precursor, by intentional genetic manipulation, such as microinjection or infection with a recombinant virus or recombinant viral vector and the like.

The term “genetic manipulation” does not include classical cross-fertilization or in vitro fertilization, but is preferably directed to the introduction of recombinant nucleic acid molecules. These nucleic acid molecules can be included in the chromosome or they can be extrachromosomal replicating DNA.

DNA constructs for homologous recombination will contain at least a portion of the nucleic acid of the present invention, where the gene has the desired genetic modification (s) and includes homology regions with a target locus. DNA constructs for arbitrary incorporation do not necessarily contain a region of homology with a recombination mediator. Markers for positive and negative selection can easily be included. Methods for producing cells having targeted gene modifications through a homologous combination are known in the art. For various methods for transfecting mammalian cells, see Keown et al., Meth. Enzymol. (1990) 185: 527-537.

For embryonic stem (ES) cells, ES cell lines can be used, or embryonic cells can be obtained directly from a host, such as a mouse, rat, guinea pig, etc. Such cells are grown on a suitable fibroblast-feeding layer or in the presence of leukemia inhibition factor (LIF). Transformed ES or embryonic cells can be used to produce transgenic animals using a suitable method described in this field.

Transgenic animals can be any non-human animals, including a non-human mammal (e.g., mouse, rat), bird or amphibian, etc., and are used in functional research, drug screening, and the like. Representative examples of the use of transgenic animals include those described below.

Transgenic plants can also be obtained. Methods for producing transgenic plant cells and plants are described in US patent No. 5767367; 5,750,870; 5,739,409; 5,689,049; 5,689,045; 5,674,731; 5656466; 5,633,155; 5,629,470; 5,595,896; 5,576,198; 5,538,879; 5,484,956; disclosures of which are incorporated herein by reference. Methods for producing transgenic plants are also discussed in Plant Biochemistry and Molecular Biology (eds. Lea and Leegood, John Wiley & Sons) (1993) pp. 275-295 and Plant Biotechnology and Transgenic Plants (eds. Oksman-Caldentey and Barz), ( 2002) 719 p.

For example, embryogenic explants containing somatic cells can be used to produce a transgenic host. After collecting cells or tissues, exogenous DNA of interest is introduced into the plant cells, and a number of different methods are known for such introduction. When isolated protoplasts are present, it is possible to introduce through DNA-mediated gene transfer protocols, including incubating protoplasts with purified DNA, such as a plasmid containing the desired exogenous sequence of interest in the presence of polyvalent cations (e.g., PEG or PLO); or electroporation of protoplasts in the presence of isolated DNA, including the target exogenous sequence. Protoplasts that successfully incorporate exogenous DNA are then selected, grown in callus, and ultimately in a transgenic plant in contact with suitable amounts and ratios of stimulatory factors such as auxins and cytokines. Other suitable plant production methods may be used, such as the use of a "gene gun", or Agrobacterium-mediated transformation, which are available to those skilled in the art.

Application methods

The proteins of the present invention are genetically encoded fluorescent proteins - photosensitizers that exhibit phototoxic properties when exposed to light, more than 1000 times the toxicity of other known green and red fluorescent proteins, including avGFP. They can be used to selectively inactivate proteins of interest and selectively suppress cell division or kill cells with targeted delivery using viral vectors and other methods.

In order to selectively inactivate the proteins of interest, a nucleic acid encoding the protein of this invention, operably linked to the protein of interest (fusion partner), must be obtained. Obtaining such designs is obvious to any person skilled in the art. For example, parts of a nucleic acid encoding different elements can be inserted into the polylinker of the vector so that there are no stop codons between the different parts in the reading frame and there are no reading frame failures. Alternatively, the desired nucleotide sequence can be assembled from the fragments using DNA ligase or HRPG with primers containing parts complementary to the terminal sequences of the joined fragments.

The resulting construct must be integrated into a vector that provides for the temporary or permanent expression of this nucleic acid in the host cells. The vector may contain elements that provide targeted delivery of the structure to the cells of interest, or is part of the particles that provide targeted delivery. After transfection of the cells with the vector and after the time necessary for the expression product (chimeric protein) to accumulate in the cells, the protein of interest can be inactivated by irradiating the cells or cell compartments with activating light.

To carry out selective killing of cells, a nucleic acid encoding a protein of the invention or a nucleic acid encoding a protein of the invention operably linked to a polypeptide capable of delivering a chimeric protein to specific cellular compartments (cell nuclei, lysosomes, mitochondria, plasma membrane) must be obtained . The resulting construct must be introduced into the cell (for example, as part of an expression vector). After the time required for the expression product to develop in the cells, the cells are irradiated with activating light of sufficient intensity to cause cell death by apoptosis or necrosis. Examples of such use are described in the experimental part below.

Sets

Kits for use in implementing one or more of the above applications are also provided in accordance with the invention. In preferred embodiments, the kits typically comprise a nucleic acid encoding a protein of the invention, preferably with elements for expression of the protein in the host cell. For example, kits may include a vector comprising a nucleic acid encoding a protein of the invention. The components of the kits are usually present in an appropriate storage medium, such as an aqueous or buffer solution, usually in an appropriate container. In separate embodiments, the kit includes many different vectors, each of which encodes the claimed protein, where the vectors are designed for expression in different environments and / or under different conditions. For example, for constitutive expression, the kit includes a vector having a strong promoter for expression in mammalian cells, and a vector with a weakened promoter. Multiple cloned site vectors for selectively inserting a promoter and / or vectors for expression in various organisms may also be included.

In addition to the components described above, the claimed kits will further include instructions for implementing the claimed methods. These instructions may be present in the claimed sets in various forms (for example, in print or on electronic media in the form of a text and / or graphic file) in an amount of one or more.

The following examples are provided as illustrative but not restrictive.

EXAMPLES

Example 1

KillerRed protein mutagenesis.

For targeted mutagenesis, nucleic acid encoding the KillerRed protein from the commercially available pKillerRed-C vector (Eurogen, Russia) was used. To obtain constructs containing the desired mutations, site-directed mutagenesis was performed using "overlap extention" of PCR products as described by Wurch et al., Methods in Molecular Biology. 12 (9), 653-657 (2004). For PCR, the Tersus PCR kit (Eurogen) PCR kit was used, according to the manufacturer's recommendations.

After amplification, the obtained fragments were purified by electrophoresis in 1% agarose gel and further extraction. DNA fragments containing the desired mutation isolated from the gel were combined into a complete construct containing point exchange by overlap PCR method (denaturation 95 ° С - 12 sec; annealing 55 ° С - 2 min; elongation 72 ° С - 1 min, 8 PCR cycles). This method implies that, as a matrix and seed, the fragments present in the reaction mixture with complementary regions are used.

Further, the whole structure obtained in the previous step was amplified by standard PCR method with the addition of primers complementary to the ends of the amplified KillerRed fragment.

The obtained nucleic acids were cloned into the pQE30 vector according to the standard protocol, the correspondence of the insert structure was checked by sequencing using standard plasmid primers.

During targeted mutagenesis, tyromine fromromophore was replaced at the 68th position of the KillerRed amino acid sequence with tryptophan (Y68W), phenylalanine (Y68F), histidine (Y68H). Of the obtained mutants, only the mutant containing the Y68W substitution retained fluorescence ability. Mutants with substitutions Y68F and Y68H did not exhibit visible fluorescence even after several rounds of random mutagenesis.

The KilerRed mutant, containing the Y68W substitution and exhibiting weak orange fluorescence, was randomly mutagenized using a random mutagenesis kit (Clontech, USA) using the methodology used by the manufacturer. After a round of random mutagenesis, clones were selected that possessed the brightest fluorescence and its fastest appearance. As a result, two clones were selected, one of which contained substitutions A33T, Y68W, F88L and E238Q, and the second - Y68W, D115G, Y223H and E238Q.

A mixture of nucleic acids encoding the selected clones was used for the second round of random mutagenesis, which was carried out as described above. Selected clones with the greatest shift of fluorescence in the green region of the spectrum and / or increased brightness of fluorescence.

Six mutant clones were selected that were tested for phototoxicity in the bacterial system when exposed to activating light.

To compare the phototoxicity of the KillerRed mutants, nucleic acids encoding the mutant variants were cloned into the pOEZO vector (Qiagen) and transformed into E. coli competent cells. The colonies were grown overnight at 37 ° C and then the plates were incubated for 3 days at 4 ° C for complete maturation of the proteins. One colony of each mutant was shaken in 0.1 ml of PBS buffer, after which half of the volume was irradiated with white light at an intensity of 1 W / cm ^{2 for} 30 min, and half was left in the dark as a control. The resulting cell suspensions were plated on Petri dishes in various dilutions and, after overnight growth at 37 ° C, the number of colonies grown was counted. Comparison of the number of colonies in the irradiated and unirradiated samples allowed us to estimate the number of cells that died from exposure to light and to select two KillerRed mutants that are highly toxic to bacterial cells. Using Sanger sequencing, the nucleotide sequences of these mutants named KillerOrange (SEQ ID NO: 01) and KillerGreen (SEQ ID NO: 03) were determined.

The amino acid sequences of these mutants are shown, respectively, in SEQ ID NO: 02 and SEQ ID NO: 04. The KillerOrange protein contains substitutions Y68W, D115G, N147S, F179L, Y223H and E238Q, and the KillerGreen protein contains V46A, Y68W, A74T, D115G, N147S , Y223H and E238Q. The KillerOrange and KillerGreen proteins were isolated using metal affinity chromatography. E. coli cells expressing these proteins were pelleted for 20 minutes at 4000 rpm in the cold (4 C0) and suspended in buffer I (125 mM NaCl, 40 mM Tris-HCl, 5 mM 2-mercaptoethanol, pH 7.5) at the rate of 1.5 ml of buffer I per precipitate obtained from 50 ml of cells in liquid LB. Then the cells were destroyed by ultrasound using a Sonic Dismembrator (Fisher Scientific), power 10 W, centrifuged for 10 min at a speed of 12000 rpm and a temperature of 4 C0. The supernatant was transferred into tubes with TALON metal-affinity resin (Clontech) pre-equilibrated with buffer I at the rate of 500 μl of resin per 1000-1200 μl of supernatant. The tubes were placed in a shaker (speed 40 rpm) and incubated for 20-25 minutes. After that, the resin was washed 3-4 times with 1000 μl of buffer I. Buffer II was used for elution (125 mM NaCl, 40 mM Tris-HCl, 5 mM 2-mercaptoethanol, 200 mM imidazole, pH 7.5). Elution was carried out for 20-25 min in a shaker, protein with 500 μl of resin was eluted with 300-350 μl of buffer II. All manipulations were carried out in the cold, all buffers and resin were pre-cooled. The fluorescence spectra of the isolated KillerOrange and KillerGreen proteins were obtained using a Varian Sagu Eclipse Fluorescence Spectrophotometer spectrofluorimeter. Protein activation was performed using blue light with a wavelength of 440-470 nm and an intensity of 1 W / cm ² using an Olympus SZX12 fluorescence binocular

It was shown that the KillerOrange protein prior to activation of its phototoxic properties has fluorescence excitation maxima at 510 and 490 nm with a shoulder at 463 nm and emission maximum at 551 nm with a weak shoulder at 585 nm (Fig. 2), and KillerGreen protein has fluorescence excitation maxima at 452 and 508 nm and the maximum emission at 483-507 nm (figure 3).

After activation, the KillerOrange protein has a maximum fluorescence excitation at 460 nm (shoulders at 490 and 510 nm) and a maximum emission at 547 nm. In this case, there is a general decrease in the fluorescence level of the solution containing KillerOrange.

After activation of the KillerGreen protein, a decrease in the fluorescence level of the solution was also observed, however, no changes in the position of the excitation maxima and fluorescence emission were detected.

Example 2

Comparison of the photoxic properties of KillerOrange, KillerGreen and KillerRed. Nucleic acids encoding the proteins KillerOrange, KillerGreen, KillerRed were obtained as described above in Example 1. A nucleic acid encoding the non-phototoxic protein DsRed2 (SEQ ID NO: 10, 11) was obtained from a commercially available vector (Clontech). Nucleic acids were cloned into the pQE30 vector (Qiagen) and transformed into competent E. coli cells. The colonies were grown overnight at 37 ° C and then the plates were incubated for 3 days at 4 ° C for complete maturation of the proteins.

One colony of each mutant was shaken in 0.15 ml of PBS buffer, after which one third of the suspension volume was irradiated for 30 min with blue light (wavelength 440-470 nm), one third with green light (wavelength 540-580 nm), and the residue was left in the dark as a control. After this, several dilutions of cell suspensions were prepared and plated on Petri dishes. After overnight growth at 37 ° C, the number of colonies grown was counted. The measurement results are presented in the table.

The results of measuring the phototoxicity of fluorescent proteins when activated by blue and green light.

Table

The data obtained indicate a high phototoxicity of the KillerOrange and KillerGreen proteins when activated by blue light and a level of toxicity comparable to the non-phototoxic protein DsRed2 when activated by green light. The overall level of phototoxicity of KillerOrange and KillerGreen when activated by blue light is close to the level of phototoxicity of KillerRed protein when activated by green light.

Example 3

Expression of KillerOrange and KillerGreen in mammalian cells.

To express KillerOrange and KillerGreen in mammalian cells, the nucleic acids encoding these proteins were cloned into the commercially available vectors pKillerRed-C, pKillerRed-dMito and pKillerRed-mem (Eurogen) instead of the KillerRed sequence. In the pKillerRed-dMito vector, these proteins turned out to be operatively fused with the localization signal in the mitochondria, and in the pKillerRed-mem vector, with the localization signal on the plasma membrane.

The resulting constructs were used for transient transfection of mammalian cells (HEK293, HeLa, HeLa Kyoto lines). Three days after transfection, fluorescence microscopy of mammalian cells showed the appearance of fluorescence in the expected cell compartments: in the cytoplasm in the case of the pKillerRed-C vector, on the membrane in the case of the pKillerRed-dMito vector and in mitochondria in the case of the pKillerRed-dMito vector. Counting the number of mitoses for a certain period of time (using fluorescence microscopy) showed that transfected cells divide at approximately the same frequency as non-transfected ones. Thus, the tested proteins did not show significant cytotoxicity (in the unactivated state).

Example 4

Preparation of stably transfected mammalian cell lines.

HeLa Kyoto cell lines stably expressing the KillerOrange and KillerGreen proteins were obtained by lentiviral transduction. For this, the open reading frames KillerOrange and KillerGreen were cloned into the lentiviral vector pRRLSIN.EFl.WPRE. Transduction was performed according to standard protocols. For each protein, a population of cells with the highest fluorescence intensity was selected using a MoFlo flow-through fluorescence cell sorter (Dako). The resulting lines were stably expressed by KillerOrange and KillerGreen in the cytoplasm. Analysis of the distribution of cells by the stages of the cell cycle (G1, S and G2 / M) using DNA staining with propidium iodide and flow cytofluorimetry showed a normal number of dividing cells in the obtained cell lines, similar to that in the original HeLa Kyoto line.

Example 5

Phototoxicity analysis of KillerOrange and KillerGreen in mammalian cells.

The phototoxic effect of KillerOrange and KillerGreen was studied using fluorescence microscopy. In the first series of experiments, HeLa Kyoto cells temporarily expressing KillerOrange and KillerGreen in different cell localization and obtained as described in Example 3 were irradiated with green or blue light using a fluorescence microscope (540-580 nm, 0.5 W / cm ² , 2 min and 450-490 nm, 0.5 W / cm ² , 2 min, respectively). Then continued monitoring of the irradiated cells using a fluorescence microscope for 24 hours. In the case of blue light irradiation, cells with localization of the tested proteins on the plasma membrane and mitochondria showed signs of cell death (detachment from the substrate, compression of the cytoplasm, blebing (blistering) and destruction of the membrane). The phototoxic effect on cells with localization of proteins in the cytoplasm was less pronounced and was manifested only in individual cells. In the case of green light exposure, a pronounced phototoxic effect was not observed.

Claims (6)

1. A nucleic acid encoding a functional phototoxic fluorescent protein, the amino acid sequence of which is selected from the group of SEQ ID NO: 02 and SEQ ID NO: 04. 2. The expression cassette, which is integrated into the genome of the cell or introduced into the cell as an extrachromosomal element provides expression of a phototoxic fluorescent protein and contains (a) a transcription initiation region that is functional in the host cell; (b) the nucleic acid according to claim 1; and (c) a transcription termination region functional in the host cell. 3. An expression cassette that, when integrated into the cell genome or introduced into the cell as an extrachromosomal element, provides the expression of a phototoxic fluorescent protein in a particular cell compartment and contains the nucleic acid of claim 1, operatively fused to a nucleic acid encoding an intracellular localization signal and regions initiation and termination of transcription, functional in the host cell. 4. A cell that is not a human embryonic cell and contains the expression cassette according to claim 2 as part of an extrachromosomal element or integrated into the cell genome as a result of the introduction of the indicated cassette into the specified cell, where the specified cell expresses a phototoxic fluorescent protein. 5. A cell that is not a human embryonic cell and contains the expression cassette according to claim 3 as part of an extrachromosomal element or integrated into the cell genome as a result of the introduction of the indicated cassette into the specified cell, where the specified cell expresses a phototoxic fluorescent protein in a particular cell compartment. 6. Isolated functional phototoxic fluorescent protein having an amino acid sequence selected from the group of SEQ ID NO: 02 and SEQ ID NO: 04.

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