JP5265491B2 - Monomer and dimer fluorescent protein variants and methods of making the variants - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescent protein variant. <P>SOLUTION: A polynucleotide sequence encodes a Discosoma red-colored fluorescent protein variant (DsRed) having low oligomerization tendency, comprising &ge;1 amino acid substitution in the AB interface, AC interface or both of the AB and AC interfaces of a wild type DsRed amino acid sequence, reduces the tendency for forming the tetramer of the DsRed variant by the amino acid substitution, and shows the fluorescence having at least one detectable red-colored wavelength by the above variant. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  This patent application is filed on US patent application Ser. No. 10 / 121,258, filed Apr. 10, 2002, “Fluorescent Protein Variants and Methods for Making Same,” and July 2002. No. 10 / 209,208, filed on May 29th, claiming the priority of “Monomeric and Dimeric Fluorescent Protein Variants and Methods for Making Same” All of which are incorporated herein by reference.

  The present invention relates generally to fluorescent protein variants, and more specifically to the anthozoan fluorescent protein variant, which has a low tendency to oligomerize and forms a monomer and / or dimer structure . The invention also relates to methods of making and using such fluorescent protein monomers and dimers. In particular, the present invention is generally a red fluorescent protein (RFP) variant, more specifically an anthozoan fluorescent protein (AnFP) variant, having such an amino acid modification. The RFP mutants described above provide for more efficient maturation than the corresponding wild-type protein or other RFP mutant from which the body was derived. The invention further relates to RFP variants that have a low tendency to tetramerize, i.e., mainly form monomeric and / or dimeric structures. The invention also relates to methods of making and using such RFP variants.

  The identification and isolation of fluorescent proteins in various organisms (including marine organisms) provides a useful tool for molecular biology. For example, Aequorea victoria green fluorescent protein (GFP) examines a variety of cellular processes, including regulation of gene expression, localization and interaction of cellular proteins, pH of intracellular compartments, and enzyme activity Therefore, it has become a commonly used reporter molecule.

  Due to the usefulness of Aequorea GFP, a number of other fluorescent proteins have been identified in an attempt to obtain proteins with various useful fluorescent properties. In addition, spectral variants of Aequorea GFP have been generated, thus obtaining proteins that are excited or fluorescent at different wavelengths, at different times, and under different conditions. The red fluorescent protein from the Discosoma coral (named dsRed or drFP583) is of great interest as it is identified and cloned to be able to fluoresce at red wavelengths.

  DsRed (Non-Patent Document 1) from Discosoma is a significant spectroscopic companion or substitution of green fluorescent protein (GFP) from Aequorea jellyfish, which is significant in biotechnology and cell biology Hope is given (Non-Patent Document 2). GFP and its blue, cyan, and yellow variants are extensively used as genetically encoded indicators to track gene expression and protein localization and as donor / acceptor pairs for fluorescence resonance energy transfer (FRET) Applications have been found. Extending the available color spectrum to the red wavelength can provide a clear new label for multicolor tracking of fusion proteins and is currently preferred when used with GFP (or a suitable variant). It must be possible to provide a new FRET donor / acceptor pair that is superior to the cyan / yellow pair used (Non-Patent Document 3).

  One problem associated with the use of DsRed as a fluorescent reporter is its slow and inefficient chromophore maturation. Most of the previous attempts to improve the speed and / or extent of DsRed maturity (Non-Patent Document 4 and Non-Patent Document 5) provide modest improvements, including commercial DsRed2 (CLONTECH, Palo Alto, CA). I just did it. Recently, an artificially produced mutant of DsRed known as T1 (shown in FIG. 2A) has become available, effectively solving the problem of slow maturation (Non-Patent Document 6). However, this mutant still has the problem of incomplete maturation, and thus, like DsRed, a significant fraction of the protein remains in the mature tetramer as a green fluorescent intermediate.

  All of the coelenterate fluorescent proteins that have been cloned so far display a type of quaternary structure, such as the weak dimerization tendency of Aequorea green fluorescent protein (GFP), the reluctance of Renilla GFP. Examples include dimerization (obligate dimerization) and obligate tetramerization of Discosoma coral (Discosoma) DsRed (Non-patent document 7 and Non-patent document 8). The weak dimerization of Aequorea GFP does not interfere with its acceptance as an essential tool in cell biology, whereas the obligatory tetramerization of DsRed is a robust tool that can be generally applied from scientific interests. Development (most notably as a fusion tag encoded by genetic manipulation) has been greatly hindered.

DsRed tetramerization is an obstacle for researchers trying to image the intracellular localization of red fluorescent chimeras, because how much fusion of tetramerized DsRed and the target protein is to the localization and function of the latter This is because there is a doubt whether it will have an effect. Further, in some cases, a result may be due to, for example, a specific interaction of two proteins under study, or a recognized interaction linked to each of the two proteins under study. It can be difficult to ascertain whether this is an artifact caused by protein oligomerization. Multiple published reports (see, for example, Non-Patent Document 3 and Non-Patent Document 9) and numerous unpublished research case communications show that DsRed chimeras form intracellular aggregates and their biological activity. It states that it was lost. DsRed also suffers from slow and incomplete maturity (Non-Patent Document 7).
One study to overcome these shortcomings has continued research on DsRed congeners in corals and sea anemones; some studies have obtained multiple red-shift proteins (10) and 11). ). However, the basic problem of tetramerization has not been overcome. The only publicly available advancement to reduce the oligomeric state of red fluorescent protein is the artificially created DsRed homologue, marketed as HcRed1 (CLONTECH), which is a single interface mutation. It is converted into a dimer (Non-patent Document 12). HcRed1 has the additional advantage that it is 35 nm red shifted from DsRed, but its advantages are limited by the low extinction coefficient (20,000 M-1 cm-1) and quantum yield (0.015) (Non-patent Document 13). Is a problematic protein for use in

  Methionine is found at position 66 of the tetrameric non-fluorescent chromoprotein from Anemonia sulcata that has been converted to a fluorescent protein through the introduction of two mutations (Non-Patent Document 11). The introduction of methionine at position 66 clearly improves the fluorescence properties of both green (zFP506) and cyan (amFP486) tetramer fluorescent proteins, but the details have not been published (Non-Patent Document 14).

  Similarly, most conventional attempts to improve the speed and / or extent of DsRed maturation (Non-Patent Document 4 and Non-Patent Document 5) are modest, including commercially available DsRed2 (CLONTECH, Palo Alto, CA). It just provided a nice improvement. Recently, an artificially produced mutant of DsRed known as T1 (shown in FIG. 2A) has become available, effectively solving the problem of slow maturation (Non-Patent Document 6). However, this mutant still has the problem of incomplete maturation, and thus, like DsRed, a significant fraction of the protein remains in the mature tetramer as a green fluorescent intermediate.

  Accordingly, there is a need in the art for the development of red fluorescent polypeptides that can be utilized for scientific applications without technical limitations due to oligomerization, particularly tetramerization and inefficient and slow chromophore maturation. There is a need for methods of making fluorescent proteins that are less prone to oligomerization, particularly tetramerization. There is a need for a method of making red fluorescent protein (RFP) that exhibits more efficient chromophore maturation than wild-type RFP or other RFP variants. Furthermore, there is a need for RFP that has a low tendency to oligomerize such as tetramerization. Most importantly, there is a need for RFP variants that have improved maturation efficiency and exhibit useful fluorescence in the monomeric state in experimental systems. Most importantly, there is a need for a method of producing fluorescent proteins that exhibit useful fluorescence in the monomer state in experimental systems. The present invention fulfills these needs and provides further advantages.

Matz et al., Nature Biotechnology 17: 969-973 [1999] Tsien, Ann. Rev. Biochem., 67: 509-544 [1998] Mizuno et al., Biochemistry 40: 2502-2510 [2001] Verkhusha et al., J. Biol. Chem., 276: 29621-29624 [2001] Terskikh et al., J. Biol. Chem., 277: 7633-7636 [2002] Bevis and Glick, Nat. Biotechnol., 20: 83-87 [2002] Baird et al., Proc. Natl. Acad. Sci. USA 97: 11984-11989 [2000] Vrzheshch et al., FEBS Lett., 487: 203-208 [2000] Lauf et al., FEBS Lett., 498: 11-15 [2001] Fradkov et al., FEBS Lett., 479: 127-130 [2000] Lukyanov et al., J. Biol. Chem., 275: 25879-25882 [2000] Gurskaya et al., FEBS Lett., 507: 16-20 [2001] CLONTECH Laboratories Inc., (2002) “Living Colors User Manual Vol. II: Red Fluorescent Protein” [Becton, Dickinson and Company], p.4 Yanushevich, Y. G., Staroverov, D. B., Savitsky, A. P., Fradkov, A. F., Gurskaya, N. G., Bulina, M. E., Lukyanov, K. A. & Lukyanov, S. A. (2002) FEBS Lett. 511, 11-14

  The present invention relates to a red fluorescent protein (RFP) variant with a low tendency to oligomerize. For example, the natural form of RFP tends to form a tetrameric structure, whereas the RFP variants of the present invention tend to form monomers and dimers.

  The present invention relates to a red fluorescent protein (RFP) mutant comprising at least one amino acid modification that results in more efficient chromophore maturation than the corresponding wild-type or mutant RFP. The present invention relates to RFP variants that are less prone to form tetramers in addition to exhibiting more efficient maturation. Thus, certain forms of RFP of the present invention have a tendency to form monomers and dimers, whereas the natural form of RFP tends to form a tetrameric structure.

  In one aspect, the present invention is an Anthozoan fluorescent protein (AnFP) having a low tendency to oligomerize, tetramerizing the fluorescent protein within the wild-type AnFP amino acid sequence and / or optionally Relates to the above AnFP comprising at least one mutation that reduces or eliminates the ability to dimerize. Preferably, AnFP is a Discosoma red fluorescent protein (DsRed) of SEQ ID NO: 1, but is not limited thereto. In some embodiments, the invention includes at least one amino acid substitution within the AB and / or AC interface of the fluorescent protein (eg, DsRed) that reduces or eliminates the degree of oligomerization of the fluorescent protein. Anthozoan fluorescent protein (AnFP), for example DsRed.

  In other embodiments, a mutant AnFP with a low tendency to oligomerize (eg, DsRed) introduces a mutation that prevents oligomerization (including dimerization), eg, a substitution, and if necessary, A monomer that disrupts the interface between oligomer subunits by introducing additional mutations necessary to restore or improve fluorescence that may be partially or completely lost as a result of disrupting subunit interactions.

  In particular, in addition to DsRed, the present invention includes dimeric and monomeric variants of other fluorescent proteins, such as fluorescent proteins from other species and fluorescent proteins having fluorescent emission spectra at wavelengths other than red. For example, green fluorescent protein and fluorescent protein from Renilla species find equal use with the present invention. Furthermore, fluorescent proteins that normally have a tendency to form tetramers and / or dimers find equal use with the present invention.

  In certain embodiments, the fluorescent protein is DsRed, and a DsRed variant that has a low tendency to oligomerize (in this case, tetramerization) is first selected at least 1 in the AC and / or AB interface of the wild-type protein. It is prepared by replacing one important residue, thereby creating a dimer or monomeric form, and then introducing further mutations that restore or improve the red fluorescence properties.

  The present invention includes, but is not limited to, fluorescent protein variants including DSRed, which contain amino acid substitutions compared to their respective wild type, and the substitutions have advantageous properties for polypeptide variants. Provided is the above fluorescent protein variant. These amino acid substitutions may be present at any position within the polypeptide and are not particularly limited to any type of substitution (conservative or non-conservative). In one embodiment, the mutation that restores or improves fluorescence is an amino acid substitution in the chromophore plane of the fluorescent protein and / or just above and / or just below the chromophore plane.

  In one aspect, the invention provides a polynucleotide sequence encoding a Discosoma red fluorescent protein (DsRed) variant having a low tendency to oligomerize, comprising an AB interface of the wild type DsRed amino acid sequence of SEQ ID NO: 1. Contains one or more amino acid substitutions in the AC interface, or AB and AC interfaces, the substitution reduces the tendency to form tetramers of the DsRed mutant, and the mutant displays at least one detectable red wavelength fluorescence There is provided a polynucleotide sequence encoding a variant characterized by the above. In one embodiment, the protein sequence has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 1. In other embodiments, the fluorescent protein has detectable fluorescence that matures at a rate of at least about 80% of the rate of fluorescence maturation of wild-type DsRed of SEQ ID NO: 1, while in other embodiments the protein is Improves fluorescence maturation compared to DsRed of SEQ ID NO: 1. In yet other embodiments, the protein substantially retains the fluorescent properties of DsRed of SEQ ID NO: 1.

  In some embodiments, fluorescent protein variants have a tendency to form dimers. Some proteins contain substitutions in the AB interface and form AC dimers.

  In some embodiments, the fluorescent protein variant has at least 9 amino acid substitutions at residues 2, 5, 6, 21, 41, 42, 44, 117, and 217, and a substitution at residue 125 of SEQ ID NO: 1. It further includes at least one or more substitutions. The protein may optionally further comprise at least one additional amino acid substitution at residues 71, 118, 163, 179, 197, 127, or 131 of SEQ ID NO: 1. In some embodiments, any one or more of the above substitutions is optionally R2A, K5E, N6D, T21S, H41T, N42Q, V44A, V71A, C117T, F118L, I125R, V127T, S131P, K163Q / M, Selected from S179T, S197T, and T217A / S.

  The present invention provides fluorescent protein variants that may be protein dimer 1, dimer 1.02, dimer 1.25, dimer 1.26, dimer 1.28, dimer 1.34, dimer 1.56, dimer 1.61, or dimer 1.76 given in FIGS. 20A-D To do. In one preferred embodiment, the protein variant is dimer 2 (SEQ ID NO: 6).

  In some embodiments, the fluorescent dimer protein variant has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, and in other embodiments, the protein has the amino acid sequence of SEQ ID NO: 1. Have at least about 95% sequence identity.

  In yet other embodiments, the fluorescent protein variant is a monomer. In this embodiment, the amino acid substitution is at the AB interface and the AC interface.

  In some embodiments, the monomeric protein variant has at least 14 amino acid substitutions at residues 2, 5, 6, 21, 41, 42, 44, 71, 117, 127, 163, 179, 197, and 217, As well as at least one or more substitutions including a substitution at residue 125 of SEQ ID NO: 1. In other embodiments, the monomeric protein variants are optionally residues 83, 124, 125, 150, 153, 156, 162, 164, 174, 175, 177, 180, 192, 194 of SEQ ID NO: 1. , 195, 222, 223, 224, and 225, further including at least one substitution. In still other embodiments, the substitution in the monomeric protein is R2A, K5E, N6D, T21S, H41T, N42Q, V44A, V71A, K83L, C117E / T, F124L, I125R, V127T, L150M, R153E, V156A, H162K, K163Q / M, L174D, V175A, F177V, S179T, I180T, Y192A, Y194K, V195T, S197A / T / I, T217A / S, H222S, L223T, F224G, L225A.

  In other embodiments, the monomeric protein variants are mRFP0.1, mRFP0.2, mRFP0.3, mRFP0.4a, mRFP0.4b, mP11, mP17, m1.01, m1.02 given in FIGS. 20A-20D. , MRFP0.5a, m1.12, mRFP0.5b, m1.15, m1.19, mRFP0.6, m124, m131, m141, m163, m173, m187, m193, m200, m205 and m220. In a preferred embodiment, the monomer variant is mRFP1 (SEQ ID NO: 8).

  In some embodiments, the monomeric variant has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, and in other embodiments, the protein has at least about 95 amino acid sequence with SEQ ID NO: 1. % Sequence identity.

  The present invention also provides a tandem dimer form of DsRed comprising DsRed protein variants operably linked by a peptide linker. The length of the peptide linker may be variable, and the peptide linker is, for example, from about 10 to about 25 amino acids in length, or from about 12 to about 22 amino acids in length. In some embodiments, the peptide linker is selected from GHGTGSTGSGSS (SEQ ID NO: 17), RMGTSSGSTKGQL (SEQ ID NO: 18), and RMGTSGSGKPGSGEGSTKGQL (SEQ ID NO: 19).

  In some embodiments, the tandem dimer subunits are dimer 1, dimer 1.02, dimer 1.25, dimer 1.26, dimer 1.28, dimer 1.34, dimer 1.56, dimer 1.61, dimer 1.76, and dimer 2 given in FIGS. 20A-20D. Selected from. The tandem dimer may be a homodimer or a heterodimer. In a preferred embodiment, the tandem dimer comprises at least one copy of dimer 2 (SEQ ID NO: 6).

  The invention also provides fusion proteins between any protein of interest operably linked to at least one fluorescent protein variant of the invention. The fusion protein optionally contains a peptide tag, which may optionally be a polyhistidine tag.

  The present invention provides a polynucleotide encoding each fluorescent protein variant described or taught herein. Furthermore, the present invention provides fluorescent protein variants encoded by polynucleotides corresponding to any of those described or taught herein. Such polypeptides may include dimer variants, tandem dimer variants, or monomer variants.

  In other embodiments, kits are provided that include at least one polynucleotide sequence encoding a fluorescent protein variant of the invention. Alternatively or additionally, the kit provides the fluorescent protein variant itself.

  In other embodiments, the present invention provides vectors encoding fluorescent protein variants described or taught herein. Such vectors may encode dimer mutants, tandem dimer mutants, or monomeric mutants, or fusion proteins containing these mutants. The present invention also provides suitable expression vectors. In other embodiments, the present invention provides host cells comprising these vectors.

  In another embodiment, the present invention provides a method for making a dimeric or monomeric variant of a fluorescent protein having a tendency to tetramerize or dimerize, comprising mutating at least one amino acid residue in the fluorescent protein. Inducing, if the protein has a propensity to tetramerize, producing a dimer variant, and if the protein has a propensity to dimerize, producing a monomeric variant; and mutation of at least one additional amino acid residue To obtain a dimer or monomer variant that retains the qualitative ability to fluoresce in the same wavelength region as the fluorescent protein that was not mutagenized.

  In any variation of this method, additional mutations may be introduced essentially into the dimer variant produced from the fluorescent protein that has a tendency to form tetramers, adding further steps to produce monomeric variants. . In some embodiments, this additional step may be performed after the initial mutagenesis step.

  In some embodiments, the methods of the invention can provide dimer or monomer variants with improved fluorescence intensity or fluorescence maturation as compared to fluorescent proteins that have not been mutagenized.

  Mutagenesis used in the methods of the present invention is by multiple overlap extension using semi-degenerate primers, error-prone PCR, site directed mutagenesis. Or a combination thereof. As a result of this mutagenesis, protein variants that tend to produce dimers or monomers can be produced.

  In some embodiments of the methods of the invention, the fluorescent protein is an Anthozoan fluorescent protein, and in some cases, the Anthozoan fluorescent protein fluoresces at a red wavelength. The Anthozoan fluorescent protein may be Discosoma DsRed.

  In other embodiments, the fluorescent protein variants of the invention can be utilized in a variety of applications. In one embodiment, the invention is a method of detecting transcriptional activity, comprising a host cell comprising a vector encoding a DsRed fluorescent protein variant operably linked to at least one expression control sequence, and the above Provided is the above method using a means of assaying fluorescent protein variant fluorescence. In this method, the fluorescence of a fluorescent protein variant produced by a host cell is assayed to show transcriptional activity.

  In other embodiments, the present invention also provides polypeptide probes suitable for use in fluorescence resonance energy transfer (FRET) comprising at least one fluorescent protein variant of the present invention.

  In yet another embodiment, the invention is a method for analyzing in vivo localization or tracking of a polypeptide of interest, wherein the fluorescent fusion protein of the invention is utilized in a host cell or tissue, Provided is the above method of visualizing a fusion protein in a host cell or tissue.

  In a further aspect, the present invention relates to a further improved variant of red fluorescent protein (RFP) that is less prone to oligomerization. In particular, the present invention not only has a tendency to form monomers and dimers when the natural form of RFP has a tendency to form a tetrameric structure, but also more efficient maturation than the corresponding non-oligomerized mutant from which they were derived. Relates to an RFP variant further characterized by: Such mutants are typically brighter than the corresponding non-oligomerized mutants, where brightness is typically expressed as the product of extinction count (EC) and quantum yield (QY) at the desired red wavelength. Is done.

  The present invention further provides a polynucleotide encoding a variant of red fluorescent protein (RFP) that has a tendency to form a tetrameric structure, wherein the variant is more efficient from an immature green species to a desired red species. Provided is the above polynucleotide, comprising at least one amino acid modification that results in maturation and at least one amino acid modification that results in a reduced tendency to tetramerize. Amino acid modifications may be substitutions, insertions and / or deletions, and substitutions are preferred.

  Thus, in one aspect, the invention relates to a polynucleotide encoding a variant of red fluorescent protein (RFP) that has a tendency to form a tetramer, wherein said variant obtains a higher fluorescence intensity at a red wavelength. It relates to a polynucleotide as described above, which comprises an amino acid modification and at least one amino acid modification that results in a reduced tetramerization tendency.

  In certain embodiments, the polynucleotide encodes a Discosoma red fluorescent protein (DsRed) variant.

  In other embodiments, the polynucleotide encodes a DsRed variant in which the amino acid substitution that results in more efficient maturation is at position 66 of wild-type DsRed of SEQ ID NO: 1. A particular substitution is the Q66M substitution within SEQ ID NO: 1. In other embodiments, the polynucleotide encodes a DsRed variant in which the amino acid substitution that results in more efficient maturation is at position 147 of wild-type DsRed of SEQ ID NO: 1. The substitution at position 147 is preferably a T147S substitution, although other substitutions at this position are possible and are specifically included within the scope of the present invention. In a further embodiment, the polynucleotide of the invention encodes a DsRed variant comprising a substitution at both position 66 and position 147 within SEQ ID NO: 1. In a preferred embodiment, the polynucleotide of the invention encodes a DsRed variant comprising Q66M and T147S substitutions.

  A polynucleotide of the present invention encoding a DsRed variant comprising at least one amino acid modification that results in improved chromophore maturation efficiency to the desired red form, such as a Q66M substitution, further comprises a codon for any of the other amino acid substitutions. , Alone or in any combination discussed above and in connection with other embodiments throughout the present disclosure. In particular, such polynucleotides (eg, encoding a DsRed variant having a Q66M mutation alone or in combination with a T147S substitution) may further comprise one or more substitutions in the AB of the wild type DsRed amino acid sequence of SEQ ID NO: 1. A DsRed variant may be encoded that is included at the interface, at the AC interface, or at the AB and AC interfaces, the substitution resulting in a reduced tendency to form a tetramer of the DsRed variant.

  In certain embodiments, such polynucleotides are 42, 44, 71, 83, 124, 150, 163, 175, 177, 179, 195, 197, 217, 2 within the wild-type DsRed amino acid sequence of SEQ ID NO: 1. 1 at an amino acid position selected from the group consisting of 5, 6, 125, 127, 180, 153, 162, 164, 174, 192, 194, 222, 223, 224, 225, 21, 41, 117, and 156 It encodes a DsRed variant further comprising the above substitutions. Possible substitutions at the indicated positions include, but are not limited to, N42Q, V44A, V71A, K83L, F124L, L150M, K163M, V175A, F177V, S179T, V195T, S197I within the wild-type DsRed amino acid sequence of SEQ ID NO: 1. , T217A, R2A, K5E, N6D, I125R, V127T, I180T, R153E, H162K, A164R, L174D, Y192A, Y194K, H222S, L223T, F224G, L225A, T21S, H41T, C117E, and V156A Contains one or more substitutions.

  Accordingly, the following substitutions within the wild type DsRed amino acid sequence of SEQ ID NO: 1: N42Q, V44A, V71A, K83L, F124L, L150M, K163M, V175A, F177V, S179T, V195T, S197I, T217A, R2A, K5E, N6D, I125R Polynucleotides encoding DsRed variants, including V127T, I180T, R153E, H162K, A164R, L174D, Y192A, Y194K, H222S, L223T, F224G, L225A, T21S, H41T, C117E, and V156A, are clearly Is in range. In a preferred embodiment, the present invention relates to a polynucleotide encoding mRFP1.1 shown in FIG. 30 (SEQ ID NO: 79).

  In other embodiments, the present invention encodes a fusion protein comprising at least one DsRed protein variant encoded by the above-described polynucleotide operably linked to at least one other polypeptide of interest. A polynucleotide. In certain embodiments, such fusion proteins may contain both Q66M or T147S substitutions or as tandem dimers.

  The invention further relates to polypeptides encoded by the polynucleotides discussed above, vectors containing such polynucleotides (including expression vectors), and recombinant host cells transformed with such polynucleotides or vectors.

  In a different aspect, the present invention relates to a kit comprising at least one of the above polynucleotides or polypeptides.

In yet another aspect, the present invention provides a method for detecting transcriptional activity comprising:
(a) including at least one amino acid modification operably linked to at least one expression control sequence that results in higher fluorescence intensity at the red wavelength and at least one additional amino acid modification that results in a reduced tendency to tetramerize Providing a host cell comprising a vector comprising a nucleotide sequence encoding a DsRed fluorescent protein variant, as well as means for assaying the fluorescent protein variant fluorescence;
(b) the method comprising the step of assaying the fluorescence of the fluorescent protein variant produced by the host cell, wherein the fluorescent protein variant fluorescence exhibits transcriptional activity.

  In a further aspect, the present invention relates to a method for detecting protein-protein interactions comprising the detection of energy transfer from a fluorescent or bioluminescent protein fusion as discussed above to a fusion protein.

In yet a further aspect, the invention is a method for analyzing in vivo localization or tracking of a polypeptide of interest comprising:
(a) a polynucleotide encoding a fusion protein comprising at least one DsRed protein variant encoded by a polynucleotide discussed above, operably linked to at least one other polypeptide of interest, and a host Providing a cell or tissue, and
(b) The above method comprising the step of visualizing the fusion protein expressed in the host cell or tissue.

  There is a need in the art for the development of red fluorescent polypeptides that can be used in scientific applications without technical limitations due to oligomerization, particularly tetramerization and inefficient and slow chromophore maturation. There is a need for methods of making fluorescent proteins that are less prone to oligomerization, particularly tetramerization. There is a need for a method of making red fluorescent protein (RFP) that exhibits more efficient chromophore maturation than wild-type RFP or other RFP variants. Furthermore, there is a need for RFP that has a low tendency to oligomerize such as tetramerization. Most importantly, there is a need for RFP variants that have improved maturation efficiency and exhibit useful fluorescence in the monomeric state in experimental systems. Most importantly, there is a need for a method of producing fluorescent proteins that exhibit useful fluorescence in the monomer state in experimental systems. The present invention fulfills these needs and provides further advantages.

FIG. 1 illustrates the tetrameric form of DsRed (PDB identification code 1G7K). Like the A-B and CD interfaces, the A-C and BD interfaces are equivalent. 2A-2C are graphical representations of DsRed tetramer, dimer and monomer types, respectively, based on the DsRed X-ray crystal structure. Residues 1-5 were not observed in the crystal structure but were added arbitrarily for completeness. The DsRed chromophore was represented in red and the four chains of the tetramer were labeled according to the convention of Yarbrough et al. (Yarbrough et al., Proc. Natl. Acad. Sci. USA 98: 462-467 [2001]). FIG. 2A shows the tetramer of DsRed, showing residues mutated in T1 with the blue residue on the outside and the green residue on the inside of the β barrel. FIG. 2B shows the DsRed AC dimer, all mutations present in dimer 2 are represented as in FIG. 2A, and the intersubunit linker present in the t dimer (12) is indicated by a dotted line. FIG. 2C shows the DsRed monomer and all mutations present in mRFP1 are represented as in FIG. 2A. FIGS. 3A-3C show the results of analytical ultracentrifugation analysis of DsRed, Dimer 2, and mRFP0.5a polypeptide, respectively. The equilibrium radius absorption profile at 20,000 rpm was modeled using a theoretical curve that can change only the molecular weight. The DsRed absorption profile (Figure 3A) best fits the apparent molecular weight of 120 kDa, consistent with the tetramer. The dimer 2 absorption profile (FIG. 3B) best matched the apparent molecular weight of 60 kDa and was consistent with the dimer. The mRFP0.5a absorption profile (FIG. 3C) best matched the apparent molecular weight of 32 kDa, consistent with the monomer containing the N-terminal polyhistidine affinity tag. FIG. 4A shows the fluorescence and absorption spectra of DsRed and mRFP1. The absorption spectrum is indicated by a solid line, the excitation is indicated by a dotted line and the emission is indicated by a broken line. FIG. 4B shows the fluorescence and absorption spectra of T1 and mRFP1. The absorption spectrum is indicated by a solid line, the excitation is indicated by a dotted line and the emission is indicated by a broken line. FIG. 4C shows the fluorescence and absorption spectra of dimer 2 and mRFP1. The absorption spectrum is indicated by a solid line, the excitation is indicated by a dotted line and the emission is indicated by a broken line. FIG. 4D shows the fluorescence and absorption spectra of t-dimer 2 (12) and mRFP1. The absorption spectrum is indicated by a solid line, the excitation is indicated by a dotted line and the emission is indicated by a broken line. FIG. 5 shows the red fluorescence maturation time course for DsRed, T1, dimer 2, t-dimer 2 (12) and mRFP1. The profile was colored as indicated on the key. A log phase culture of E. coli expressing the desired construct was rapidly purified at 4 ° C. Maturation at 37 ° C. was monitored starting 2 hours after harvest. The initial decrease in mRFP1 fluorescence is due to slight quenching when warming from 4 ° C to 37 ° C. FIGS. 6A-6F show light and fluorescence microscopy images of HeLa cells expressing Cx43 fused to T1, dimer 2 or mRFP1. Images 6A, 6C and 6E were obtained using excitation at 568 nm (55 nm bandwidth) and emission at 653 nm (95 nm bandwidth) and using additional transmitted light. Lucifer yellow fluorescence (images 6B, 6D and 6F) was obtained using excitation at 425 nm (45 nm bandpass) and emission at 535 nm (55 nm bandpass). FIG. 6A shows two contacted cells transfected with Cx43-mRFP1 and connected by a single large gap junction. FIG. 6B shows one cell microinjected at the point marked Lucifer yellow with an asterisk, and the dye passes rapidly through neighboring cells (1-2 seconds). FIG. 6C shows four adjacent cells transfected with Cx43-dimer2. The bright line between the two rightmost cells is the result of having two contacting phosphor films and not a gap junction. FIG. 6D shows that the microinjected dye slowly passes to neighboring cells (observed approximately one third of that time). FIG. 6E shows that two neighboring cells transfected with Cx43-T1 and typical perinuclear localized aggregation are displayed. FIG. 6F shows that the dye did not pass between adjacent cells. FIG. 7 shows a schematic diagram of the directed evolution strategy of the present invention. Although randomization in 2 positions is shown, this technique has been used with up to 5 fragments. 8A and 8B show SDS-PAGE analysis of DsRed, T1, dimer 2, t-dimer 2 (12), and mRFP1 polypeptide. The oligomeric state of each protein is demonstrated by running both unboiled and boiled proteins (20 μg) on a 12% SDS-PAGE Tris-HCl precast gel (BioRad). FIG. 8A shows the gel before Coomasie staining, which was imaged using excitation at 560 nm and emission at 610 nm. Tandem dimer or t-dimer 2 (12) has a small tetramer component due to the small amount of covalent tandem pairs participating in intermolecular dimer pairs. Non-boiling fluorescent proteins do not necessarily move to their expected molecular weight. FIG. 8B shows the same gel as FIG. 8A after Coomassie staining. The ˜20 kDa band results from partial hydrolysis of the main acrylic chain of the protein containing the red chromophore. Figures 9A-9D show fluorescent images of red fluorescent protein expressed in E. coli. E. coli strain JM109 (DE3) was transfected with DsRed, T1, dimer 2 or mRFP1, seeded on LB / agar supplemented with ampicillin and at 37 ° C. for 12 hours, then 20 ° C. For 8 hours, and then the plate was imaged using a digital camera. In FIG. 9A, when excited at 540 nm and imaged using a 575 nm (long pass) emission filter, the quadrants corresponding to T1, dimer 2, and mRFP1 all appear to have similar brightness. Fluorescence is not visible to E. coli transformed with equivalently treated DsRed. In FIG. 9B, when excited at 560 nm and imaged using a 610 nm (long pass) emission filter, mRFP1 becomes brighter due to its 25 nm red shift. In FIG. 9C, monomer RFP1 does not contain a green fluorescent component, so when excited at 470 nm, a wavelength suitable for EGFP excitation, it is very weak compared to T1 and dimer 2. FIG. 9D shows a digital color photograph of the same plate taken at room temperature after 5 days, which represents the orange and purple shades of T1 and mRFP1, respectively. FIG. 10A shows a table describing protocols and multiple libraries created during the evolution of Dimer 1 and mRFP1 and other intermediate types. The template, the method of mutagenesis, the targeted location within the DsRed polypeptide, and the resulting clone are shown. FIG. 10B shows a table describing protocols and multiple libraries created during the evolution of Dimer 1 and mRFP1 and other intermediate types. The template, the method of mutagenesis, the targeted location within the DsRed polypeptide, and the resulting clone are shown. FIGS. 11A-11C show a primer pair used in the mutagenesis protocol, as well as a table giving the key to the target codon position. FIG. 11B shows a table that gives the primer pair used in the mutagenesis protocol, as well as a key for the target codon position. FIG. 11C shows the primer pair used in the mutagenesis protocol, as well as a table giving the key to the target codon position. FIG. 12A provides the PCR primer sequences listed in FIGS. 11A-11C. FIG. 12B provides the PCR primer sequences listed in FIGS. 11A-11C. FIG. 13 shows a table describing the results of a series of experiments testing the functionality of various DsRed chimeric molecules. The chimeric molecule comprises a DsRed sequence and a Cx43 polypeptide. Plasmids encoding the fusion polypeptide were transfected into HeLa cells, and the ability of the expressed fusion polypeptide to form a functional gap junction was assayed by microinjection of Lucifer yellow dye. Passage of the dye from one HeLa cell to adjacent HeLa cells indicates a functional gap junction and thus the presence of a functional fusion polypeptide. FIG. 14 shows various biophysical properties of wild-type DsRed, T1, dimer 2, t-dimer 2 (12), and mRFP1 polypeptide. FIG. 15 shows a table giving excitation / emission wavelength values, relative maturation rates and red / green ratio values for red and green fluorescent protein species. FIG. 16 provides the nucleotide sequence of the Discosoma species wild-type red fluorescent protein open reading frame (DsRed). FIG. 17 provides the amino acid sequence of the wild type red fluorescent protein (DsRed) of Discosoma species. FIG. 18 provides the nucleotide sequence of a discosoma species mutant fast T1 red fluorescent protein. FIG. 19 provides the amino acid sequence of a mutant fast T1 red fluorescent protein of Discosoma species. FIG. 20A provides a table showing the amino acid substitutions identified during the construction of the mutant DsRed protein. Also shown are the substitutions originally contained in the fast T1 DsRed mutant. FIG. 20B provides a table showing the amino acid substitutions identified during the construction of the mutant DsRed protein. Also shown are the substitutions originally contained in the fast T1 DsRed mutant. FIG. 20C provides a table showing the amino acid substitutions identified during the construction of the mutant DsRed protein. Also shown are the substitutions originally contained in the fast T1 DsRed mutant. FIG. 20D provides a table showing the amino acid substitutions identified during the construction of the mutant DsRed protein. Also shown are the substitutions originally contained in the fast T1 DsRed mutant. FIG. 21 provides the nucleotide sequence of the Discosoma red fluorescent protein variant dimer 2 open reading frame. FIG. 22 provides the amino acid sequence of Discosoma red fluorescent protein mutant dimer 2. FIG. 23 provides the nucleotide sequence of the Discosoma red fluorescent protein variant mRFP1 open reading frame. FIG. 24 provides the amino acid sequence of the Discosoma red fluorescent protein mutant mRFP1. FIG. 25 provides the nucleotide sequence of the Discosoma wild-type red fluorescent protein open reading frame modified using humanized codon usage. FIG. 26 illustrates the maturation of Q66M DsRed compared to wild type DsRed protein. FIG. 27 shows the excitation and emission spectra of wild type DsRed (dsRed w.t.) and Q66M DsRed (dsRED Q66M), plotting the relative luminance as a function of wavelength. FIG. 28 shows a Coomassie stained band on SDS-polyacrylamide gel representing wild type DsRed, Q66M DsRed and K83DsRed after hydrolysis at pH1. FIG. 29 shows the absorption spectra of mRFP1, mRFP Q66M, and mRFP1.1. Absorption spectra were normalized to a 280 nm peak that should approximate the total protein concentration. The emission spectrum (measured by excitation at 550 nm) was normalized to its respective absorption maximum for expression. FIG. 30 provides the amino acid sequence of mRFP1.1. FIG. 31 gives the nucleotide sequence of mRFP1.1.

Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, any method or material similar or equivalent to the methods or materials described herein can be used in the practice of the present invention. For purposes of the present invention, the following terms are defined:

  The term “nucleic acid molecule” or “polynucleotide” means a single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymer and can function similarly to a naturally occurring nucleotide unless otherwise specified. Includes polynucleotides containing known analogs of naturally occurring nucleotides. When a nucleic acid molecule is represented by a DNA sequence, this is also understood to include an RNA molecule having a corresponding RNA sequence (where “U” (uridine) replaces “T” (thymidine)).

  The term “recombinant nucleic acid molecule” refers to a non-naturally occurring nucleic acid molecule that contains two or more linked polynucleotide sequences. Recombinant nucleic acid molecules can be produced by recombinant methods, particularly genetic engineering techniques, or can be produced by chemical synthesis methods. The recombinant nucleic acid molecule can encode a fluorescent protein variant of the invention linked to a fusion protein, eg, a polypeptide of interest. The term “recombinant host cell” means a cell containing a recombinant nucleic acid molecule. Thus, a recombinant host cell can express a polypeptide from a “gene” that is not found in native (non-recombinant) cells.

  Reference to a polynucleotide "encoding" a polypeptide means that the polypeptide is produced by transcription of the polynucleotide and translation of the mRNA produced therefrom. A coding polynucleotide is considered to include both the coding strand (its nucleotide sequence is identical to the mRNA) and its complementary strand. It will be appreciated that such coding polynucleotides are considered to comprise degenerate nucleotide sequences that encode the same amino acid residue. Nucleotide sequences that encode polypeptides can include polynucleotides that contain introns as well as coding exons.

  The term “expression control sequence” means a nucleotide sequence operably linked to a polynucleotide that regulates transcription or translation of the polynucleotide or localization of the polypeptide. Expression control when the expression control sequence controls or regulates transcription of the nucleotide sequence and, where appropriate, translation (ie, transcription or translation control element, respectively) or localization of the encoded polypeptide to a particular compartment of the cell. Sequences are “operably linked”. Thus, expression control sequences target promoters, enhancers, transcription terminators, initiation codons (ATG), splicing signals for intron cleavage and correct reading frame, stop codons, ribosome binding sites, or polypeptides to specific locations Target sequences (eg, polypeptides to the cytosol, nucleus, plasma membrane, endoplasmic reticulum, mitochondrial membrane or matrix, chloroplast membrane or chloroplast cavity, intermediate trans-Golgi squamous sac, or lysosomes or endosomes) Possible cell compartmentalization signal). Cell compartmentalization domains are well known in the art, for example, amino acid residues 1-81 of galactosyltransferase, a human type II membrane anchored protein, or amino acid residue 1 of the presequence of subunit IV of cytochrome c oxidase. (See also Hancock et al., EMBO J. 10: 4033-4039, 1991; Buss et al., Mol. Cell. Biol. 8: 3960-3963, 1988; US Pat. No. 5,776,689). Each of which is incorporated herein by reference).

  The terms “operably linked” or “operably linked” or “operably linked” and the like, when used to describe a chimeric protein, are physically and functionally related to each other. Means a polypeptide sequence arranged in a genetic relationship. In the most preferred embodiment, the function of the polypeptide component of the chimeric molecule is unchanged compared to the functional activity of the isolated portion. For example, the fluorescent protein of the present invention can be fused with a polypeptide of interest. In this case, it is preferred that the fusion molecule retains its fluorescence and that the polypeptide of interest retains its original biological activity. In some embodiments of the invention, the activity of the fluorescent protein or protein of interest may be reduced compared to their isolated activity. Such fusions can also utilize the present invention. As used herein, a chimeric fusion molecule of the invention may be in a monomeric state or a multimeric state (eg, a dimer).

  In another example, a tandem dimeric fluorescent protein variant of the invention comprises two “functionally linked” fluorescent protein units. The two units are linked in such a way that each maintains fluorescence activity. The first and second units of the tandem dimer need not be identical. In other embodiments of this example, the third polypeptide of interest may be operably linked to a tandem dimer, thereby forming a three-part fusion protein.

  The term “oligomer” means a complex formed by the specific interaction of two or more polypeptides. A “specific interaction” or “specific association” is one that is relatively stable under certain conditions (eg, physiological conditions). Reference to a “trend” that a protein oligomerizes indicates that the protein can form dimers, trimers, tetramers, etc. under certain conditions. In general, fluorescent proteins such as GFP and DsRed have a tendency to oligomerize under physiological conditions, but as disclosed herein, fluorescent proteins also have pH conditions other than physiological conditions, for example There are cases where oligomerization occurs even under. Conditions under which the fluorescent protein oligomerizes or has a tendency to oligomerize are measured using well-known methods disclosed herein (see Examples 1 and 3) or other methods known in the art. can do.

  As used herein, a molecule having a “low oligomerization tendency” is a molecule that has a low tendency to form a structure with multiple subunits to form a structure with fewer subunits. For example, molecules that can form a tetrameric structure under physiological conditions usually show a low tendency to oligomerize if the molecule is modified to have a preference for forming a monomer, dimer or trimer as a result. In general, molecules that can form a dimer structure under physiological conditions show a low tendency to oligomerize if the molecule is modified to have a preference to form monomers as a result. Thus, “low oligomerization tendency” usually applies equally to proteins that are dimers and proteins that are usually tetramers.

  The term “non-tetramerized” as used herein refers to a protein type that produces trimers, dimers and monomers but does not produce tetramers. Similarly, “non-dimerized” means a protein type that remains monomeric.

  As used herein, the term “(chromophore) maturation efficiency” referring to red fluorescent protein (RFP) has matured from a species with a green fluorescent protein (GFP) -like absorption spectrum to the last RFP absorption spectrum. The percentage of protein is shown. Thus, maturation efficiency is determined after sufficient time for the maturation process to be practically complete (eg,> 95%). Preferably, the resulting RFP, such as DsRed, is at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 98%. Most preferably, it may contain at least about 99% red fluorescent species.

  As used herein, the term “luminance” referring to a fluorescent protein is measured as the product of the extinction coefficient (EC) and the fluorescence quantum yield (QY) at a given wavelength.

  The term “probe” means a substance that specifically binds to another substance (“target”). Probes include, for example, antibodies, polynucleotides, receptors and their ligands, and can generally be labeled to provide a means to identify or isolate molecules to which the probe has specifically bound. The term “label” means a composition that can be detected with or without a device, eg, by visual inspection, spectroscopy, or photochemistry, biochemistry, immunochemistry, or chemical reaction. Useful labels include, for example, phosphorus-32, fluorescent dyes, fluorescent proteins, electron density reagents, enzymes (eg, those commonly used in ELISA), small molecules (eg, biotin), digoxigenin, or other haptens or peptides (In contrast, antisera or antibodies (which may be monoclonal antibodies) can be used). The fluorescent protein variants of the present invention are themselves detectable proteins, but nevertheless, for example, to facilitate identification of the protein respectively during expression and isolation of the expressed protein It will be appreciated that the labeling may be such that it can be detected by means other than fluorescence, such as by incorporating a radionuclide label or peptide tag into the protein. Labels useful for the purposes of the present invention generally produce a measurable signal (eg, radioactive signal, fluorescence, enzyme activity, etc.), both to quantify, for example, the amount of fluorescent protein variant in a sample. Can be used for

  The term “nucleic acid probe” refers to a polynucleotide that binds to a specific nucleotide sequence or subsequence of a second (target) nucleic acid molecule. A nucleic acid probe is generally a polynucleotide that binds to a target nucleic acid molecule through complementary base pairing. It is understood that a nucleic acid probe can also specifically bind to a target sequence that is less than perfectly complementary to the probe sequence, and that binding specificity can depend in part on the stringency of the hybridization conditions. Will be done. Nucleic acid probes are labeled with radionuclides, chromophores, lumiphores, chromogens, fluorescent proteins, or small molecules such as, for example, biotin (which can itself be bound by, for example, streptavidin complexes) Thus providing a means of isolating the probe, including the target nucleic acid molecule to which the probe specifically binds. By assaying for the presence or absence of the probe, the presence or absence of the target sequence or subsequence can be detected. The term “labeled nucleic acid probe” refers to a covalent or stable non-covalent bond (eg, directly or through a linker molecule, so that the presence of the probe can be identified by detecting the presence of the label bound to the probe. By ionic bond, van der Waals bond or hydrogen bond) is meant a nucleic acid probe bound to a label.

  The term “polypeptide” or “protein” means a polymer of two or more amino acid residues. This term applies to natural amino acid polymers as well as amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding natural amino acid. The term “recombinant protein” refers to a protein produced from a recombinant DNA molecule by expression of a nucleotide sequence that encodes the amino acid sequence of the protein.

  The term “isolated” or “purified” means a material that is substantially or essentially free from components that normally accompany the material in its native state in nature. Purity or homogeneity is generally measured using analytical chemistry techniques such as polyacrylamide gel electrophoresis, high performance liquid chromatography. A polynucleotide or polypeptide is considered isolated when it is the predominant molecular species present in the preparation. Generally isolated protein or nucleic acid molecules represent more than 80% of the macromolecular species present in a preparation when examined using conventional methods to determine the purity of such molecules, often all present More than 90% of macromolecular species, usually more than 95% of macromolecular species, and in particular poly, which has been purified to intrinsic homogeneity and is the only molecular species to be detected A peptide or polynucleotide.

  The term “natural” is used to mean a naturally occurring protein, nucleic acid molecule, cell, or other substance. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses). Natural substances may be naturally occurring types and may be modified, eg, isolated, by hand.

The term “antibody” refers to a polypeptide substantially encoded by one or more immunoglobulin genes, or an antigen-binding fragment thereof, that specifically binds and recognizes an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Antibodies can be produced by digesting with peptidase, or using recombinant DNA methods, complete immunoglobulins and well-characterized antigen-binding fragments of antibodies. Can exist). Such antigen binding fragments of antibodies include, for example, Fv, Fab ′, and F (ab ′) ₂ fragments. The term “antibody” as used herein includes antibody fragments produced by modification of whole antibodies, or those newly synthesized using recombinant DNA methods. The term “immunoassay” refers to an assay that utilizes an antibody that specifically binds to an analyte. An immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and / or quantify the analyte.

  The term “identical” means residues in a sequence that are the same when aligned for maximum match when used with more than one polynucleotide sequence or more than one polypeptide sequence. . When percent sequence identity is used for a polypeptide, it is recognized that one or more residue positions that are not originally identical may differ by conservative amino acid substitutions, where the first amino acid residue Is replaced with another amino acid residue having similar chemical properties (eg, similar charge or hydrophobicity or hydrophilicity) and thus does not alter the functionality of the polypeptide. Where polypeptide sequences differ in conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Such adjustments are made using well-known methods, for example, conservative substitutions can be scored as partial mismatches rather than complete mismatches, thus increasing the percent sequence identity. Thus, for example, when the same amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and one. Conservative substitution scoring can be calculated using any well-known algorithm (eg, Meyers and Miller, Comp. Appl. Biol. Sci. 4: 11-17, 1988; Smith and Waterman, Adv. Appl. Math. 2: 482, 1981; Needleman and Wunsch, J. Mol. Biol. 48: 443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85: 2444 (1988); Higgins and Sharp, Gene 73 : 237-244, 1988; Higgins and Sharp, CABIOS 5: 151-153, 1989; Corpet et al., Nucl. Acids Res. 16: 10881-10890, 1988; Huang et al., Comp. Appl. Biol. Sci. 8: 155 -165, 1992; see Pearson et al., Meth. Mol. Biol., 24: 307-331, 1994). Alignment can also be done by simple visual inspection and manual alignment of the sequences.

The term “conservatively altered change” when used with respect to a particular polynucleotide sequence means a different polynucleotide sequence that encodes the same or essentially the same amino acid sequence, or the polynucleotide is an amino acid. If no sequence is encoded, it means essentially the same sequence. Because of the degeneracy of the genetic code, a large number of functionally identical polynucleotides encode a given polypeptide. For example, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. That is, at every position where arginine is defined by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleotide sequence changes are “silent mutations”, which can be considered as one of “conservatively altered changes”. Accordingly, it will be appreciated that each polynucleotide sequence disclosed herein as encoding a fluorescent protein variant also describes all possible silent mutations. In addition, each codon in a polynucleotide (usually excluding AUG, which is the only codon of methionine, and UUG, which is usually the only codon of tryptophan) is modified by standard techniques to provide functionally identical molecules. It will also be understood that can be provided. Accordingly, each silent variation of a polynucleotide that does not change the sequence of the encoded polypeptide is silently described herein. In addition, a single amino acid or a small percentage of amino acids in the encoded sequence (typically less than 5%, and in general, subject to modification replacing amino acids with chemically similar amino acids It will be appreciated that each substitution, deletion, or addition that modifies, adds, or deletes (less than 1%) can be considered a conservatively modified change. Conservative amino acid substitutions that give functionally similar amino acids are well known in the art and include the following six groups, each of which contains amino acids that are considered conservative substitutions for each other:
1) Alanine (Ala, A), Serine (Ser, S), Threonine (Thr, T);
2) Aspartic acid (Asp, D), glutamic acid (Glu, E);
3) Asparagine (Asn, N), glutamine (Gln, Q);
4) Arginine (Arg, R), Lysine (Lys, K);
5) Isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), valine (Val, V); and
6) Phenylalanine (Phe, F), tyrosine (Tyr, Y), tryptophan (Trp, W).

  Two or more amino acid sequences or two or more nucleotide sequences are “substantially identical” if the amino acid sequences or nucleotide sequences share at least 80% sequence identity with each other or with a reference sequence over a predetermined comparison window, or It is considered “substantially similar”. Thus, substantially similar sequences include, for example, those having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity.

  A subject nucleotide sequence is considered “substantially complementary” to a reference nucleotide sequence if the complement of the subject nucleotide sequence is substantially identical to the reference nucleotide sequence. The term “stringent conditions” refers to the temperature and ionic conditions used in a nucleic acid hybridization reaction. Stringent conditions are sequence-dependent and depend on different environmental parameters. Generally, stringent conditions are selected to be about 5 ° C. to 20 ° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Tm is the temperature at which 50% of the target sequence hybridizes with a perfectly matched probe under a defined ionic strength and pH.

  The term “allelic variant” means a polymorphism of a gene at a particular locus, as well as cDNA derived from the mRNA transcripts of the gene, and the polypeptides encoded thereby. The term “preferred mammalian codon” refers to the subset of codons most often used in proteins expressed in mammalian cells from among codon sets encoding amino acids and is selected from the following list: Gly (GGC, Glu); Glu (GAG); Asp (GAC); Val (GUG, GUC); Ala (GCC, GCU); Ser (AGC, UCC); Lys (AAG); Asn (AAC); Met (AUG); (AUC); Thr (ACC); Trp (UGG); Cys (UGC); Tyr (UAU, UAC); Leu (CUG); Phe (UUC); Arg (CGC, AGG, AGA); Gln (CAG); His (CAC); and Pro (CCC).

Fluorescent molecules are useful in fluorescence resonance energy transfer (FRET) involving donor and acceptor molecules. In order to optimize the efficiency and detectability of FRET between donor and acceptor molecules, several factors need to be balanced. In order to maximize the overlap integral, the emission spectrum of the donor must overlap as much as possible with the excitation spectrum of the acceptor. Also, the quantum yield of the donor moiety and the extinction coefficient of the acceptor must be as high as possible to maximize R 2 _O (which represents the distance where the energy transfer efficiency is 50%). However, the fluorescence that results from direct excitation of the acceptor can be difficult to distinguish from the fluorescence that results from FRET, so that you can find a wavelength region that can efficiently excite the donor without directly exciting the acceptor. Donor and acceptor excitation spectra should not overlap as much as possible. Similarly, the emission spectra of the donor and acceptor should not overlap as much as possible so that the two emissions can be clearly distinguished. When the emission from the acceptor is measured as a single reading or as part of the emission ratio, a high fluorescence quantum yield of the acceptor moiety is preferred. One factor to consider when choosing a donor and acceptor pair is the efficiency of fluorescence resonance energy transfer between them. Preferably, the efficiency of FRET between the donor and acceptor is at least 10%, more preferably at least 50%, and even more preferably at least 80%.

  The term “fluorescence properties” refers to the molar extinction coefficient at the appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation or emission spectrum, the excitation wavelength maximum and the emission wavelength maximum, the ratio of the excitation amplitude at two different wavelengths, two By ratio of emission amplitude at different wavelengths, excited state lifetime, or fluorescence anisotropy. A measurable difference in any one of these properties between wild-type Aequorea GFP and a spectral variant or mutant thereof is useful. A measurable difference can be determined by measuring any quantitative fluorescence characteristic (eg, the amount of fluorescence at a particular wavelength, or the integration of fluorescence over the emission spectrum). The ratioing process provides an internal standard and eliminates excitation source absolute brightness, detector sensitivity, and variations in light dispersion or quenching due to the sample, so excitation amplitudes at two different wavelengths Or the measurement of the ratio of the emission amplitudes (“excitation amplitude ratio measurement” and “emission amplitude ratio measurement”, respectively) is particularly advantageous.

  As used herein, the term “fluorescent protein” refers to any protein that can fluoresce when excited by appropriate electromagnetic radiation, with the exception of chemically tagged proteins with chemical tags, And a polypeptide that fluoresces only by the presence of certain amino acids such as tryptophan or tyrosine whose emission peaks at the ultraviolet wavelength (ie, less than about 400 nm) is not considered a fluorescent protein for the purposes of the present invention. In general, the fluorescent protein useful in the preparation of the composition of the invention or the fluorescent protein used in the method of the invention is a protein that induces its fluorescence by autocatalytically forming a chromophore. A fluorescent protein can contain a naturally occurring or artificially created amino acid sequence (ie, a variant or a mutant). The term “mutant” or “variant” when used in reference to a fluorescent protein means a protein that is different from a reference protein. For example, Aequorea GFP spectral variants can be derived from naturally occurring GFP by artificially creating mutations such as amino acid substitutions in a reference GFP protein. For example, ECFP is a spectral variant of GFP that contains substitutions for GFP (compare SEQ ID NOs: 10 and 11).

  Many cnidarians use green fluorescent protein as an energy transfer acceptor in bioluminescence. The term “green fluorescent protein” is used broadly herein to mean a protein that fluoresces green light, such as Aequorea GFP (SEQ ID NO: 10). GFP has been isolated from Pacific Northwest jellyfish, Aequorea victoria, Renilla reniformis, and Phialidium gregarium (Ward et al., Photochem Photobiol. 35: 803-808, 1982; Levine et al., Comp. Biochem. Physiol. 72B: 77-85, 1982, each of which is incorporated herein by reference). Similarly, in this specification, “red fluorescent protein” that emits red fluorescence, “cyan fluorescent protein” that emits cyan fluorescence, and the like are mentioned. For example, RFP has been isolated from the coral form Discosoma (Matz et al., Matz et al., Nature Biotechnology 17: 969-973 [1999]). The term “red fluorescent protein”, or “RFP” is used in the broadest sense, and specifically the red fluorescent protein from Discosoma RFP (DsRed) and any other species such as corals and sea anemones As well as their variants as long as they retain the ability to fluoresce red light.

  The term “coral” as used herein encompasses species within the Anthozoa class, and specifically includes both coral and corallimorph.

  A variety of Aequorea GFP-related fluorescent proteins with useful excitation and emission spectra have been artificially produced by modifying the amino acid sequence of native GFP from A. victoria (Prasher et al., Gene 111: 229-233, 1992; Heim et al., Proc. Natl. Acad. Sci., USA 91: 12501-12504, 1994; US Pat. No. 5,625,048; International Patent Application PCT / US95 / published as PCT WO96 / 23810 14692, each of which is incorporated herein by reference). As used herein, reference to a “related fluorescent protein” means a fluorescent protein having a substantially identical amino acid sequence as compared to a reference fluorescent protein. Generally, related fluorescent proteins have a contiguous sequence of at least about 150 amino acids that share at least about 85% sequence identity with the reference fluorescent protein as compared to the reference fluorescent protein sequence, and in particular, with the reference fluorescent protein Have a contiguous sequence of at least about 200 amino acids sharing at least about 95% sequence identity. That is, in the present specification, reference is made to the “equorea” exemplified by various spectral variants and GFP mutants having substantially the same amino acid sequence as GFP (SEQ ID NO: 10) of A. victoria. (Aequorea-related fluorescent protein) or “GFP-related fluorescent protein”, “Discosoma-related fluorescent protein”, exemplified by various mutants having an amino acid sequence substantially identical to that of DsRed (SEQ ID NO: 1) Or “DsRed related fluorescence related proteins”, as well as their concomitant, eg, Renilla related fluorescent proteins or Phialidium related fluorescent proteins.

  The term “mutant” or “variant” is also used herein in the sense of a fluorescent protein containing a mutation relative to the corresponding wild-type fluorescent protein. Further referred to herein as a “spectral variant” or “spectral mutant” of a fluorescent protein, which refers to a mutant fluorescent protein having a different fluorescence characteristic of the corresponding wild-type fluorescent protein. Show. For example, CFP, YFP, ECFP (SEQ ID NO: 11), EYFP-V68L / Q69K (SEQ ID NO: 12) and the like are GFP spectral variants.

  Examples of Aequorea GFP-related fluorescent proteins include wild-type (native) Aequorea victoria GFP (Prasher et al., Supra, 1992; see also SEQ ID NO: 10), allelic variants of SEQ ID NO: 10, Eg variants with Q80R substitution (Chalfie et al., Science 263: 802-805, 1994, which is incorporated herein by reference); and spectral variants of GFP, eg CFP, YFP, and enhanced forms thereof and others (See US Pat. Nos. 6,150,176; 6,124,128; 6,077,707; 6,066,476; 5,998,204; and 5,777,079, each of which is incorporated herein by reference) GFP-related fluorescent proteins with the folding mutation of, and fragments of these proteins that are fluorescent, such as A. victoria G from which the two N-terminal amino acid residues have been removed Includes FP. Some of these fluorescent proteins contain different aromatic amino acids in the central chromophore and fluoresce at a clearly shorter wavelength than the wild-type GFP species. For example, artificially produced GFP proteins called P4 and P4-3 contain the substitution Y66H in addition to other mutations, and artificially produced GFP proteins called W2 and W7 In addition, Y66W is contained.

  The term “non-tetramerized fluorescent protein” is used broadly herein to refer to a normally tetrameric fluorescent protein that has been modified to have a low tendency to tetramerize compared to the corresponding unmodified fluorescent protein. Broadly mean. As such, unless specifically indicated otherwise, the term “non-tetramerized fluorescent protein” encompasses dimeric fluorescent proteins, tandem dimeric fluorescent proteins, as well as fluorescent proteins that remain monomeric.

  As used herein, the term “aggregation” means the tendency of the expressed protein to form insoluble precipitates or visible spots and is distinguished from “oligomerization”. In particular, mutations that reduce aggregation, eg, increase protein solubility, do not necessarily reduce oligomerization, ie, do not convert tetramers into dimers or monomers.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides fluorescent protein variants that can be derived from fluorescent proteins that have a dimerization or tetramerization tendency. As disclosed herein, in one embodiment of the present invention, the fluorescent protein variant of the present invention can be derived from a naturally occurring fluorescent protein or from a spectral variant or mutant thereof, It contains at least one mutation that reduces or eliminates the tendency of the fluorescent protein to oligomerize. In particular, the present invention provides dimer and monomeric red fluorescent protein (RFP) and RFP variants with a low tendency to oligomerize. As disclosed herein, in further embodiments of the present invention, fluorescent proteins with improved maturation effects are provided. In particular, the present invention provides dimer and monomeric red fluorescent protein (RFP) and RFP variants with improved maturation effects. In an embodiment of the invention, the fluorescent maturation effect in the protein variant is reduced compared to other variants, including the parent protein, containing at least one mutation that reduces or eliminates the tendency of the fluorescent protein to oligomerize Provided are fluorescent protein variants containing at least one mutation that improves.

  The cloning of red fluorescent protein from Discosoma (DsRed) is of great interest due to its great potential as a tool for advancing cell biology. However, careful study of the properties of this protein reveals several issues that make DsRed a genetically encoded indicator for tracking gene expression and for fluorescence resonance energy transfer (FRET) It has been found that, like Aequorea GFP and its blue, cyan and yellow variants, which have found wide use as both donor / acceptor pairs, can prevent wide acceptance. Broadening the spectrum of available colors can provide a clear new label for multicolor tracking of fusion proteins and, together with GFP, a new FRET donor / acceptor that is superior to the currently preferred cyan / yellow pair Pairs can be provided.

  The three most urgent problems for 28 kDa DsRed are its strong oligomerization tendency, its slow maturation, and inefficient maturation from species with its GFP-like spectrum to the final RFP spectrum.

  A variety of techniques have been used to confirm that DsRed is an obligate tetramer both in vitro and in vivo. For a number of reasons, the oligomeric state of DsRed is problematic for application to fuse it with the protein of interest to monitor the latter's tracking or interaction. Using purified protein, it was shown that DsRed requires more than 48 hours to reach> 90% of its maximum red fluorescence (see below). During the maturation process, the green intermediate accumulates first and then slowly converts to the final red form. However, the conversion to the green component does not proceed until complete, so some fractions with aged DsRed remain green. The main disadvantage due to incomplete maturation is that the excitation spectrum spreads considerably into the green wavelength due to energy transfer between the green and red species within the tetramer. This is a particularly serious problem because it overlaps the excitation spectrum of potential FRET partners such as GFP.

An early report of DsRed cloning reported an in vivo application to mark the behavior of Xenopus blastomeres one week after development (Matz et al., Nature Biotechnology 17: 969-973 [1999]). As disclosed herein, DsRed characterization is the time it takes for red fluorescence to appear, the pH sensitivity of the chromophore, the intensity of the fluorescence that absorbs the chromophore light, and the ease with which the protein can be photobleached. , And whether the protein is usually present as a monomer or oligomer in the solution. The results demonstrate that DsRed provides a useful complement or alternative to GFP and its spectral mutants. In addition, DsRed mutants that are non-fluorescent or have a blocked or delayed green to red emission conversion and mutants whose final fluorescence is substantially red shifted from wild-type DsRed are characterized. (Baird et al . , Proc. Natl. Acad. Sci., USA 97: 11984-11989, 2000; Gross et al . , Proc. Natl. Acad. Sci., USA 97: 11990-11995, 2000, respectively. Incorporated by reference).

Red fluorescent protein variants with improved maturation effect The present invention is an RFP variant that exhibits more effective chromophore maturation than a control wild type RFP or control RFP variant, which is a control (wild type or variant) The RFP variants are provided as a result of at least one amino acid modification within the sequence.

  Wild type RFP protein typically consists of contamination by about 70% red protein and about 30% green immature protein. Through careful mass spectrometry and biochemical studies, it has been confirmed that the Cα-N bond of Q66 in DsRed is oxidized when the protein matures to the red form, and then the amino acid at position 66 involved in chromophore maturation. Further research on the role proceeded. By site-directed mutagenesis, substitution of methionine (M) for native glutamine (Q) at amino acid position 66 shows a deeper pink color than wild-type protein and immature green-type content than wild-type DsRed It was confirmed that a protein with a small amount was obtained. Furthermore, the Q66M DsRed mutant was found to mature more rapidly than the wild type DsRed. Further experiments have shown that the Q66M mutation retains its advantageous properties when introduced into non-tetramerized (ie, dimer or monomer) DsRed mutants that contain additional mutations. Further details of these findings are illustrated in the examples below. Thus, the RFP variants of the present invention can be derived from naturally occurring (wild-type) RFPs or from spectral variants or mutants thereof, and more effectively at least one chromophore maturation. Contain mutations.

  While the present invention has been described with reference to the DsRed Q66M mutation, it will be understood that it is not so limited. Mutations of other amino acids within the wild-type DsRed sequence that play a role in chromophore structure, orientation and / or maturation can also result in DsRed variants with improved maturation effects. Similarly, RFP variants that show improved maturation effects can also be produced by amino acid modifications (eg, substitutions) at corresponding (homologous) positions or regions in other RFPs. All such variants are specifically within the scope of the invention, either alone or in combination with other mutations (substitutions, insertions and / or deletions) within the wild type RFP sequence. That is, an example of a further amino acid modification that would improve the maturation of both wild-type DsRed protein and DsRed variants including Q66M DsRed is a substitution at amino acid position 147 of wild-type DsRed. The preferred substitution at this position is T147S, but other substitutions that provide similar improvements in spectral properties and, in particular, maturation effects and potential rates are possible. In particular, substitution of amino acids with properties similar to threonine (T) is expected to produce such variants.

  In certain embodiments, the present invention relates to RFP variants that have a reduced tendency to tetramerize with improved maturation efficiency as a result of one or more additional mutations within the RFP molecule. In particular, in this embodiment, the invention relates to non-tetramerized DsRed mutants such as dimers or monomers that exhibit improved maturation efficiency compared to the corresponding non-tetramerized DsRed mutant. Further details on the design and preparation of such variants are provided below.

  Briefly, the RFP variants of the present invention can be derived from RFPs that have a tendency to dimerize or tetramerize. As disclosed herein, the RFP variants of the present invention can be derived from naturally occurring RFPs or from spectral variants or from mutants thereof, and at least one mutation that improves maturation efficiency , And optionally contains a mutation that eliminates at least one additional RFP oligomerization tendency.

  The fluorescent protein variants of the present invention may be derived from any fluorescent protein known to oligomerize, such as, for example, Aequorea victoria GFP (SEQ ID NO: 10), Renilla (Renilla reniformis) GFP, green fluorescent protein (GFP) such as Phialidium gregarium GFP; red fluorescent protein (RFP) such as Discosoma RFP (SEQ ID NO: 1); or related to GFP or RFP Examples include fluorescent proteins. Thus, the fluorescent proteins are cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), enhanced GFP (EGFP; SEQ ID NO: 13), enhanced CFP (ECFP; SEQ ID NO: 14), enhanced YFP (EYFP; SEQ ID NO: 15). ); DsRed fluorescent protein (SEQ ID NO: 1), any other homologue, or a mutant or variant of such fluorescent protein.

  As disclosed herein, the tendency of the fluorescent protein variants of the present invention to oligomerize is reduced or eliminated. There are two basic approaches to reduce the tendency to form intermolecular oligomers of fluorescent proteins such as RFP: (1) introducing oligomerization into appropriate regions of fluorescent proteins such as RFP molecules Can be reduced or eliminated, and (2) the two subunits of the fluorescent protein can be operably linked, eg, RFPs can be linked to each other by a linker such as a peptide linker . If it is reduced or eliminated by the following procedure (1), usually to restore fluorescence that is lost or very impaired as a result of typically introducing mutations into the oligomer interface It is necessary to introduce further mutations into the molecule.

Red fluorescent protein mutants with reduced tendency to oligomerize The present invention has been reduced or eliminated by the introduction of amino acid substitutions that reduce or abolish the tetramerization tendency of the component monomers. Fluorescent protein variants are provided. In one embodiment, the resulting structure has a tendency to dimerize. In other embodiments, the resulting structure tends to remain monomeric.

  Various dimer molds can be produced. For example, an AB oriented dimer may be formed, or an AC oriented dimer may be formed. However, with the production of dimer molds, fluorescence or fluorescence maturation rates may be lost. The present invention provides a method of making a dimer mold that displays detectable fluorescence and, moreover, fluorescence with an advantageous rate of maturation.

  In one embodiment, the dimer may be an intermolecular dimer. Furthermore, the dimer may be a homodimer (including two molecules of the same species) or a heterodimer (including two different types of molecules). In a preferred embodiment, the dimer can spontaneously form at physiological conditions. Molecules that form such types of structures as used herein are said to have a reduced tendency to oligomerize because the ability of the monomer units to form tetrameric intermolecular oligomers is low or absent.

  A non-limiting illustrative example of such a dimer red fluorescent protein variant is described herein and named “dimer 2”. The dimer 2 nucleotide sequence is listed in SEQ ID NO: 7 and FIG. The dimer 2 polypeptide is listed in SEQ ID NO: 6 and FIG.

  In an attempt to create an even more advantageous form of the DsRed mutant dimer, we have devised a new strategy to synthesize “tandem” DsRed mutant dimers. This approach utilizes covalent tethering of two prepared monomeric DsRed units to obtain DsRed dimer forms with advantageous properties. The basic strategy was to fuse two copies of the AC dimer with a polypeptide linker so that important dimer interactions are satisfied via intermolecular contact with tandem partners encoded within the same polypeptide. . As used herein, such functionally linked homodimers or heterodimers are referred to as “tandem dimers”, which are substantially less prone to form a tetrameric structure.

  Illustrative examples of such tandem red fluorescent protein variant dimers include, but are not limited to, covalently linked by a peptide linker (preferably about 9 to about 25, more preferably about 9 to 20 amino acid residues long). 2 monomer units of dimer 2 (SEQ ID NO: 6) linked in a functionally functional manner. Examples of such a linker that can be used in the present invention include, but are not limited to, for example, a 9-residue linker RMGTGSGQL (SEQ ID NO: 16), a 12-residue linker GHGTGSTGSGSS (SEQ ID NO: 17), a 13-residue linker RMGSTSGSTKGQL ( SEQ ID NO: 18), or the 22-residue linker RMGSTSGSGKPGSGEGSTKGQL (SEQ ID NO: 19). As described above, such tandem dimer subunits preferably contain mutations compared to the wild-type DsRed sequence of SEQ ID NO: 1 for the purpose of preserving / restoring fluorescence properties. An illustrative example of a tandem red fluorescent protein dimer herein is a mutant DsRed in which at least one of the monomers has the amino acid sequence of SEQ ID NO: 6, preferably from about 9 to about 25, more preferably from about 10 to A dimer consisting of two monomers operably linked by a peptide linker, including any of the 9, 12, 13, and 22 residue linkers described above, approximately 20 amino acid residues long. Yet another illustrative example of a tandem red fluorescent protein dimer herein consists of two identical or different DsRed mutant monomer subunits, at least one of which is within the DsRed polypeptide of SEQ ID NO: 1: Substitutions: N42Q, V44A, V71A, F118L, K163Q, S179T, S197T, T217S (internal mutation in β-barrel); R2A, K5E and N6D (mutation that reduces aggregation); I125R and V127T (AB interface mutation) And tandem dimers containing T21S, H41T, C117T and S131P (miscellaneous surface mutations). Just like other illustrative dimers, the two monomer subunits are peptide linkers, preferably about 9 to about 25, more preferably about 10 to about 25 amino acid residues in length, such as 9, 12, 13, and It can be fused by any of the 22 above residue linkers. Short linkers are generally preferred over long linkers unless they significantly retard affinity maturation or otherwise interfere with dimer fluorescence and spectral properties. As described above, the two monomer subunits in the dimer may be the same or different. Thus, for example, one subunit can be a wild-type DsRed monomer of SEQ ID NO: 1 operably linked to a mutant DsRed polypeptide, eg, a DsRed listed above or otherwise disclosed herein. Any of the mutants may be used. Monomers must be linked such that important dimer interactions are satisfied through intermolecular contact with tandem partners. The peptide linker is preferably protease resistant. The peptide linkers specifically disclosed herein are for illustration only. One skilled in the art will appreciate that other peptide linkers, preferably protease resistant linkers, are also suitable for the purposes of the present invention. See, for example, Whitlow et al., Protein Eng 6: 989-995 (1993).

In one embodiment disclosed in more detail in the following example, a novel approach is used to link wild type DsRed intermolecular oligomers by linking the C terminus of the A subunit and the N terminus of the B subunit. The trend was overcome by making a tandem dimer via a flexible linker. Based on the crystal structure of the DsRed tetramer, a 10-20 residue linker, such as an 18 residue linker (Whitlow et al . , Prot. Eng. 6: 989-995, 1993, supra, which is incorporated herein by reference) Is expected to be long enough to extend from the C terminus of the A subunit to the N terminus of the C subunit (approximately 30 cm), but not long enough to extend to the N terminus of the B subunit (greater than 70 cm). It was. Thus, “oligomerization” in a tandem dimer is an intramolecular, ie, tandem dimer of DsRed (tDsRed), eg, encoded by a single polypeptide chain. Furthermore, another dimer red fluorescent protein was obtained by the combination of tDsRed and the I125R mutant (tDsRed-I125R). This strategy is generally known that the distance between the N-terminus of one protein and the C-terminus of a dimer partner can be linked in a functional manner with monomers using a linker of appropriate length. It should be appreciated that it can be applied to any protein system. In particular, this strategy uses other targeted fluorescent mutagenesis methods disclosed herein to form obligate dimers that have interesting spectral properties but are difficult to destroy. It can be used to modify it otherwise.

Mutagenesis strategy to produce dimeric and monomeric red fluorescent proteins The present invention has a tendency to form reduced tetramer oligomers due to the presence of one or more mutations in the fluorescent protein (ie, form tetramers) Fluorescent protein variants are provided that have a reduced or eliminated tendency. As disclosed herein, mutations are introduced into DsRed to identify DsRed mutants with reduced oligomerization activity, including, for example, DsRed-I125R, a DsRed mutant of SEQ ID NO: 20. did. Strategies that produce DsRed mutants introduce a mutation in DsRed that is expected to interfere with the dimer interface (AB or AC, see FIGS. 1 and 2) to prevent tetramer formation Is involved. This strategy results, for example, in the production of DsRed mutants with reduced propensity for tetramer formation by disrupting the AB interface using a single substitution of isoleucine 125 with arginine (I125R).

  A strategy to reduce the oligomeric state of DsRed has been to replace key dimer interface residues with charged amino acids, preferably arginine. Dimer formation appears to require a target residue to interact via symmetry with the same residue of the dimer partner. The interaction must be destroyed at the expense of high energy resulting from the close placement of the two positive charges. Initial attempts to break the DsRed AC interface (see Figure 2A) with single mutations T147R, H162R, and F224R consistently gave non-fluorescent proteins. However, the AB interface was somewhat less elastic and proved to be able to be destroyed by a single mutation I125R, suffering from the increase in green component and over 10 days to full maturation, resulting in a poor red fluorescent dimer.

  Further illustrative examples of mutations (amino acid substitutions) that can improve the fluorescence properties of I125R include mutations in at least one of amino acid positions 163, 179 and 217 within SEQ ID NO: 1. In preferred embodiments, the I125R variant comprises at least one of the K163Q / M, S179T and T217S substitutions. Further illustrative variants may contain additional mutations at positions N42 and / or C44 within SEQ ID NO: 1. Yet another group of illustrative DsRed dimers includes additional mutations in at least one of residues I161 and S197 within SEQ ID NO: 1. Specific examples of DsRed mutants obtained by this mutagenesis procedure include DsRed-I125R, S179T, T217A, and DsRed-I125R, K163Q, T217A.

  We point out that there is a discrepancy in the naming convention of DsRed subunits in the prior art. As shown in FIG. 1, one convention designates an ABCD subunit. However, the different arrangement shown in FIG. As shown in the model of FIG. 1, the AC interface in this figure is equivalent to the AB interface shown in FIG. The meaning of the subunit interface of the present invention is based on the convention used in FIG. 2, except for FIG.

  A similar directed mutagenesis strategy starting from T1-I125R (see FIG. 10A, library D1) was performed and finally dimer 1 was identified. Dimer1 was slightly better than wt DsRed in both luminance and maturation rate, but had a substantially green peak comparable to that of T1. Dimer1 was also slightly blue shifted with an excitation maximum at 551 nm and an emission maximum at 579 nm. Error-prone PCR of dimer 1 (Figure 10A, library D2) finds dimer 1.02 containing mutant V71A in the hydrophobic core of the protein and effectively non-green component in the excitation spectrum Obtained. The second round of random mutagenesis (Figure 10A, library D3) identified a mutant K70R that further reduced green excitation, S197A that redshifts the dimer back to the DsRed wavelength, and T217S that greatly improved the maturation rate. . Unfortunately, K70R and S197A were relatively slow to mature and T217S had a green peak comparable to DsRed. Two more rounds of directed mutations were performed using dimer 1.02 as a template; first focused on the three positions identified above (FIG. 11A, library D3), and the second was C117, F118. , F124, and V127 were focused (FIG. 10A, library D4).

  For all four generations, we continued the directed evolution strategy to produce the optimal dimer mutant and named it dimer 2 (illustrated in Figure 2B). This variant contains 17 mutations, 8 of which are inside the β-barrel (N42Q, V44A, V71A, F118L, K163Q, S179T, S197T and T217S), 3 were found in T1 Mutations that reduce aggregation (R2A, K5E and N6D; Bevis and Glick, Nat. Biotechnol., 20: 83-87 [2002]; and Yanushevich et al., FEBS Lett., 511: 11-14 [2002] (See) 2 are AB interface mutations (I125R and V127T) and 4 are miscellaneous surface mutations (T21S, H41T, C117T and S131P). The dimer 2 nucleotide sequence is given in SEQ ID NO: 7 and FIG. The dimer 2 polypeptide is provided in SEQ ID NO: 6 and FIG.

  The final product of the mutagenesis approach described herein is a monomeric red fluorescent protein termed mRFP1 that contains the following mutations within the wild-type DsRed sequence of SEQ ID NO: 1: N42Q, V44A, V71A , K83L, F124L, L150M, K163M, V175A, F177V, S179T, V195T, S197I, T217A, R2A, K5E, N6D, I125R, V127T, I180T, R153E, H162K, A164R, L174D, Y192A, Y194K, H222S, L223T L225A, T21S, H41T, C117E, and V156A. Of these, the first 13 mutations are inside the β-barrel. Of the remaining 20 external mutations, 3 are aggregation-reducing mutations (R2A, K5E, and N6D), 3 are AB interface mutations (I125R, V127T, and I180T), and 10 is an AC interface mutation (R153E, H162K, A164R, L174D, Y192A, Y194K, H222S, L223T, F224G, and L225A), and four are additional beneficial mutations (T21S, H41T, C117E, and V156A). The mRFP1 nucleotide sequence is given in SEQ ID NO: 9 and FIG. The mRFP1 polypeptide is provided in SEQ ID NO: 8 and FIG.

  Although mRFP1 is believed to be optimized in many respects, those skilled in the art will recognize that other mutations in these and other regions of the wild-type DsRed amino acid sequence (SEQ ID NO: 1) are also qualitative for the wild-type DsRed protein. It will be appreciated that monomeric DsRed variants can be generated that retain unique red fluorescence properties. Thus, mRFP1 serves merely as an illustration and embodiments of the present invention are in no way limited to this particular monomer.

  In particular, monomeric DsRed variants herein, such as mRFP1, can be further modified to alter the spectral and / or fluorescent properties of DsRed. For example, based on experience with GFP, it is known that in the excited state, the electron density of the chromophore tends to shift from the phenolate to the carbonyl end. Thus, substitutions that increase the positive charge at the carbonyl terminus of the chromophore tend to decrease the energy of the excited state and cause a red shift in protein absorption and emission wavelength maxima. Decreasing the positive charge near the carbonyl end of the chromophore has the opposite effect and tends to cause a blue shift in protein wavelength. Similarly, mutations have been introduced in DsRed to produce mutants with altered fluorescent properties.

  Charged (ionized D, E, K, and R), bipolar (H, N, Q, S, T, and uncharged D, E, and K), and polarizable side chains (eg, C, F, H, Amino acids with M, W and Y) are useful for modifying the oligomerization ability of fluorescent proteins, particularly when they are replaced with amino acids having uncharged, nonpolar or nonpolarizable side chains.

  Similarly, monomers of other oligomerized fluorescent proteins can be prepared according to similar mutagenesis strategies, and these are intended to be within the scope of the present invention.

Anthozoan Fluorescent Protein Variants The mutagenesis methods provided by the present invention can be utilized to create advantageous fluorescent protein variants with reduced oligomerization (ie, tetramerization) ability. It is possible and also intended to find uses similar to those of the Discosoma DsRed mutant protein. It is known in the art that DsRed proteins are members of a family of highly related cognate proteins that share a high degree of amino acid identity and protein structure (see, eg, Labas et al., Proc. Natl. Acad. Sci. USA 99: 4256-4261 [2002]; and Yanushevich et al., FEBS Letters 511: 11-14 [2002]). These alternative fluorescent proteins are further advantageous because they have the ability to fluoresce at a different wavelength than does Discosoma DsRed. If dimeric or monomeric forms of these proteins can be produced, they can have great experimental potential as fluorescent markers.

  Anthozoan species for which the relevant fluorescent proteins have been identified include, but are not limited to, Anemonia species, Clavularia species, Condylactis species, Heteractis species , Renilla species, Ptilosarcus species, Zoonthus species, Scolymia species, Montastraea species, Ricordea species, Goniopara species, and the like.

A fluorescent protein fused with a fusion protein target protein containing a tandem dimer and a monomer can be prepared using, for example, a recombinant DNA method and used as a marker for identifying the position and amount of the target protein to be produced. Accordingly, the present invention provides a fusion protein comprising a fluorescent protein variant moiety and a polypeptide of interest. The polypeptide of interest can be of any length, for example, approximately, provided that the fluorescent protein component of the fusion protein can fluoresce or can be induced to fluoresce when exposed to electromagnetic radiation of the appropriate wavelength. The length may be 15 amino acid residues, about 50 residues, about 150 residues, or up to about 1000 or more amino acid residues. The polypeptide of interest may be, for example, a peptide tag such as a polyhistidine sequence, a c-myc epitope, a FLAG epitope; to perform a function in a cell expressing a fusion protein containing an enzyme or contains a fusion protein May be an enzyme that can be used to identify cells that do; a protein for testing the ability to interact with one or more proteins in the cell, or disclosed or otherwise desired herein. Any other protein may be used.

  DsRed, a Discosoma (coral) red fluorescent protein disclosed herein, can be used as a complement or alternative to GFP or its spectral variants. In particular, the present invention encompasses fusion proteins of any of the tandem dimers and monomeric DsRed fluorescent proteins discussed above with altered spectral and / or fluorescent properties, and variants thereof.

  Also provided is a fusion protein comprising a fluorescent protein variant operably linked to one or more of the polypeptides of interest. The polypeptide of the fusion protein may be linked via a peptide bond, or the fluorescent protein variant may be linked to the polypeptide of interest via a linker molecule. In one embodiment, the fusion protein is a recombinant nucleic acid containing a polynucleotide encoding a fluorescent protein variant operably linked to one or more polynucleotides encoding one or more polypeptides of interest. Expressed from the molecule.

  The polypeptide of interest may be any polypeptide, eg, a peptide tag such as a polyhistidine peptide, or a cellular polypeptide such as an enzyme, G-protein, growth factor receptor, or transcription factor; Or one of two or more proteins forming a complex. In one embodiment, the fusion protein is a donor fluorescent protein variant, an acceptor fluorescent protein variant, and a tandem fluorescent protein variant construct comprising a peptide linker moiety that binds the donor and the acceptor. The amino acid emits characteristic light of the donor, and when the donor is excited, the donor and the acceptor exhibit fluorescence resonance energy transfer, and the linker moiety substantially does not emit light that excites the donor. The above construction may be used. Thus, the fusion proteins of the present invention may include two or more functionally linked fluorescent protein variants, which may be directly or indirectly linked, and further for one or more purposes. The polypeptide may be included.

Preparation of DsRed Dimers and Monomers The present invention is also operatively linked to a protein wherein the protein is a dimeric fluorescent protein, a tandem dimeric fluorescent protein, a fluorescent protein variant that may be a monomeric protein, or one or more polypeptides of interest. Also provided are polynucleotides that encode fusion proteins comprising fluorescent proteins. In the case of a tandem dimer, the entire dimer can be encoded by one polynucleotide molecule. If the linker is a non-peptide linker, the two subunits can be separately encoded and encoded by separate polynucleotide molecules and then linked by methods known in the art.

  The present invention further relates to vectors containing polynucleotides and the like, and host cells containing polynucleotides or vectors. Also provided is at least one polynucleotide recombinant nucleic acid molecule that encodes a fluorescent protein variant operably linked to one or more other polynucleotides. One or more other polynucleotides may be, for example, transcriptional regulatory elements such as promoters or polyadenylation signal sequences, or translational regulatory elements such as ribosome binding sites. Such recombinant nucleic acid molecules can be contained in a vector, which can be an expression vector, and the nucleic acid molecule or vector can be contained in a host cell.

Vectors generally contain the elements necessary for replication in prokaryotic or eukaryotic host systems or both, if desired. Such vectors, including plasmid vectors and viral vectors such as bacteriophages, baculoviruses, retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Semliki forest viruses and adeno-associated virus vectors are known and can be purchased from commercial sources. (Promega, Madison WI; Stratagene, La Jolla CA; GIBCO / BRL, Gaithersburg MD) or can be constructed by those skilled in the art (eg, Meth. Enzymol. , Vol. 185, Goeddel, Ed. (Academic Press, Inc. 1990); Jolly, Canc . Gene Ther. 1: 51-64, 1994; Flotte, J. Bioenerg. Biomemb. 25: 37-42, 1993; Kirshenbaum et al., J. Clin. Invest. 92: 381-387, 1993; each of which is incorporated herein by reference).

  The vector containing the polynucleotide encoding the fluorescent protein variant may be a cloning vector or an expression vector, and may be a plasmid vector, a viral vector, or the like. In general, a vector contains a selectable marker that does not depend on what the polynucleotide of the invention encodes, and further contains a transcriptional or translational regulatory element, and is tissue-specific for a polynucleotide operably linked thereto. A promoter sequence capable of conferring expression, which is not required, but may be a polynucleotide encoding a fluorescent protein variant (eg, a tandem dimer fluorescent protein), thus introducing the introduced vector and Provides a means for selecting a particular type of cell from a mixed population of cells containing the recombinant nucleic acid molecule contained therein.

  If the vector is a viral vector, it can be selected based on its ability to infect one or several specific cell types with relatively high efficiency. For example, viral vectors can also be derived from viruses that infect specific cells of the organism of interest (eg, vertebrate host cells such as mammalian host cells). Viral vectors have been developed for use in certain host systems, particularly mammalian systems, such as retroviral vectors such as those based on human immunodeficiency virus (HIV), Adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, vaccinia virus vectors, etc. (Miller and Rosman, Bio Techniques 7: 980-990, 1992; Anderson et al., Nature 392: 25-30, Addendum, 1998; Verma And Somia, Nature 389: 239-242, 1997; Wilson, New Engl. J. Med. 334: 1185-1187 (1996), each of which is incorporated herein by reference).

  Recombinant production of a fluorescent protein variant (which may be a component of a fusion protein) involves expressing a polypeptide encoded by the polynucleotide. Polynucleotides encoding fluorescent protein variants are useful starting materials. Polynucleotides encoding fluorescent proteins are disclosed herein or known in the art and are obtained using routine methods, and then the propensity for oligomerization of the encoded fluorescent protein is observed. It can be modified to lack. For example, a polynucleotide encoding GFP can be isolated by PCR of cDNA from A. victoria using primers based on the DNA sequence of Aequorea GFP (SEQ ID NO: 21). A polynucleotide encoding red fluorescent protein (DsRed) from Discosoma can also be isolated by PCR of cDNA of Discosoma coral or obtained from commercially available DsRed2 or HcRed1 (CLONTECH). PCR methods are well known in the art and routinely performed (eg, US Pat. No. 4,683,195; Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51: 263, 1987; edited by Erlich, “PCR Technology (PCR technology, Stockton Press, NY, 1989)). A mutant form of the fluorescent protein can then be generated by site-directed mutagenesis of the polynucleotide encoding the fluorescent protein. Similarly, is a tandem fluorescent protein expressed from a polynucleotide prepared by PCR, for example, using a primer that can encode a peptide linker that functionally links the first monomer and at least a second monomer of the fluorescent protein? Or by other methods.

  Construction of the expression vector and expression of the polynucleotide in the transfected cells also involves the use of molecular cloning techniques well known in the art (Sambrook et al., “Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989); edited by Ausubel et al., “Current Protocols in Molecular Biology” (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. 1990, and addendum) )). The expression vector contains, for example, an expression control sequence operably linked to the polynucleotide sequence of interest encoding the fluorescent protein variant shown above. Expression vectors can be adapted to function in prokaryotes or eukaryotes by including appropriate promoters, replication sequences, markers, and the like. The expression vector can be transfected into a recombinant host cell for expression of the fluorescent protein variant, and the host cell can be selected for high levels of expression, eg, to obtain large amounts of isolated protein. it can. Host cells can be maintained in cell culture or can be cells in vivo in an organism. Fluorescent protein variants can be produced by expression from a polynucleotide encoding the protein in a host cell such as E. coli. Aequorea GFP-related fluorescent protein is best expressed, for example, by cells cultured at about 15-30 ° C, although higher temperatures (eg, 37 ° C) may be used. After synthesis, the fluorescent protein is stable at higher temperatures and can be used for assays at such temperatures.

  The expressed fluorescent protein variant (which may be a tandem dimer fluorescent protein or a non-oligomerized tandem fluorescent protein) may be operably linked to the first polypeptide of interest, and further to the second of interest. To a polypeptide tag, such as a peptide tag (which can be used to facilitate the isolation of fluorescent protein variants containing any other polypeptide linked thereto). For example, a polyhistidine tag containing 6 histidine residues is incorporated at the N-terminus or C-terminus of a fluorescent protein variant, which is then isolated in a single step using nickel-chelate chromatography. be able to. An additional peptide tag comprising a c-myc peptide, a FLAG epitope, or any ligand (or cognate receptor) comprising any peptide epitope (or antibody or antigen-binding fragment thereof that specifically binds to the epitope) is Are well known in the art and can be used as well (see, eg, Hopp et al., Biotechnology 6: 1204 (1988); US Pat. No. 5,011,912, each of which is hereby incorporated by reference) .

Kits of the Invention The present invention also provides kits to facilitate and / or standardize the use of the composition and to facilitate the methods of the invention. Materials and reagents for performing these various methods can be provided in the kit. As used herein, the term “kit” means a combination of articles that facilitates a process, assay, analysis or manipulation.

  The kit contains chemical reagents (eg, polypeptides or polynucleotides) as well as other components. In addition, the kit of the present invention may also include, for example, but not limited to, devices and reagents for sample collection and / or purification, devices and reagents for product collection and / or purification, reagents for bacterial cell transformation Pre-transformed or transfected host cells, sample tubes, holders, trays, racks, dishes, plates, kit user instructions, solutions, buffers or other chemical reagents, suitable for use for standardization Samples, normalizations, and / or control samples may be included. The kit of the present invention may also be packaged, for example, in a ribbed box for convenient storage and safe shipping.

  In some embodiments, for example, the kit of the invention comprises a fluorescent protein of the invention, a polynucleotide vector (eg, a plasmid) encoding the fluorescent protein of the invention, a bacterial cell line suitable for propagating the vector, And reagents for purifying the expressed fusion protein. Alternatively, the kits of the invention may provide the reagents necessary to perform mutagenesis of Anthozoan fluorescent protein to produce protein variants with reduced tendency to oligomerize.

  A kit may comprise one or more polynucleotides encoding one or more fluorescent protein variants, or polypeptides thereof, which may be part of one or more compositions of the invention, eg, a fusion protein. Good. The fluorescent protein variant may be a fluorescent protein with a low tendency to oligomerize, such as a non-oligomerized monomer, or may be a tandem dimer fluorescent protein and the kit contains multiple fluorescent protein variants The plurality may be a plurality of sudden fluorescent protein mutant variants or a plurality of tandem dimeric fluorescent proteins, or a combination thereof.

  The kits of the invention may also contain one or more recombinant nucleic acid molecules that in part encode a fluorescent protein variant, which may be the same or different, and may further include, for example, a restriction end A nuclease recognition site or recombinase recognition site, or a functionally linked second polynucleotide containing or encoding any polypeptide of interest may be included. The kit may further comprise instructions for using the components of the kit, particularly the composition of the present invention contained in the kit.

  Such kits may be particularly useful when one of skill in the art can conveniently select one or more proteins with favorable fluorescent properties for a particular application and thus provide a plurality of different fluorescent protein variants. . Similarly, kits containing multiple polynucleotides that encode different fluorescent protein variants provide many advantages. For example, a polynucleotide is made to contain a convenient restriction endonuclease or recombinase recognition site, thus facilitating functional linkage of the polynucleotide to a regulatory element or to a polynucleotide encoding a polypeptide of interest. Or, if desired, two or more polynucleotides encoding fluorescent protein variants can be operably linked to each other.

Uses of fluorescent protein mutants Fluorescent protein mutants having the characteristics of the present invention are useful in any method using fluorescent proteins. That is, fluorescent protein variants, including monomeric, dimeric, and tandem dimeric fluorescent proteins, can be used in many methods where fluorescent markers are already used (eg, for use in detection assays such as immunoassays or hybridization assays, or cellular Coupling of fluorescent protein variants to antibodies, polynucleotides or other receptors to track the movement of proteins in them) is useful as a fluorescent marker. For intracellular tracking studies, a first (or other) polynucleotide encoding a fluorescent protein variant is fused to a second (or other) polynucleotide encoding a protein of interest, if desired. For example, the construct can be inserted into an expression vector. When expressed in cells, it is possible to localize a protein of interest based on fluorescence without worrying that the localization of the protein is an artifact caused by oligomerization of the fluorescent protein component of the fusion protein. it can. In one embodiment of this method, the two proteins of interest are independently fused with two fluorescent protein variants having different fluorescent properties.

  Fluorescent protein variants having the properties of the present invention are useful in systems for detecting the induction of transcription. For example, a nucleotide sequence encoding a non-oligomerized monomer, dimer or tandem dimer fluorescent protein can be fused to a promoter of interest or other expression control sequence (which can be contained in an expression vector) and the construct can be Can be transfected, and the induction of a promoter (or other regulatory element) can be measured by detecting the presence or amount of fluorescence, thereby responsiveness of the receptor-to-promoter signaling pathway A means for observing is possible.

  The fluorescent protein variants of the invention are also useful in applications involving FRET, which can detect events as a function of the movement of fluorescent donors and acceptors towards or away from each other. One or both of the donor / acceptor pair may be a fluorescent protein variant. Such donor / acceptor pairs provide a wide separation between donor excitation and emission peaks and provide good overlap between donor emission and acceptor excitation spectra. The mutant red fluorescent protein or red shift mutant disclosed herein is disclosed herein as such a pair of acceptors.

  FRET can be used to detect cleavage of a substrate with a donor and acceptor bound to the substrate facing away from the cleavage site. When the substrate is cleaved, the donor / acceptor pair is physically separated and FRET is eliminated. Such assays can be performed, for example, by contacting a substrate with a sample and measuring the qualitative or quantitative change in FRET (see, eg, US Pat. No. 5,741,657, which is incorporated herein by reference). Incorporated in the description). The fluorescent protein variant donor / acceptor pair may also be part of a fusion protein bound by a peptide having a proteolytic cleavage site (see, eg, US Pat. No. 5,981,200, which is incorporated by reference). FRET can also be used to detect potential changes across the membrane. For example, placing the donor and acceptor on opposite sides of the membrane and causing them to move across the membrane in response to a voltage change thereby results in a measurable FRET (see, eg, US Pat. No. 5,661,035, which Incorporated by reference).

In other embodiments, the fluorescent proteins of the invention are fluorescent sensors for protein kinase and phosphatase activity or Ca ²⁺ , Zn ²⁺ , cyclic 3 ′, 5′-adenosine monophosphate, and cyclic 3 ′, 5 ′. -Useful for making small ionic and molecular indicators such as guanosine monophosphate.

  Fluorescence in a sample is typically measured using a fluorometer, where excitation radiation from an excitation source having a first wavelength passes through excitation optics, which causes the excitation radiation to be sampled. Will be excited. In response, the fluorescent protein variant in the sample emits radiation having a wavelength different from the excitation wavelength. A light collection system collects light emitted from the sample. The device may include a temperature controller for maintaining the sample at a specific temperature while scanning, and a microtiter plate holding multiple samples for placement so that different wells are exposed. You may have a multi-axis movement stage to move. The multi-axis translation stage, temperature controller, autofocus function, and electronics related to imaging and data collection can be managed by a properly programmed digital computer, which collects the data collected during the assay. , Convert to another format for display. This process can be miniaturized and automated to allow screening of thousands of compounds in a high speed, high throughput mode. These and other methods of performing assays with fluorescent materials are well known in the art (eg, Lakowicz, “Principle of Fluorescence Spectroscopy”) (Plenum Press, 1983); Fluorescence microscopy of living cells in culture ("Fluorescence Microscopy of Living Cells in Culture") Herman, "Resonance energy transfer microscopy" in Part B, Meth. Cell Biol. 30 219-243 (Taylor and Wang; Academic Press) 1989); Turro, "Modern Molecular Photochemistry" (Benjamin / Cummings Publ. Co., Inc. 1978), pages 296-361 References, each of which is incorporated herein by reference).

  Thus, the present invention provides a method for identifying the presence of a molecule in a sample. Such a method is performed, for example, by binding a fluorescent protein variant of the present invention to a molecule and detecting fluorescence by the fluorescent protein variant in a sample suspected of containing the molecule. The molecule to be detected may be a polypeptide, polynucleotide, or any other molecule (eg, antibody, enzyme, receptor) and the fluorescent protein variant may be a tandem dimeric fluorescent protein.

  The sample to be tested can be any sample and can be a biological sample, an environmental sample, or any other sample where it is preferable to determine whether a particular molecule is present therein. Preferably the sample comprises cells or extracts thereof. The cells can be obtained from vertebrates (including mammals such as humans), or non-vertebrates, and can be cells from plants or animals. Cells can be obtained from cultures of such cells (eg, cell lines) or isolated from organisms. Thus, the cells may be contained in a tissue sample, which can be obtained from an organism by any means commonly used to obtain a tissue sample (eg, a human biopsy). When the method is performed using intact living cells or freshly isolated tissue or organ samples, the presence of the molecule of interest in the living cells is identified, for example, to determine the intracellular distribution of that molecule Means are provided. The use of the fluorescent protein variants of the present invention for such purposes has substantial benefits in that the potential for ectopic identification or localization by oligomerization of the fluorescent protein is greatly reduced.

  A fluorescent protein variant can bind to the molecule, either directly or indirectly, using any bond that is stable under the conditions to which the protein-molecule complex is exposed. That is, the fluorescent protein and the molecule may be linked by a chemical reaction between the protein and a reactive group present on the molecule, or the bond contains a linker moiety (which contains a specific reactive group for the fluorescent protein and the molecule. ). It will be appreciated that the appropriate conditions for binding the fluorescent protein variant to the molecule are selected depending on, for example, the chemical nature of the molecule and the desired binding type. If the molecule of interest is a polypeptide, a convenient means of binding the fluorescent protein variant to the molecule is a recombinant nucleic acid molecule (eg, tandem dimer fluorescence operably linked to a polynucleotide encoding the polypeptide molecule). Including the polynucleotide encoding the protein) and expressing them as fusion proteins.

  Also provided are methods of identifying substances or conditions that control the activity of expression control sequences. Such methods include, for example, recombinant nucleic acid molecules (including polynucleotides encoding fluorescent protein variants operably linked to expression control sequences) to control expression of the polynucleotide from the expression control sequences. This is done by exposure to a substance or condition suspected of being able to be detected and detecting the fluorescence of the fluorescent protein variant resulting from such exposure. Such methods identify, for example, chemicals or biological substances (including cellular factors involved in tissue-specific expression from control elements) that contain cellular proteins that can regulate expression from expression control sequences. Useful to do. As such, the expression control sequence may be a transcriptional control element such as a promoter, enhancer, silencer, intron splicing recognition site, polyadenylation site, or the like, or a translational control element such as a ribosome binding site.

  Fluorescent protein variants having the properties of the present invention are also useful in methods for identifying specific interactions between a first molecule and a second molecule. Such a method includes, for example, a first molecule bound to a first fluorescent protein variant that is a donor and a second molecule bound to a second fluorescent protein variant that is an acceptor. Contacting under conditions that allow specific interaction of the molecule with a second molecule; exciting the donor; detecting fluorescence or emission resonance energy transfer from the donor to the acceptor, thereby causing the first molecule to This can be done by identifying the specific interaction of the two molecules. Such interaction conditions may be any conditions in which molecules are predicted or suspected of being able to interact specifically. In particular, if the molecule to be tested is a cellular molecule, the conditions are generally physiological conditions. As such, the method may be performed in vitro using conditions such as buffer, pH, ionic strength, etc. that mimic physiological conditions, or the method may be performed in a cell or cell extraction. You may carry out using a thing.

  Luminescent resonance energy transfer requires energy transfer from a chemiluminescent, bioluminescent, lanthanide, or transition metal donor to the red fluorescent protein moiety. Because the excitation wavelength of red fluorescent protein is longer, it is possible to transfer energy from various donors and over longer distances than is possible for green fluorescent protein variants. Also, because of the longer emission wavelength, it can be detected more efficiently with solid-phase photodetectors, and is particularly valuable for in vivo applications because red light penetrates tissues much more favorably than shorter wavelengths. . Chemiluminescent donors include, but are not limited to, luminol derivatives and peroxysuccinate systems. Bioluminescent donors include, but are not limited to, aequorin, obelin, firefly luciferase, Renilla luciferase, bacterial luciferase, and variants thereof. Lanthanide donors include, but are not limited to, terbium chelates containing an ultraviolet absorbing sensitizer chromophore that is linked to multiple ligand groups to shield metal ions from solvent water. Transition metal donors include, but are not limited to, ruthenium and osmium chelates of oligopyridine ligands. Chemiluminescent and bioluminescent donors do not require excitation light but are energized by the addition of a substrate, whereas metal-based systems require excitation light, but give a longer excited state lifetime and do not want background Facilitates time-gated detection to distinguish fluorescence and scatter.

  The first molecule and the second molecule are cellular proteins and are studied to determine whether these proteins interact specifically or to confirm such interactions; Good. Such a first cellular protein and a second cellular protein may be the same, for example when examining oligomerization ability, for example when testing as a specific binding partner involved in an intracellular pathway, Those proteins may be different. The first molecule and the second molecule may also be polynucleotides and polypeptides, for example, transcriptional regulatory element activity is known or polynucleotide and transcription factor activity to be investigated is known or It may be a polypeptide to be investigated. For example, the first molecule may comprise a plurality of nucleotides, which may be random or may be a variant of a known sequence, for testing sequence transcription regulatory element activity, and the second molecule Are transcription factors and such methods may be useful for identifying novel transcriptional regulatory elements having the desired activity.

  The present invention also provides a method for determining whether a sample contains an enzyme. Such a method can be performed, for example, by contacting a sample with a tundam fluorescent protein variant of the invention; exciting the donor and measuring the fluorescence properties in the sample, in which case the enzyme in the sample The degree of fluorescence resonance energy transfer changes due to the presence of. Similarly, the present invention relates to a method for measuring the activity of an enzyme in a cell. Such a method provides, for example, a cell that expresses a tandem fluorescent protein variant construct in which the peptide linker moiety that connects the donor and the acceptor comprises a cleavage recognition amino acid sequence specific for the enzyme; It can be carried out by measuring the degree of fluorescence resonance energy transfer in the cell. In this case, the degree of fluorescence resonance energy transfer changes due to the presence of enzyme activity in the cell.

  A method for measuring the pH of a sample is also provided. Such methods include, for example, contacting a sample with a first fluorescent protein variant (which may be a non-oligomerized tandem fluorescent protein), where the emission intensity of the first non-oligomerized fluorescent protein is pH By changing the pH between pH 5 and pH 10); exciting the indicator; and measuring the intensity of light emitted by the first fluorescent protein variant at the first wavelength, wherein: The emission intensity of the fluorescent protein variant 1 indicates the pH of the sample. A first fluorescent protein variant useful in this method or any other method of the invention may comprise the two DsRed monomers set forth in SEQ ID NO: 8. It will be appreciated that such fluorescent protein variants are also useful alone or in combination for the various disclosed methods of the present invention.

  The sample used in the method for determining the pH of the sample can be any sample, including, for example, a biological tissue sample, or a cell or fraction thereof. Further, in this method, the sample is brought into contact with the second fluorescent protein variant (here, the luminescence intensity of the second fluorescent protein variant changes as the pH changes from 5 to 10, and the second fluorescent protein variant changes). The protein variant emits light at a second wavelength that is distinctly different from the first wavelength); excites the second fluorescent protein variant; the light emitted by the second fluorescent protein variant at the second wavelength The fluorescence at the second wavelength is compared with the fluorescence at the first wavelength. The first (or second) fluorescent protein variant comprises a targeting sequence, eg, a cell compartmentalization domain, eg, a fluorescent protein variant in a cell, that is cytosolic, endoplasmic reticulum, mitochondrial matrix, chloroplast cavity, inner trans-Golgi. It may include a domain that targets the sac, lysosomal cavity, or endosomal cavity. For example, the cell compartmentalization domain can comprise amino acid residues 1 to 81 of galactosyltransferase, a human type II membrane anchored protein, or amino acid residues 1 to 12 of the presequence of subunit IV of cytochrome c oxidase. .

  In order to further illustrate certain specific embodiments and aspects of the invention, the following examples are provided. The examples are not intended to limit the scope of any aspect of the invention. Although specific reaction conditions and reagents are described, one of ordinary skill in the art will understand alternative or equivalent conditions that also utilize the invention, in which case the alternative or equivalent conditions will depart from the scope of the invention. It is clear that it is not.

Example 1
Construction of dimer and monomer red fluorescent protein
Materials and methods
DsRed mutagenesis and screening
The DsRed gene is amplified from the vector pDsRed-N1 (CLONTECH, Palo Alto, Calif.) Or T1 mutant (provided by BS Glick, University of Chicago) and pRSET _B (Invitrogen ^™ ; Baird et al., Proc. Natl. Acad Sci. USA 97: 11984-11989 [2000]) (4) subcloned. The pRSET _B vector produces a 6xHis-tagged fusion protein, where an N-terminal polyhistidine tag having the following sequence: MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDP (SEQ ID NO: 22) is coupled to an appropriately subcloned sequence.

The resulting construct was used as a template to introduce the I125R mutant, and the QuikChange ^™ site-directed mutagenesis kit (Stratagene®) was used according to the manufacturer's instructions. The complete DsRed wild type cDNA and polypeptide sequence is given in GenBank accession number AF168419. This nucleotide sequence is also given in FIG. 16 and SEQ ID NO: 2. Variants of this nucleotide sequence are also known (Clontech) where the various nucleotide positions have been corrected to accommodate mammalian codon usage preferences. This nucleotide sequence is given in FIG. 25 and SEQ ID NO: 23. The corresponding polypeptides encoded by both of these nucleotide sequences are given in FIG. 17 and SEQ ID NO: 1.

  Similarly, the DsRed T1 variant cDNA nucleotide sequence is provided in FIG. 18 and SEQ ID NO: 5. The corresponding polypeptide is given in FIG. 19 and SEQ ID NO: 4.

  The DsRed amino acid numbering used herein is consistent with the wild-type sequence of GFP, where residues 66-68 of wild-type DsRed (Gln-Tyr-Gly) are GFP (Ser-Tyr-Gly) Homologous to chromophore forming residues 65-67. The amino terminal polyhistidine tag was numbered -33 to -1.

Error Prone PCR PCR prone to mutagenesis errors was performed essentially as described in Griesbeck et al. (J. Biol. Chem., 276: 29188-29194 [2001]). Briefly, the cDNA encoding DsRed in the vector pRSET _B (Invitrogen ^™ ) was treated with error-prone PCR using Taq DNA polymerase. The 5 'primer contains a BamHI site, ends with a starting Met of DsRed, and the 3' primer contains an EcoRI site, ends with a stop codon, except for the initiator methionine, all base mutagenesis of the DsRed open reading frame Made theoretically possible. PCR reaction (38 cycles of annealing at 55 ° C.), 10 μL of 10 × Mg ^{2+ in} PCR buffer (Roche Molecular Biochemicals), 150 μM Mn ²⁺ , 250 μM of 3 nucleotides, 50 μM of residual nucleotides, and 5 ng of template DNA Performed in four 100 μL batches containing.

Mutagenized PCR products were combined, purified by agarose gel electrophoresis, digested with BamHI and EcoRI, and isolated on a QIAGEN® QIAquick ^™ DNA purification spin column according to the manufacturer's instructions. The resulting fragment was ligated into pRSET _B and the crude ligation mixture was transformed by electroporation into E. coli BL21 (DE3) Gold (Stratagene®).

Overlapping extension PCR mutagenesis Semi-random mutations at multiple remote locations were introduced by overlapping extension PCR using multiple fragments essentially as described in Ho et al., Gene 77: 51-59 (1989). . Briefly, PCR amplification of DsRed templates was performed using 2-4 pairs of sense and antisense oligonucleotide primers (Invitrogen ^TM or GenBase) with a semi-degenerate codon at the position of interest (Stratagene®). )) In each reaction. The resulting overlap fragments were gel purified using a QIAGEN® gel extraction kit and recombined by overlap extension PCR with Pfu or Taq DNA polymerase (Roche).

The full-length gene was digested with BamHI / EcoRI (New England BioLabs®) and ligated with T4 ligase (New England BioLabs®) in pRSET _B. Chemically competent E. coli JM109 (DE3) was transformed and grown on LB / agar at 37 ° C.

Bacterial fluorescence screening Bacteria spread on LB / agar plates were screened essentially as described by Baird et al., Proc. Natl. Acad. Sci. USA 96: 11241-11246 (1999). Briefly, the bacterial plate was subjected to a 470 nm (40 nm bandwidth), 540 nm (30 nm bandwidth), or 560 nm (40 nm bandwidth) excitation filter and 530 nm (40 nm bandwidth), 575 nm (long pass) with a 150-W Xe lamp. ) Or 610 nm (long pass) emission filter. Fluorescence was imaged with a cooled charge coupled device camera (Sensys Photometrics, Tucson, AZ) and processed using Metamorph software (Universal Imaging, West Chester, PA).

  The desired fluorescent colonies were cultured overnight in 2 ml of LB supplemented with ampicillin. Bacteria were pelleted by centrifugation and photographed again to confirm that the protein was favorably expressed in culture. For fast mature protein, a fraction with cell pellet was extracted using B-per II (Pierce) to obtain a complete spectrum. DNA was purified from the remaining pellet by QIAGEN® QIAprep® plasmid isolation spin column according to the manufacturer's instructions and DNA sequencing was performed. To confirm the oligomeric state of the DsRed mutant, a single colony of E. coli was restreaked on LB / agar and matured at room temperature. Two to two weeks later, the bacteria were scraped from the plate, extracted using B-per II, analyzed by SDS-PAGE (BioRad) (not boiled), and the gel was imaged using a digital camera. .

Bacterial transformation and DsRed protein purification ligation mixture was transformed into Escherichia coli BL21 (DE3) Gold (Stratagene) by electroporation in 10% glycerol with ligation mixture (0.1 cm cuvette, 12.5 kV / cm, 200Ω, 25μF).

The protein was expressed and purified essentially as described by Baird et al., Proc. Natl. Acad. Sci. USA 96: 11241-11246 (1999). Briefly, when cultured for protein expression, the transformed bacteria are grown in LB containing 100 mg / l ampicillin to an OD ₆₀₀ of 0.6, at which time 1 mM isopropyl β-D-thiogalactoside is used. And induced. The bacteria were allowed to express the recombinant protein for 6 hours at room temperature and then overnight at 4 ° C. The bacteria were then pelleted by centrifugation, resuspended in 50 mM Tris.HCl / 300 mM NaCl, and lysed by a French press. The bacterial lysate was centrifuged at 30,000 × g for 30 minutes, and the protein was purified from the supernatant using Ni-NTA resin (QIAGEN®).

Spectroscopic analysis of the purified protein was performed on a fluorescence spectrometer (Fluorolog-2, Spex Industries), typically in 100 mM KCl, 10 mM MOPS, pH 7.25. All DNA sequencing can be performed using Molecular Pathology Shared Resource, University of California, San Diego, Cancer Center).

Construction of mammalian cell expression constructs including DsRed tandem dimer and chimeric constructs
To construct a tandem dimer of DsRed protein, dimer 2 in pRSET _B was amplified in two separate PCR reactions. The first reaction introduced 5 ′ BamHI and 3 ′ SphI sites, while the second reaction introduced 5 ′ SacI and 3 ′ EcoRI sites. The construct was assembled with a four-part ligation containing the digested dimer 2 gene, a synthetic linker with a phosphorylated and sticky end, and digested pRSET _B. Four different linkers encoding polypeptides of various lengths were used.

哺乳動物細胞で発現するために、DsRed変異体をpRSET_Bから、KpnI制限酵素切断部位およびKozak配列をコードする5'プライマーを用いて増幅した。PCR産物を消化し、pcDNA3中にライゲートし、これを用いて大腸菌（E. coli）DH5αを形質転換した。

For expression in mammalian cells, the DsRed mutant was amplified from pRSET _B using a 5 ′ primer encoding a KpnI restriction enzyme cleavage site and Kozak sequence. The PCR product was digested, ligated into pcDNA3 and used to transform E. coli DH5α.

  A gene encoding a chimeric fusion polypeptide containing DsRed and connexin 43 (Cx43) was constructed. To produce these fusions, Cx43 was first amplified using a 3 'primer encoding a 7 residue linker ending at the BamHI site. The construct was assembled with a three-part ligation containing KpnI / BamHI digested Cx43, BamHI / EcoRI digested enhanced GFP, and digested pcDNA3. For all other fusion proteins (Cx43-T1, -dimer 2, -t dimer 2 (12) and -mRFP1), the gene for the fluorescent protein was ligated into the BxHI / EcoRI digested Cx43-GFP vector.

DsRed protein variant production and characterization
The DsRed mutant was expressed essentially as described in Baird et al., Proc. Natl. Acad. Sci. USA 96: 11241-11246 (1999). All proteins were purified by Ni-NTA chromatography (QIAGEN®) and dialyzed into phosphate buffered saline supplemented with 10 mM Tris, pH 7.5 or 1 mM EDTA. All biochemical characterization experiments were performed essentially as described in Baird et al., Proc. Natl. Acad. Sci. USA 97: 11984-11989 (2000).

The maturation time course was measured using a monochromator (TECAN, Austria) on a Safire 96 well plate reader. All photobleaching (photobleaching) measurements in microdroplets under paraffin oil, Zeiss Axiovert 35 fluorescence with 540nm to (25 nm bandpass) excitation filter that delivered light of 40 × target and 4.5 W / cm ² This was performed using a microscope.

Analytical ultracentrifugated purified recombinant DsRed was dialyzed extensively against PBS, pH 7.4 or 10 mM Tris, 1 mM EDTA, pH 7.5. Sedimentation equilibrium experiments were performed on a Beckman Optima XL-I analytical ultracentrifuge at 20 ° C., and the absorbance at 558 nm was measured as a function of radius. Samples of DsRed were normalized to 3.57 μM (0.25 absorbance units), from which 125 μL aliquots were fed into 6 channel cells. Data were analyzed by nonlinear least squares analysis using the ORIGIN software package supplied by Beckman at 10K, 14K, and 20K rpm overall. The goodness of the fit was assessed on the basis of the amount and randomness of the residues expressed as the difference between the experimental data and the theoretical curve, and also by checking each fit parameter for physical rationality.

Absorption / fluorescence spectra and extinction coefficient fluorescence spectra were measured using a Fluorolog spectrofluorometer (Spex Industries, Edison, NJ). The absorption spectrum of the protein was measured using a Cary UV-Vis spectrometer. For quantum yield measurements, the fluorescence of a solution of DsRed or DsRed mutant in PBS was compared to an equal absorbing solution of rhodamine B and rhodamine 101 in ethanol. The quantum yield calculation included a correction for the refractive index difference between ethanol and water. As for the extinction coefficient measurement, native protein absorption was measured using a spectrometer, and protein concentration was measured by the BCA method (Pierce).

Mammalian cell imaging and microinjection
HeLa cells were transfected via the use of Fugene 6 transfection reagent (Roche) with DsRed mutant or Cx43-DsRed fusion in pcDNA3. Transfected cells were grown for 12 hours to 2 days in DMEM at 37 ° C., and then cells in glucose-supplemented HBSS were imaged at room temperature using a Zeiss Axiovert 35 fluorescence microscope. Individual cells expressing Cx43 fused to the DsRed mutant or contact untransfected cells for control experiments were microinjected with a 2.5% solution of Lucifer Yellow (Molecular Probes, Eugene, OR). Images were acquired and processed with the Metafluor software package (Universal Imaging, West Chester, PA).

result
Stepwise Evolution of DsRed Molecules The present invention provides a method for stepwise evolution of tetramer DsRed into a dimer and then either into a two-copy genetic fusion of proteins, ie a tandem dimer or to a true monomer called mRFP1 To do. Disrupting each subunit interface by insertion of arginine initially impairs the resulting protein, but the red fluorescence is a random and directed mutation that reaches a total of 17 substitutions in dimer and 33 substitutions in mRFP1. I was able to rescue by triggering. The fusion of the gap junction protein connexin 43 to mRFP1 formed a fully functional junction, but similar fusions to tetramers and dimers failed. mRFP1 has a slightly lower extinction coefficient, quantum yield and photostability than DsRed, but mRFP1 matures more than 10 times (> 10 ×) and thus exhibits similar brightness in living cells. Furthermore, the excitation and emission peaks of mRFP1, 584 and 607 nm, are approximately 25 nm red shifted from DsRed, which should give greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.

  The consensus view is that the monomeric form of DsRed will be essential if one always tries to reach its full potential as a genetically encoded red fluorescent tag (Remington, Nat. Biotechnol., 20: 28-29 [2002]). The present invention provides directed evolution and preliminary characterization of the first monomeric red fluorescent protein. The present invention provides an independent alternative to GFP in the construction of fluorescence-tagged fusion proteins.

Directed and random evolution of DsRed dimers
The basic strategy for reducing the oligomeric state of DsRed has been to replace critical hydrophobic residues in the dimer interface with charged residues such as arginine. The high energy price of burying charged residues within a non-polar hydrophobic interface or placing two positive charges in close proximity must disrupt the interaction. Initial attempts to break and release the DsRed AC interface (see FIG. 2A) with single mutations T147R, H162R, and F224R consistently gave a non-fluorescent protein. However, for the AB interface, it was shown that it was slightly less resilient and could be broken by a single I125R to give a poor red fluorescent dimer, which requires more than 10 days for increased green content and full maturation. I was troubled.

  In order to reconstruct the red fluorescence of DsRed-I125R, the protein was subjected to an iterative cycle of evolution. This accelerated evolution strategy can utilize either random mutagenesis or semi-directed mutagenesis to create a library of mutant molecules and screen for the desired properties. The directional evolution strategy of the present invention is shown in FIG. Each cycle of mutagenesis begins with random mutagenesis and identifies positions that affect either the maturity or brightness of these red fluorescent proteins. Once several residues were identified, an extended library was constructed and several of these key positions were simultaneously mutated in the library to make multiple substitutions (see FIGS. 10-12). These directed libraries combine the benefits of improved mutant gene shuffling with an efficient method of overcoming the limited number of substitutions accessible during random mutagenesis by error-prone PCR. Most in vitro recombination methods rely on random gene fragmentation. In contrast, the method of the present invention can generate a designed fragment using PCR and reassemble it to give a full-length shuffled gene.

  A library of mutant red fluorescent proteins was screened in E. coli colonies and evaluated for both their amount of red fluorescence under direct excitation at 540 nm and the ratio of emission intensity at 540 nm / 470 nm excitation. The former constraint selected very bright or fast mature mutants, while the latter constraint selected a reduced 470 nm excitation or red shifted excitation spectrum. Using multiple cycles of random mutagenesis, sequence positions that affect protein maturation and brightness are found, then a library of mutations extended at these positions is generated and recombination is performed to achieve optimal permutation. I found it.

  Initial random mutagenesis of DsRed-I125R identified multiple beneficial mutations including K163Q or M, S179T and T217S. These three positions were included in our original directed library in which all 7 residues were mutated simultaneously to multiple rational substitutions. Additional positions targeted in the first directed library included N42 and V44, residues important for the fast phenotype of T1 (Bevis and Glick, Nat. Biotechnol., 20: 83-87 [2002]) . Also included were I161 and S197, where specific mutations contributed to mild improvement of DsRed2 (CLONTECH) and the very similar “E57” (Terskikh et al., J. Biol. Chem., 277: 7633). -7636 [2002]). From this library, multiple clones such as DsRed-I125R, S179T, T217A and DsRed-I125R, K163Q, T217A were identified, but the improvement was not significant.

  As an alternative strategy, the DsRed mutant fast tetramer T1 (Bevis and Glick, Nat. Biotechnol., 20: 83-87 [2002]) was also studied. Introduction of the I125R mutation into this protein (T1 DsRed-I125R polypeptide sequence is set forth in SEQ ID NO: 24) yields a dimer that matures in just a few days, compared to the best DsRed dimer produced at that time It was possible. Furthermore, a significant improvement was observed in our original library by targeting the location that helped rescue DsRed-I125R.

  A similar directed mutagenesis strategy (see FIG. 10A, library D1) starting from T1-I125R was performed and finally dimer 1 was identified. Dimer 1 was somewhat better in both luminance and maturation speed than wt DsRed, but had a substantial green peak comparable to T1. Dimer 1 was also somewhat blue shifted with an excitation maximum of 551 nm and emission maximum of 579 nm. The error-prone PCR of dimer 1 (Figure 10A, library D2) led to the discovery of dimer 1.02, which contains the mutation V71A in the hydrophobic core of the protein and is effective in the green component in the excitation spectrum Does not contain. The second round of random mutagenesis (FIG. 10A, library D3) is mutant, K70R with further reduced green excitation, S197A with red shift of dimer back to DsRed wavelength, and T217S that greatly improves maturation rate. Was identified. Unfortunately, K70R and S197A were relatively slowly matured and T217S had a green peak comparable to DsRed. Two or more rounds of directed mutagenesis were performed using dimer 1.02 as a template: first at the three positions identified above (Figure 11A, library D3), and secondly C117, F118, F124, And V127 (FIG. 10A, library D4).

  Continued directed evolution strategy for all four generations to produce the optimal dimer mutant, termed dimer 2 (illustrated in Figure 2B). This variant contains 17 mutations, 8 of which (N42Q, V44A, V71A, F118L, K163Q, S179T, S197T and T217S) are inside the β barrel, 3 (R2A, K5E and N6D) See mutations found in T1 that reduce aggregation (Bevis and Glick, Nat. Biotechnol., 20: 83-87 [2002]; and Yanushevich et al., FEBS Lett., 511: 11-14 [2002]. 2 (I125R and V127T) are AB interface mutations and 4 (T21S, H41T, C117T and S131P) are miscellaneous surface mutations. The dimer 2 nucleotide sequence is set forth in SEQ ID NO: 7 and FIG. The dimer 2 polypeptide is set forth in SEQ ID NO: 6 and FIG.

Building a tandem dimer for DsRed
In an attempt to produce even more advantageous forms of DsRed, an alternative novel strategy for synthesizing a more stable DsRed dimer was devised. This approach utilized two covalently tethered monomeric DsRed units made artificially to obtain DsRed dimer forms with advantageous properties. The basic strategy was to fuse two copies of the AC dimer using a polypeptide linker so that important dimer interactions are satisfied via intermolecular contact with tandem partners encoded within the same polypeptide.

  Based on the crystal structure of DsRed tetramer (Yarbrough et al., Proc. Natl. Acad. Sci. USA 98: 462-467 [2001]; and Wall et al., Nature Struct. Biol., 7: 1133-1138 [2000]) The 10-20 residue linker can extend from the C terminus of the A subunit to the N terminus of the C subunit (approximately 30 Å, see Figure 2B), but not to the N terminus of the B subunit (> 70 Å) Intended. Utilizing optimized dimer 2 and using linkers of various lengths (9, 12, 13, or 22 amino acids), known protease resistant linkers (Whitlow et al., Protein Eng., 6: 989-995 [1993] ) Produced a series of four tandem constructs containing sequences similar to.

  Of the four constructs, only one tandem construct with a 9-residue linker was noted with slightly slower maturation. The other three constructs could not be practically distinguished in this regard and could be used equally well in the present invention. The tandem dimer construct with a 12-residue linker was named tdimer2 (12) and was used in all subsequent experiments. As expected, dimer 2 and t-dimer 2 (12) have identical excitation and emission maxima and quantum yields (see FIG. 14). However, the extinction coefficient of t-dimer 2 (12) is twice that of dimer 2, which is due to the presence of two equal absorbing chromophores per polypeptide chain.

In an attempt to create an improved DsRed dimer that can well withstand the destruction of the remaining interface of the monomer DsRed, a library was constructed that incorporated mutations into the t-dimer (12) that disrupt the AC interface. The initial dimer library was reassembled with 3 ′ primers encoding mutations H222G and F224G (FIG. 10A, library D5). These two residues form a dimer contact bulk at the C-terminal tail of DsRed, which is trapped around the C-terminal tail of the dimer partner. The best two unique clones from this library, HF2Ga and HF2Gb, are very similar in sequence to dimer 1 with the main difference being the mutation F124L present in both clones, K163H present in HF2Gb, and It was a replacement for H222G and F224G. When fed to a 12% SDS-PAGE gel without boiling, both HF2Ga and HF2Gb migrate as fluorescent dimers, and therefore they must maintain a stable dimer interface.

At the same time, a more direct approach was taken to disrupt the AC interface through the introduction of mutations that disrupt the dimer. Let dimer 1 be the template for the first such library (FIG. 10A, library M1), where we target nine different positions, which contain two important AC interface residues, H162 and A164, These were replaced by lysine or arginine, respectively. The brightest colonies from this library were difficult to distinguish from the background red fluorescence of E. coli colonies even after extended imaging with a digital camera. Suspicious colonies were restreaked on LB / agar, matured at room temperature for 2 weeks and the crude protein preparation was analyzed by SDS-PAGE. Gel imaging represented a single pale band, consistent with the expected mass of the monomer. Therefore, we named this type mRFP0.1 (m onomeric R ed F luorescent P rotein ( monomer red fluorescent protein)). Sequencing of this clone showed that mRFP0.1 was equivalent to Dimer 1 with mutations E144A, A145R, H162K, K163M, A164R, H222G and H224G.

  A much brighter mRFP0.2 was generated by random mutagenesis on mRFP0.1 (Figure 10A, library M2), which gave clear red fluorescence and monomer bands by SDS-PAGE, and a single additional It contained the mutation Y192C. Both mRFP0.1 and mRFP0.2 displayed green fluorescence at least 3 times brighter than red fluorescence, but as expected for the monomer, there was no FRET between the green and red components.

  Considering that mutations that would benefit dimers could also benefit monomers, a template mixture containing mRFP0.2, dimer 1.56, HF2Ga and HG2Gb was treated with PCR-based template shuffling and directed mutagenesis ( FIG. 10A, library M3). The top clone identified in this library, mRFP0.3, was relatively bright and the green fluorescence component was significantly reduced. Furthermore, mRFP0.3 showed a nearly 10 nm red shift from DsRed and was derived primarily from dimer 1.56.

  The goal of the next directed library (Figure 10B, library M4) was to study the effect of mutations in K83, which has previously been shown to produce a red shift in DsRed (Wall et al. , Nature Struct. Biol., 7: 1133-1138 [2000]). The top two clones, named mRFP0.4a and mRFP0.4b, each contained a K83I or L mutation, shifted 25 nm red compared to DsRed, and had very similar maturation rate and brightness values. Unlike all previous generations of monomers, E. coli colonies transformed with mRFP0.4a were excited with 540 nm light and observed through a red filter within 12 hours after transformation. Gave red fluorescence.

  The template mixture of mRFP0.4a and mRFP0.4b was treated with random mutagenesis (FIG. 10B, library M5) and the resulting library was screened thoroughly. The five fastest mature clones from this library were derived from mRFP0.4a, which contained the individual mutations L174P, V175A (2 clones), F177C and F177S. The F177S clone or mRFP0.5a was seen to mature slightly faster and had a minimal green peak in the absorption spectrum. One colony isolated from this library was exceptionally bright when grown on LB / agar, but very poorly expressed when grown in liquid culture. This clone, named mRFP0.5b, was derived from mRFP0.4b and contained two new mutations; L150M inside the barrel and V156A outside.

  The next library (FIG. 10B, library M6) was intended to optimize the region around residues V175 and F177 in both mRFP0.5a and mRFP0.5b with increased diversity. The top clone in this library, named mRFP0.6, was derived from mRFP0.5b, but three other clones, one from mRFP0.5b, one from mRFP0.5a, and 1 One appears to have resulted from multiple crossovers between the two templates. The final library (Figure 10B, library M7) targeted a residue near L150 because it was the only significant remaining mutation that was derived from random mutagenesis and was not reoptimized Because it was a mutation. The top clone had a combination of mutations at all target positions, but the clone with the single mutation R153E was found to be slightly better expressed in culture. This clone was further modified through deletion of an unnecessary V1a insertion and replacement of the cysteine at position 222 with serine.

  This final clone, named mRFP1, contained all 33 mutations (see Figure 2C) compared to wild type DsRed. Of these mutations, 13 (N42Q, V44A, V71A, K83L, F124L, L150M, K163M, V175A, F177V, S179T, V195T, S197I and T217A) are inside the β-barrel. Of the 20 remaining outer mutations, 3 (R2A, K5E and N6D) are mutations with reduced aggregation from T1. Three (I125R, V127T and I180T) are AB interface mutations, 10 (R153E, H162K, A164R, L174D, Y192A, Y194K, H222S, L223T, F224G and L225A) are AC interface mutations, and four (T21S, H41T, C117E and V156A) are additional mutations. The nucleotide and polypeptide sequences of mRFP1 are set forth in SEQ ID NOs: 9 and 8, respectively.

Characterization of dimer 2, t-dimer 2 (12) and mRFP1
Initial confirmation for the monomer structure of mRFP1 and its precursor is based on SDS-PAGE results (see FIG. 8) and the lack of FRET between the green and red fluorescent components in the early generation. Therefore, analytical ultracentrifugation was performed on DsRed, Dimer 2, and mRFP0.5a (an evolutionary precursor of mRFP1). The mRFP0.5a polypeptide sequence is set forth in FIGS. 20A-20D. Analytical equilibrium ultracentrifugation analysis demonstrated the expected tetramer, dimer, and monomer configurations of the species tested (see FIGS. 3A-3C).

  In fluorescence and absorption spectrum analysis, DsRed, T1, and dimer 2 all have a fluorescent component that contributes to the excitation spectrum at 475-486 nm by FRET between oligomer partners (see FIGS. 4A-4C). In this analysis, the T1 peak is clearly distinct (FIG. 4B), but in dimer 2 (FIG. 4C), the excitation shoulder near 480 nm is almost obscured by the 5 nm blue-shifted excitation peak. The 25 nm red-shifted monomer mRFP1 (Figure 4D) also has a peak at 503 nm in the absorption spectrum, but in contrast to the mutant, this species is a non-fluorescent mutant, so what is the excitation spectrum collected at the emission wavelength? Also not shown. When a 503 nm absorbing species is directly excited, negligible fluorescence emission is observed at any wavelength.

As shown in FIG. 5, the maturation rate of dimer 2, t dimer 2 (12) and mRFP1 is accelerated more than that of DsRed, but only mRFP1 matures at least as fast as T1. Based on data collected at 37 ° C., t _0.5 for mRFP1 and T1 maturation is less than 1 hour. E. coli colonies expressing either dimer 2 or mRFP1 display a similar or brighter level of fluorescence after overnight incubation at 37 ° C. than those expressing T1 (FIG. 9). See).

Expression of dimer 2, t-dimer 2 (12) and mRFP1 in mammalian cells The fluorescence of dimer 2, t-dimer 2 (12) and mRFP1 protein in the environment of mammalian cells was examined. Mammalian expression vectors encoding dimer 2, t-dimer 2 (12) and mRFP1 were expressed in transiently transfected Hela cells. Within 12 hours the cells displayed strong red fluorescence evenly distributed throughout the nucleus and cytoplasm (data not shown).

  Looking at this result, it was tested whether RFP-fusion polypeptides can be made in which the RFP moiety retains its fluorescence and the fusion polypeptide partner retains native biological activity. Although this experiment was performed using the gap junction protein connexin 43 (Cx43), it would be possible to demonstrate the advantages of monomeric red fluorescent protein if the fused Cx43 polypeptide retains its biological activity. A series of constructs consisting of Cx43 fused to either GFP, T1, dimer 2, t-dimer 2 (12) or mRFP1 were expressed in HeLa cells that do not express endogenous connexin. After transfection, the red fluorescence of the cells was observed using a fluorescence microscope. The results of this experiment are shown in FIGS. 6A, 6C and 6E. As previously reported (Lauf et al., FEBS Lett. 498: 11-15 [2001]), the Cx43-GFP fusion protein was properly transported to the membrane and assembled into a functional gap junction (data shown) Cx43-DsRed tetramer (ie, T1 tetramer) formed a red fluorescent mass that consistently localized around the nucleus (FIG. 6E). Both Cx43-t dimer 2 (12) (not shown) and Cx43-dimer 2 (FIG. 6C) were properly transported to the membrane, but neither construct formed a visible gap junction. In contrast, the Cx43-mRFP1 construct behaved similarly to Cx43-GFP and a large number of red gap junctions were observed (FIG. 6A).

  In other experiments, lucifer yellow was microinjected into transfected cells to assess gap junction functionality (see FIGS. 6B, 6D and 6F; and FIG. 13). Cx43-mRFP1 gap junctions passed quickly and undoubtedly (FIG. 6B), but neither Cx43-T1 transformed cells (FIG. 6E) nor non-transformed cells (not shown) passed the dye. Both the Cx43-dimer 2 and Cx43-t dimer 2 (12) constructs slowly passed the dye through the contacting transforming neighbors for approximately one third of the contact time (FIG. 6D).

Discussion The monomer mRFP1 simultaneously overcomes three important problems associated with the wild-type tetramer form. Specifically, mRFP1 is a monomer, matures rapidly and has minimal emission when excited at a wavelength suitable for GFP. Due to these properties, mRFP1 is a red fluorescent protein suitable for the construction of fusion proteins and multicolor labeling in combination with GFP. As demonstrated using the gap junction-forming protein Cx43, the mRFP1 fusion protein is functional and is transported in the same way as its GFP analog.

  The extinction coefficient and fluorescence quantum yield result in a decrease in brightness compared to DsRed for all mature mRFP1, but this does not interfere with the use of mRFP1 for imaging experiments because it exceeds 10 times the mRFP1 maturation time The shortening is even more than compensating for the decrease in brightness. The mutant RFP polypeptides of the invention, such as t-dimer 2 (12), also have uses as FRET-based sensors. This species is bright enough and displays FRET with all variants of Aequorea GFP.

  The present invention provides a method for making even more advantageous RFP species. These methods use a multi-stage evolution strategy involving single or multiple rounds of evolution with a small number of mutation steps per cycle. These methods are also useful for converting other oligomeric fluorescent proteins into advantageous monomeric or dimeric forms.

Example 2
Preparation and Characterization of Fluorescent Protein Variants This example demonstrates that mutations can be introduced into GFP spectral variants to reduce or eliminate protein oligomerization ability.

The dimer interface ECFP (SEQ ID NO: 14) and EYFP-V68L / Q69K (SEQ ID NO: 12) were subcloned into the bacterial expression vector pRSET _B (Invitrogen Corp., La Jolla CA) to obtain ECFP (SEQ ID NO: 14) and EYFP. An N-terminal His ₆ tag was created on -V68L / Q69K (SEQ ID NO: 12), which allowed purification of bacterially expressed proteins on a nickel-agarose (Qiagen) affinity column. Mutants associated with all dimers in the cDNA were generated by site-directed mutagenesis using the QuickChange mutagenesis kit (Stratagene) and then expressed and purified as described above. All cDNAs were sequenced to confirm that only the desired mutation was present.

EYFP-V68L / Q69K (SEQ ID NO: 12) was mutagenized using the QuickChange kit (Stratagene). Overlapping mutation primers are called “top” for the 5 ′ primer and “bottom” for the 3 ′ primer and named according to the specific mutation introduced (Table 1). All primers had a melting point above 70 ° C. Mutations were made as close to the center of the primers as possible, and all primers were purified by polyacrylamide gel electrophoresis. Primers are shown in the 5 'to 3' direction with the mutagenized codon underlined (Table 1).

For protein expression, plasmids containing various EYFP-V68L / Q69K (SEQ ID NO: 12) mutant cDNAs were transformed into E. coli strain JM109 and contained 100 μg / ml ampicillin. Were grown in LB to an OD ₆₀₀ of 0.6, at which time they were induced with 1 mM isopropyl-β-D-thiogalactoside. The protein is expressed by the bacteria for 6-12 hours at room temperature and then overnight at 4 ° C., then the bacteria are centrifuged to a pellet, which is reconstituted in phosphate buffered saline (pH 7.4). It was suspended and lysed with a French press. The bacterial lysate was clarified by centrifugation at 30,000 × g for 30 minutes. The protein in the clarified lysate was affinity purified with Ni-NTA-agarose (Qiagen).

All GFPs used in these experiments were 238 amino acids long. The cDNA encoding GFP was subcloned into pRSET _B and an additional 33 amino acids were fused to the N-terminus of GFP. The sequence of this tag is MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDP (SEQ ID NO: 22). That is, the total length of the EYFP-V68L / Q69K (SEQ ID NO: 12) mutant expressed from this cDNA was 271 amino acids. The His6 tag was removed using EKMax (Invitrogen) to see if the association properties measured for GFP were affected by the presence of the N-terminal His6 tag. A dilution series of enzyme and His6-tagged GFP was prepared, and the conditions necessary for complete removal of the His6-tag were determined. The purity of all expressed and purified proteins was analyzed by SDS-PAGE. In all cases, the expressed protein was highly pure, no contaminating protein was detected significantly, and all were of the correct molecular weight. Furthermore, the removal of the His6 tag was very efficient, as indicated by the presence of a single band migrating with a molecular weight less than His6-EYFP-V68L / Q69K.

By spectroscopic analysis of the purified proteins, EYFP-V68L / Q69K (SEQ ID NO: 12; “wtEYFP”; Table 2) was analyzed for chromophore modifications of these proteins (Ward et al., In “Green Fluorescent Protein: Properties, Applications and Protocols (Green Fluorescent Protein: Properties, Applications and Protocols), edited by Chalfie and Kain, Wiley-Liss [1998]), or the quantum yield (EYFP-V68L / Q69K and derived from it) It was also confirmed that there was no significant change in the standard used for the mutants (fluorescein). Fluorescence spectra were obtained using a Fluorolog spectrofluorometer. Protein absorption spectra were obtained using a Cary UV-Vis spectrophotometer. The extinction coefficient is measured by the modified chromophore method (Ward et al., In “Green Fluorescent Protein: Properties, Applications and Protocols”, edited by Chalfie and Kain, Wiley-Liss [1998]). did.

  To measure the degree of dimer homoaffinity, wtEYFP and the dimer mutants derived therefrom were subjected to ultracentrifugation for sedimentation equilibrium analysis. The purified recombinant protein is dialyzed thoroughly against phosphate buffered saline (pH 7.4), and 125 μl of protein sample at a concentration ranging from 50 μM to 700 μM is centrifuged in 6 channels with an EPON centerpiece. I put it in the cell. The corresponding dialysis buffer was used as a sample blank. Sedimentation equilibrium experiments were performed using a Beckman Optima XL-1 analytical ultracentrifuge at 20 ° C. while measuring the absorbance at 514 nm. Each sample was examined at three or more of the following speeds: 8,000 rpm, 10,000 rpm, 14,000 rpm, and 20,000 rpm. Periodic absorption measurements at each rate confirmed that the sample had reached equilibrium at each rate.

Data was analyzed comprehensively at all rotor speeds by nonlinear least squares analysis using a software package (Origin) supplied by Beckman. The goodness of fit was evaluated based on the magnitude and randomness of the error expressed as the difference between the experimental data and the theoretical curve, and by checking each fit parameter for physical validity. Using Sedenterp v1.01, the molecular weight and partial specific volume of each protein were measured, and the data was introduced into the homoaffinity measurement formula (Table 3). In addition, dissociation constants (Kd) derived from data obtained by analytical ultracentrifugation are shown for several proteins (Table 4).

  For live cell experiments, ECFP (SEQ ID NO: 14; “wtECFP”) and EYFP-V68L / Q69K (SEQ ID NO: 12; “wtEYFP”) targeting the plasma membrane (PM) are expressed in mammalian expression vectors. Subcloned into certain pcDNA3 (Invitrogen Corp.) and mutagenized as described above and sequenced. Targeting the GFP variant to PM creates an N-terminal or C-terminal fusion of the GFP variant to a short peptide containing a consensus sequence for acylation and / or prenylation (post-translational lipid modification) Was done. GFP mutant cDNA targeting PM was transfected into HeLa cells or MDCK cells and expressed, and the expression pattern and the degree of association were measured using a fluorescence microscope. FRET efficiency was measured to determine the degree of interaction between PM-ECFP and PM-EYFP-V68L / Q69K. Analysis of interaction by FRET donor-dequenching method (Miyawaki and Tsien, supra, 2000) shows that wtECFP and wtEYFP interact in a manner that depends on the association of wtECFP and wtEYFP, and that this interaction is hydrophobic. It was proved that the amino acids in the contact portion were effectively eliminated by changing to any one or combination of mutations A206K, L221K, and F223R.

  These results demonstrate that the solution oligomeric state of Aequorea GFP and its spectral variants and the dimer mutants derived therefrom were accurately measured by analytical ultracentrifugation. ECFP (SEQ ID NO: 14) and EYFP-V68L / Q69K (SEQ ID NO: 12) GFP spectral variants formed homodimers with a fairly high affinity of about 113 μM. Site-directed mutagenesis was used to modify the amino acid composition to effectively eliminate dimerization and associated cell biology problems. That is, the modified fluorescent protein provides a means of using FRET to measure the association properties of a host protein fused to a modified CFP or YFP. False positives associated with dimerization of ECFP (SEQ ID NO: 14) and EYFP-V68L / Q69K (SEQ ID NO: 12), with possible misidentification of host protein organelle distribution or localization by GFP dimerization The ambiguity and possibility of FRET results has been effectively eliminated.

  Renilla GFP and Discosoma red fluorescent protein are obligate oligomers in solution. Since Aequorea GFP is also generally thought to dimerize in solution, and GFP crystallizes as a dimer, this study was designed to characterize the oligomeric state of GFP . The crystal interface between the two monomers contained many hydrophilic contacts as well as some hydrophobic contacts (Yang et al., Supra, 1996). However, it was not directly clear how much each type of interaction contributed to the formation of dimers in solution.

  As disclosed herein, the extent of GFP self-association depends on sedimentation equilibrium, analytical ultracentrifugation (which includes similar molecules (self-associating homomeric complexes) and heterologous molecules (heteromer complexes; Laue and (See Stafford, Ann. Rev. Biophys. Biomol. Struct. 28: 75-100, 1999)) which is very useful for measuring both oligomeric behaviors). Compared to X-ray crystallography, the experimental conditions used in the analytical ultracentrifugation experiments were very similar to the physiological conditions of the cells. The monomer contact sites identified by X-ray crystallography within the multimer complex are not necessarily the same as in solution. Further, as compared with the ultracentrifugation for analysis, only the X-ray crystal analysis method cannot provide definitive information on the affinity of the complex. The results of this study contain hydrophobic residues A206, L221, F223, and positively charged side chains, as determined by in vitro analytical ultracentrifugation and concentration-dependent analysis of FRET in intact cells. (A206K, L221K, and F223R) proved that dimerization was eliminated.

Example 3
Characterization of the coral red fluorescent protein, DsRed and their mutants This experiment describes the first biochemical and biological characterization of DsRed and DsRed mutants.

The coding sequence of DsRed is taken from pDsRed-N1 (Clontech Laboratories) using PCR primers (which add an N-terminal BamHI recognition site upstream of the start Met codon and a C-terminal EcoRI site downstream of the stop codon). Amplified. After restriction digestion, the PCR product was cloned between the BamHI and EcoRI sites of pRSET _B (Invitrogen) and the resulting vector was amplified in DH5α bacteria. Use the resulting plasmid as a template for error-prone PCR (Heim and Tsien, Curr. Biol. 6: 178-182, 1996, which is incorporated herein by reference). Thus, the use of primers located immediately upstream and downstream of the DsRed coding sequence theoretically allowed mutation of all coding bases (including the starting Met). The mutagenized PCR fragment was digested with EcoRI and BamHI and recloned into pRSET _B. Alternatively, directed mutations were made on the pRSET _B- DsRed plasmid using the Quick-Change mutagenesis kit (Stratagene).

  In both random and directed mutagenesis experiments, the mutagenic plasmid library was electroporated into JM109 bacteria, plated on ampicillin-containing LB plates and screened with a digital imaging device (Baird et al., Proc. Natl). Acad. Sci. USA 96: 11242-11246, 1999, which is incorporated herein by reference). The device irradiates the plate with light from a 150 watt xenon arc lamp, filtered with a bandpass excitation filter and directed to the plate with two optical fiber bundles. The fluorescence emission from the plate was imaged through an interference filter equipped with a cooled CCD camera. Images taken at different wavelengths were digitally magnified using MetaMorph software (Universal Imaging) to allow identification of spectral shift mutants. After selection, the mutant colonies were manually picked and placed in LB / Amp medium, and the culture was then used for protein or plasmid preparation. DsRed mutant sequences were analyzed using dye-terminator dideoxy sequencing.

DsRed and its mutants were purified using an N-terminal polyhistidine tag (SEQ ID NO: 22; see Example 1) provided by the pRSET _B expression vector (see Baird et al., supra, 1999). For spectroscopic characterization, Microcon-30 (Amicon) was used to microconcentrate the protein and the buffer was changed to 10 mM Tris (pH 8.5). Alternatively, microconcentration produced large protein aggregates that were dialyzed against 10 mM Tris (pH 7.5) for oligomerization studies. To test for photosensitivity of protein maturation, the entire synthesis was repeated in the dark by wrapping the culture flask in foil and all purification was performed in a dimly lit room with red light. There was no difference in protein yield or color when the protein was prepared in light or dark.

The amino acid numbering is the wild type sequence (DsRed; Matz) of residues 66-68, drFP583 where Gln-Tyr-Gly is homologous to the chromophore-forming residues of GFP (65-67, Ser-Tyr-Gly) Et al., Nature Biotechnology 17: 969-973 [1999]). Extra amino acids after the initiator Met of Clontech was introduced numbered "1a", and the residues of N-terminal polyhistidine tag ^- 33 to ^- I gave a 1 number.

Fluorescence spectra were obtained using a Fluorolog spectrofluorometer. Protein absorption spectra were obtained using a Cary UV-Vis spectrophotometer. For quantum yield measurements, the fluorescence of a solution of DsRed or DsRed K83M in phosphate buffered saline was compared to a solution of rhodamine B and rhodamine 101 in ethanol showing comparable absorption. In calculating the quantum yield, the difference in refractive index between ethanol and water was corrected. For measuring the extinction coefficient, the absorbance of the native protein was measured with a spectrophotometer, and the protein concentration was measured by the BCA method (Pierce).

  The pH sensitivity of DsRed is determined by adding 100 μl of DsRed diluted in weak buffered solution to 100 μl of strongly buffered pH solution (total 200 μl / well) three times for pH 3 to pH 12 in 96 well format. It was measured. The fluorescence of each well was measured using a 525-555 nm bandpass excitation filter and a 575 nm long pass emission filter. After obtaining 96-well fluorometer measurements, 100 μl of each pH buffered DsRed solution was analyzed with a spectrofluorometer to observe pH dependent spectral shape changes. For a DsRed maturation time trial, make a freshly synthesized and purified DsRed dilution in 10 mM Tris (pH 8.5) and store this solution in a capped cuvette (not airtight) at room temperature, The spectrum was measured regularly. For mutant maturation data, fluorescence emission spectra (excitation at 475 nm or 558 nm) were obtained immediately after synthesis and purification and after storage for more than 2 months at 4 ° C. or room temperature.

The quantum yield for photodisruption was measured separately with a microscope stage or a spectrofluorometer. Microdroplets of aqueous DsRed solution were made under oil on a microscope slide and bleached with 1.2 W / cm ² light through a 525-555 nm bandpass filter. Fluorescence was monitored over time using the same filter and a 563-617 nm emission filter. For comparison, EGFP (containing mutations F64L, S65T; SEQ ID NO: 13) and EYFP-V68L / Q69K (also containing mutations S65G, S72A, T203Y; SEQ ID NO: 12) into microdroplets, Similarly, bleaching was performed at 460 to 490 nm with 1.9 W / cm 2 and monitoring was performed at 515 to 555 nm and 523 to 548 nm, respectively.

For spectrofluorimeter photobleaching experiments, a solution of DsRed is prepared in a rectangular micro cuvette, overlaid with oil, for a total irradiation volume of 0.25 cm x 0.2 cm x 1 cm with 50 μl protein solution. To exist. The protein solution was irradiated with 0.02 W / cm ² light from a monochromator centered at 558 nm (bandwidth 5 nm). Fluorescence was measured over time with 558 nm excitation (bandwidth 1.25 nm) and 583 nm emission. The quantum yield (Φ) for photobleaching is given by the formula Φ = (ε · I · t _90% ) ^-1 (where ε is the extinction coefficient ( ^{cm 2} mol ^-1 ) and I is the intensity of the incident light (Einstein) Cm ⁻² s ⁻¹ ) and t _90% is the time (seconds) it takes for the fluorophore to be 90% bleached) (Adams et al., J. Am. Chem. Soc. 110: 3312- 3320, 1988, which is incorporated herein by reference).

  Polyhistidine tagged DsRed, DsRed K83M and wild type Aequorea GFP (SEQ ID NO: 10) were run on a non-denaturing 15% polyacrylamide gel. To prevent denaturation, a protein solution (10 mM Tris-HCl in pH 7.5) is mixed 1: 1 with 2 × SDS-PAGE sample buffer (containing 200 mM dithiothreitol) and loaded directly onto the gel without boiling. did. A pre-stained broad molecular weight marker set (BioRad) was used as a size standard. The gel was then imaged with an Epson1200 Perfection flatbed scanner.

  The purified recombinant DsRed was dialyzed thoroughly against phosphate buffered saline (pH 7.4) or 10 mM Tris, 1 mM EDTA (pH 7.5). Sedimentation equilibration experiments were performed at 20 ° C. while measuring absorbance at 558 nm as a function of radius on a Beckman Optima XL-1 analytical ultracentrifuge. A 125 μl sample (0.25 absorbance units) of 3.57 μM DsRed was loaded into a 6-channel cell. Data at 10,000, 14,000, and 20,000 rpm were comprehensively analyzed by nonlinear least squares analysis using the Origin software package (Beckman). The goodness of fit was assessed based on the magnitude and randomness of the error expressed as the difference between the experimental data and the theoretical curve, and by checking each fit parameter for physical validity.

  FRET between immature green DsRed and mature red DsRed was examined in mammalian cells. DsRed in the vector pcDNA3 was transfected into HeLa cells using lipofectin and after 24 hours the cells were imaged with a fluorescence microscope. Fluorescence of immature green species (excitation 465-495nm, 505nm dichroism, emission 523-548nm) and mature red protein (excitation 529-552nm, 570nm dichroism, emission 563-618nm) with a cooled CCD camera It was measured. After a selective photobleaching of the red component by irradiating light from a xenon lamp filtered with 570 nm dichroism only for a cumulative duration of 3, 6, 12, 24, and 49 minutes The measurement of was repeated. By the final time, about 95% of the initial red emission disappeared, while the green emission was greatly enhanced.

  A yeast two-hybrid assay was also performed. Downstream of the Gal4 activation domain (“Bait”; amino acid residues 768-881) and the DNA binding domain (“Prey”; amino acid residues 1-147) in the pGAD GH and pGBT9 vectors (Clontech), respectively. The DsRed coding region was cloned in-frame. These DsRed 2 hybrid plasmids were transformed into S. cerevisiae strain HF7C, which cannot synthesize histidine if there is no interaction between the proteins fused to the Gal4 fragment. . Yeast containing both DsRed-bait plasmid and DsRed-prey plasmid was streaked onto histidine-deficient medium and plates were visually inspected to determine growth. Alternatively, yeast was grown on filters placed on plates lacking tryptophan and leucine and selected for food and prey plasmids. After overnight growth, the filter was removed from the plate, frozen in liquid nitrogen, thawed and incubated in X-gal at 30 ° C. overnight and 4 ° C. for 2 days to give β-galactosidase activity (by blue color development). Test). In both β-galactosidase and histidine proliferation assays, the negative control consisted of yeast containing a bait and a prey plasmid, but only that bait or prey was fused to DsRed.

  Surprisingly, DsRed took several days at room temperature to reach full red fluorescence. At room temperature, the purified protein sample initially exhibits green fluorescence as the major component (excitation maxima and emission maxima of 475 nm and 499 nm, respectively), peaking in intensity in about 7 hours and almost zero in 2 days. became. On the other hand, red fluorescence reached half of its maximum fluorescence after about 27 hours, and took more than 48 hours to reach 90% of maximum fluorescence (see Baird et al., Supra, 2000).

Fully mature DsRed has an extinction coefficient of 75,000 M ⁻¹ cm ⁻¹ at its 558 nm absorption maximum and a fluorescence quantum yield of 0.7, which has been reported previously (Matz et al., Nature Biotechnology 17: 969-973 [1999]) much larger than the values of 22,500 M ^-1 cm ^-1 and 0.23. These properties make mature DsRed very similar to rhodamine dyes in terms of wavelength and luminosity. Unlike many GFP variants, DsRed has negligible pH (<10%) absorbance or fluorescence at pH 5-12 (Baird et al., Supra, 2000). However, acidification to pH 4-4.5 suppressed both absorbance and excitation at 558 nm compared to the shorter wavelength shoulder at 526 nm, while not changing the shape of the emission spectrum. DsRed was also relatively resistant to photobleaching. When exposed to 1.2 W / cm ² of about 540 nm light in a microscope stage, the DsRed microdrop under the oil takes one hour to bleach 90%, while the spectrofluorometer's It took 83 hours to fade 90% with 20 mW / cm ² 558 nm light in a micro cuvette. Microscopic and fluorometric measurements resulted in optical bleaching quantum efficiencies of 1.06 × 10 ⁻⁶ and 4.8 × 10 ⁻⁷ (average 7.7 × 10 ⁻⁷ ), respectively. Similar microscopic measurements of EGFP (S65T; SEQ ID NO: 13) and EYFP-V68L / Q69K (including SEQ ID NO: 12; Q69K) resulted in 3 × 10 ⁻⁶ and 5 × 10 ⁻⁵ , respectively.

  To investigate the properties of the red chromophore and identify DsRed mutants useful as biological indicators, abruptly, DsRed was randomly selected and for specific sites predicted by GFP sequence alignment to be near the chromophore. Mutagenesis was performed. Many mutants were identified that mature more slowly or not at all, but none that mature faster than DsRed. Random mutant screening identified mutants that displayed green or yellow, which were found to be due to the substitutions K83E, K83R, S197T, and Y120H. Green fluorescence is due to mutant species with excitation maxima and emission maxima of 475 nm and 500 nm, respectively, while yellow is not due to a single molecular species at intermediate wavelengths, this green species and DsRed-like material Of the mixture.

  The DsRed K83R mutant has the lowest percent conversion to red, and has been found to be very useful as a stable type of immature green fluorescent form of DsRed (Baird et al., Supra, 2000). Further directed mutagenesis of K83 produced more green and yellow mutants, which were impaired chromophore maturation. For many of the late mature and incomplete K83 mutants, the red peak was at a longer wavelength than DsRed. The final red fluorescent species of K83M is of particular interest because it exhibits an emission maximum at 602 nm, relatively little residual green fluorescence and a quantum yield of 0.44. But its maturation was slower than wild-type DsRed. Y120H had a red shift similar to K83M and appeared to produce brighter bacterial colonies, but maintained much more residual green fluorescence.

  The spectroscopic data of the DsRed mutant is shown in FIG. “Maturation” of a protein means the rate at which red fluorescence appears for 2 days after protein synthesis. Since some maturity occurs during synthesis and purification (which takes 1-2 days), numerical quantification is not accurate. A simple +/− evaluation method was used, where (−−) is almost unchanged, (−) is a 2-5 fold enhancement in red fluorescence, (+) is a 5-20 fold enhancement, And (++) indicate wild type enhancement (approximately 40-fold). Two months after protein synthesis, the peak emission fluorescence obtained with 558 nm excitation from the same sample was divided by the 499 nm fluorescence obtained with 475 nm excitation to determine the red / green ratio. The molar ratio of two molecular species for the difference in extinction coefficient or quantum yield between the two molecular species, or for the possibility of FRET between the two molecular species if they are in a macromolecular complex Is not corrected, the value does not represent the molar ratio of the two molecular species.

  To determine whether Lys70 or Arg95 can form an imine with the carbonyl terminus of a GFP-like chromophore (see Tsien, Nature Biotechnol. 17: 956-957, 1999), DsRed mutants K70M, K70R, and R95K was produced. K70M did not have a red component and remained completely green, while K70R slowly matured into a red species that shifted slightly to red. The spectral similarity between K70R and wild-type DsRed indicates that any amino acid is not covalently incorporated into the chromophore. No fluorescence at visible wavelengths was detected from R95K, indicating that Arg95 is conserved in GFP Arg96 (all fluorescent proteins characterized to date (Matz et al., Nature Biotechnology 17: 969-973 [1999]) would have been expected, because R95K did not form a green chromophore, so whether Arg95 is also required for reddening was tested. There wasn't.

  Aequorea GFP tends to form dimers at high concentrations in solution and in some crystalline form, and Renilla GFP is obligately dimer (Ward et al., In “Green Fluorescent Protein: Properties, The ability of DsRed to oligomerize, considering the possibility of forming “Green Fluorescent Protein: Properties, Applications and Protocols”, Ed. Chalfie and Kain, Wiley-Liss [1998]) Examined. In an initial test of the expressed protein by SDS-PAGE, the polyhistidine-tagged proteins DsRed and DsRed K83R appear to be larger than 110 kDa when mixed with 200 mM DTT and not heated prior to loading onto the gel. The molecular weight migrated as a red band and a yellow-green band, respectively, suggesting the formation of aggregates (Baird et al., Supra, 2000). In comparison, Aequorea GFP migrated as a fluorescent green band close to its predicted monomer molecular weight of 30 kDa when treated similarly. When the sample was heated briefly before electrophoresis, this high molecular weight DsRed band was not observed (Gross et al., Supra, 2000). Under these conditions, a band close to the predicted monomer molecular weight of 30 kDa was predominant, which was colorless without Coomassie staining.

  To more accurately measure the oligomerization state, the DsRed protein was subjected to analytical equilibrium centrifugation (Laue and Stafford, supra, 1999). DsRed is present in solution as an obligate tetramer (Baird et al., Supra, 2000), both at low and physiological salt concentrations, by overall curve fitting of absorbance data measured from an equilibrated DsRed radial scan Was shown to do. When modeling data with a single molecular species tetramer, the fitted molecular weight was 119,083 Da, which is in good agreement with the theoretical molecular weight of 119,068 Da for the polyhistidine-tagged DsRed tetramer. Attempts to fit a curve to another stoichiometry from monomer to pentamer did not converge or gave inappropriate values for floating variables and large non-random errors. The error for the tetramer fit was much smaller and more randomly distributed, but was improved somewhat more by extending the model so that the obligate tetramer could dimerize into an octamer. The dissociation constant was 39 μM. That is, molecular species that absorb at 558 nm appear to be tetramers over a monomer concentration range of 14 nM to 11 μM in vitro. Even the highest concentration of tetramer achieved in the ultracentrifugation cell was more than an order of magnitude less than the fitted dissociation constant, suggesting only the indication of octamer formation at the highest concentration.

  To confirm whether DsRed oligomerizes in living cells, FRET analysis in mammalian cells and a two-hybrid assay in yeast cells were performed. HeLa cells were imaged 24 hours after transfection with wild type DsRed, which contained a mixture of immature green intermediate and final red type. Green fluorescence was monitored intermittently before and during selective photobleaching of the red species with intense orange light for 49 minutes. If the two proteins were not associated, it was predicted that bleaching of the red species would have no effect on green fluorescence. In practice, however, green fluorescence increased 2.7 to 5.8 fold in different cells, corresponding to 63% to 83% of FRET efficiency. These values are the highest FRET efficiency ever observed between GFP mutants for cyan and yellow fluorescent proteins bound by zinc ion saturated zinc finger domains (Miyawaki and Tsien, supra, 2000). It is equal to or more than a certain 68%.

Additional evidence of in vivo oligomerization is provided by a directed yeast two-hybrid screen. When DsRed fusions to the Gal4 DNA binding domain and the activation domain were expressed in HF7C yeast, the yeast showed a his ⁺ phenotype and was able to grow without histidine supplementation. Indicates that a hybrid interaction has occurred. None of the fusion constructs (DsRed-DNA binding domain or DsRed-activation domain) alone produces a his ⁺ phenotype, ie, DsRed-DsRed, not a non-specific DsRed-Gal4 interaction. It shows that the interaction causes a positive result. Furthermore, his ⁺ yeast turns blue when lysed and incubated with X-gal, suggesting that the DsRed-DsRed interaction also directs the transcription of the β-galactosidase gene. That is, two separate transcription measurements of the yeast two-hybrid assay confirmed that DsRed associates in vivo.

  This study of DsRed revealed that DsRed has favorable properties as well as some non-optimal properties in that it is useful for complementing GFP or as a DNA substitute for it. The most favorable property identified is that DsRed has a much higher extinction coefficient and fluorescence quantum yield (0.7) than previously reported, thereby increasing the fluorescence brightness of the mature fully folded protein. It is comparable to rhodamine dyes and optimal GFP.

DsRed is also very resistant to photobleaching with intensity typical of spectrofluorometers (mW / cm ² ) or microscopes with arc lamp illumination and interference filters (W / cm ² ) Yes, the photobleaching quantum yield is on the order of 7 × 10 ^{−7 in} both forms. This value is significantly better than that of the two most common green and yellow GFP-specific variants, EGFP (3 × 10 ⁻⁶ ) and EYFP-V68L / Q69K (5 × 10 ⁻⁵ ). The average number of photons emitted by a single molecule before photobleaching is the ratio of fluorescence to photobleaching quantum yield, ie 1 × 10 ⁶ , 2 for DsRed, EGFP, and EYFP-V68L / Q69K, respectively. × 10 ⁵ and 1.5 × 10 ⁴ . However, if the molecule can be brought into a dark state, such as a triplet or tautomer from which fluorescence can be recovered, the apparent photobleaching quantum yield is large in strong light intensity and in a short time. Will rise. Since GFP usually exhibits a range of such dark states (Dickson et al., Nature 388: 355-358, 1997; Schwille et al., Proc. Natl. Acad. Sci. USA 97: 151-156, 2000), DsRed Cannot be expected to be so simple. The photobleaching measurements described herein are performed over minutes to hours and include sufficient time for such recovery. In contrast, fluorescence correlation spectroscopy and flow cytometry monitor a single passage of molecules with a focused laser beam within microseconds to milliseconds, so the transient dark state that lasts longer than the transit time is It is measured as photobleaching, and the apparent quantum yield of bleaching increases. Techniques such as laser scanning confocal microscopy where the identified molecules are repeatedly scanned can exhibit moderate photobleaching depending on the time scale of irradiation and recovery.

Another desired property of DsRed is negligible sensitivity to pH changes over a wide range (pH 4.5-12). Currently available bright GFP mutants are more easily quenched by acidic pH than DsRed. Such pH sensitivity can be exploited to sense pH changes under control conditions, particularly those in organelles or other specific compartments (Llopis et al . , Proc. Natl. Acad. Sci., USA 95 : 6803-6808, 1998), but this property can cause artifacts in some applications.

DsRed mutants such as K83M demonstrate that DsRed can be brought closer to longer wavelengths (564 and 602 nm excitation and emission maxima) while retaining sufficient quantum efficiency (0.44). Shift to bathochromic of 6nm and 19nm corresponds to the energy of 191cm ^-1 and 541cm ^-1, which, for a single amino acid change that does not modify the chromophore, rather large. A homologue of DsRed recently cloned from sea anemone has an absorption maximum at 572 nm, a very weak emission with quantum yield <0.001 at 595 nm, and one mutant has an emission peak at 610 nm, but is very dark and mature (Lukyanov et al., J. Biol. Chem. 275: 25879-25882, 2000, which is incorporated herein by reference).

  Less desirable features of DsRed are its slow and incomplete maturation and the ability to oligomerize. Maturity times on the order of a few days hamper the use of DsRed as a reporter in short-term gene expression studies and applications aimed at tracking fusion proteins in short-generation time or fast-growing organisms. Yes. Since GFP maturation was greatly accelerated by mutagenesis (Heim et al., Nature 373: 663-664, 1995, which is incorporated herein by reference), DsRed can be mutagenized as well. And mutants with faster maturation times could be isolated.

  Since all Lys83 mutants have become capable of at least some maturity, primary amines are unlikely to play a direct catalytic role on this residue, and this suggestion is the most chemically conservative substitution ( The observation that Lys to Arg) significantly interfered with red color development is supported. Ser197 gave similar results in that the most conservative possible substitution (Ser to Thr) also significantly delayed maturation. Mutations at Lys83 and Ser197 sites appear several times independently in separate random mutagenesis experiments, and interestingly in dsFP483, a highly homologous cyan fluorescent protein from the same Discosoma species , Lys83 and Ser197 are replaced by Leu and Thr, respectively. Either of the latter two mutations may explain why dsFP483 never turns red. Residues other than Lys83 and Ser197 also affected red maturation.

The multimeric nature of DsRed is evidenced by evidence from four distinct strains, which are slow to migrate unless pre-boiled on SDS-PAGE, analytical ultracentrifugation, and immature green form of mammalian cells. This is a directed two-hybrid assay in yeast using strong FRET to type red form and HIS3 and LacZ reporter genes. Analytical ultracentrifugation provides the clearest evidence for four obligate stoichiometry over the entire range of monomer concentrations measured (10 ^-8 to 10 ^-5 M), with octamer formation occurring at higher concentrations. I gave a hint that I could. In addition, studies in living cells have demonstrated that aggregation occurs under typical use conditions, including the cytosolic reducing environment and the presence of native protein.

  DsRed oligomerization does not preclude its use as a reporter of gene expression, but this applies when DsRed is fused to a host protein, for example to investigate host protein tracking or interaction in cells. Can give results including artifacts. For mass M host proteins that do not have their own tendency to aggregate, fusion with DsRed can cause the formation of at least four (M + 26 kDa) complexes. Furthermore, since many proteins in signaling are activated by oligomerization, fusion to DsRed and the resulting association can cause constitutive signaling. For oligomeric host proteins, fusion to DsRed can cause stoichiometric mismatches, quaternary steric hindrance, or cross-linking in large aggregates. In fact, red chameleons, a fusion of cyan fluorescent protein, calmodulin, and calmodulin-binding peptide, and DsRed have much more visible spots in mammalian cells than the corresponding yellow chameleon with yellow fluorescent protein instead of DsRed. (Miyawaki et al., Proc. Natl. Acad. Sci. USA 96: 2135-2140, 1999).

  The results disclosed above show that DsRed mutants, like GFP mutants, can be produced to reduce or eliminate the tendency of fluorescent proteins to oligomerize. DsRed variants can be constructed and tested using, for example, yeast two hybrids or other similar assays to identify and isolate non-aggregating mutants. In addition, the X-ray crystal structure of DsRed can be examined to prove that modification of optimal amino acid residues produces a type of DsRed that has a reduced tendency to oligomerize.

Example 4
DsRed variants with reduced tendency to oligomerize This example reduces the tendency of DsRed to form tetramers with mutations corresponding to those introduced into the GFP variant to reduce or eliminate oligomerization To make it possible to make it in DsRed.

  Considering the results described in Example 1 and using the DsRed crystal structure as a guideline, it was confirmed that amino acid residues may be involved in DsRed oligomerization. One of these amino acids, isoleucine-125 (I125), was chosen because in the oligomer, the I125 residues of each subunit are close together in a paired manner, ie This is because the side chain of I125 of the A subunit is about 4 angstroms from the side chain of I125 of the C subunit, and the I125 residues of the B subunit and the D subunit are located in the same manner. Furthermore, like those identified with the Aequorea GFP mutant, the hydrophobic region of the I125 side chain was demonstrated to be involved in subunit-subunit interactions. Based on these observations, a DsRed mutant containing a substitution of Lys (K) and Arg (R), which are positively charged amino acids, was produced for I125.

DsRed I125K and I125R were prepared using the QuickChange mutagenesis kit, using DsRed cDNA (SEQ ID NO: 23; Clontech) subcloned into the expression vector pRSET _B (Invitrogen) as a mutagenesis template . Primers for mutagenesis (mutant codons are underlined) are:

  Muteins were prepared according to standard methods and analyzed by polyacrylamide gel electrophoresis as described (Baird et al., Supra, 2000). For further analysis, DsRed I125R was dialyzed extensively with PBS and then diluted in PBS until the absorbance of the 558 nm solution was 0.1. This solution was centrifuged in a Beckman XL-1 analytical ultracentrifuge at 10,000 rpm, 12,000 rpm, 14,000 rpm, and 20,000 rpm in PBS. Absorbance vs. radius at 558 nm was determined and compared to a wild type tetramer DsRed control (Baird et al., Supra, 2000).

  DsRed I125K became red fluorescent and produced a protein that was a mixture of dimers and tetramers, as shown by analyzing native protein by non-denaturing polyacrylamide gel electrophoresis. Similar analysis of DsRed I125R revealed that this protein is exclusively a dimer. When the dimer state of DsRed I125R was confirmed by analytical ultracentrifugation, no residual tetramer was detected. These results indicate that the interaction between the A: C subunit and the B: D subunit can be disrupted, thereby reducing the tendency of the DsRed mutant to oligomerize. No attempt was made to destroy the contact portion of A: B and C: D. These results demonstrate that the method of reducing or eliminating oligomerization of the GFP variant described in Example 1 is generally applicable to other fluorescent proteins that have a tendency to oligomerize.

Example 5
Preparation and characterization of tandem DsRed dimers This example shows that tandem DsRed proteins can be formed by linking two DsRed monomers, and such tandem DsRed proteins maintain the emission and excitation spectra characteristic of DsRed But it does not oligomerize.

In order to construct tDsRed, 3 ′ primer 5′-CCGGATCCCCTTTGGTGCTGCCCTCTCCGCTGCCAGGCTTGCCGCTGCCGCTGGTGCTGCCAAGGAACAGATGGTGGCGTCCCTCG-3 ′ (SEQ ID NO: 37) was designed. This primer overlaps the last 25 bp of DsRed (derived from the Clontech vector pDsRed-N1), encodes the linker sequence GTSGSSGKPGSGEGSTKG (SEQ ID NO: 38), followed by the BamHI site of pRSET _B (Invitrogen) And a BamHI restriction site to be in frame. Later, it was confirmed that the primer sequence contained three mismatches in the overlap region and contained several codons that were not optimal for mammalian expression. Therefore, a new 3 ′ primer 5′-CCGGATCCCCCTTGGTGCTGCCCTCCCCGCTGCCGGGCTTCCCGCTCCCGCTGGTGCTGCCCAGGAACAGGTGGTGGCGGCCCTCG-3 ′ (SEQ ID NO: 39) was also used. The 5 ′ primer 5′-GTACGACGATGACGATAAGGATCC-3 ′ (SEQ ID NO: 40) also contained a BamHI restriction site in-frame with the BamHI site of pRSET _B.

PCR amplification of DsRed and DsRed I125R with new linkers using Taq DNA polymerase (Roche) and annealing protocol (2 cycles at 40 ° C, 5 cycles at 43 ° C, 5 cycles at 45 ° C, and 15 cycles at 52 ° C) Was used). The resulting PCR product was purified by agarose gel electrophoresis and digested with BamHI (New England Biolabs). BamHI and calf intestinal phosphatase (New England Biolabs) -treated vectors were loaded with DsRed or DsRed I125R in-frame with the His-6 tag and inserted between the 5 'BamHI and 3' EcoRI recognition sites. Prepared from pRSET _B.

  The digested PCR product and the vector were ligated with T4 DNA ligase (NEB), and the mixture was used to transform E. coli DH5α by heat shock. Transformed colonies were grown on LB agar plates supplemented with the antibiotic ampicillin. Colonies were picked randomly and plasmid DNA was isolated by standard miniprep method (Qiagen). DNA sequencing was used to confirm the correct orientation of the insert sequence.

  To express the protein, competent E. coli JM109 (DE3) was transformed with the isolated and sequenced vector. A single colony grown on LB agar / ampicillin is used to inoculate 1 liter of LB / ampicillin culture and then the OD600 of the broth is 0.5-1.0 while shaking at 225 rpm at 37 ° C. Allowed to grow. IPTG was added to a final concentration of 100 mg / l and the cultures were grown either at 37 ° C. for 5 hours (tDsRed) or at room temperature for 24 hours (RT; tDsRed I125R). Cells were harvested by centrifugation (10 min, 5000 rpm), the pellet was resuspended in 50 mM Tris (pH 7.5), and the cells were lysed by a single pass through a French press. The protein was purified by Ni-NTA (Qiagen) chromatography and stored in elution buffer or dialyzed into 50 mM Tris (pH 7.5) as described by the manufacturer.

  With respect to the excitation and emission spectra of tDsRed and tDsRed I125R and the maturation time, the protein behaved in the same way as its uncoupled partner protein. As expected, tDsRed fluoresces within about 12 hours at RT, while tDsRed I125R took several days for a significant red to appear. The maturation of tDsRed I125R lasted almost 10 days. The excitation maximum and emission maximum were constant at 558 nm and 583 nm, respectively.

  Analysis of the protein by SDS-polyacrylamide electrophoresis revealed differences in tandem dimers. Due to the high stability of the tetramer, DsRed that was not boiled migrated with an apparent molecular weight of approximately 110 kDa. In addition, the band on the gel corresponding to the DsRed tetramer retained its red fluorescence, indicating that the exact barrel structure of each monomer was complete. When boiled before loading the sample, DsRed migrated as a monomer of approximately 32 kDa because it was non-fluorescent and probably denatured.

  SDS-PAGE analysis confirmed the tandem structure of the expressed red fluorescent protein, tDsRed and tDsRed I125R. Non-boiled tDsRed migrated with the same apparent molecular weight (approximately 110 kDa) as non-boiled normal DsRed. Only when the samples were boiled (denatured) before being loaded onto the gel, the difference in their molecular structure was apparent. The boiled tDsRed migrated with an apparent molecular weight of about 65 kDa, which was about the same as the molecular weight of the two DsRed monomers, while the boiled DsRed migrated with a molecular weight of 32 kDa for the monomer.

  A similar comparison was made for DsRed-I125R and tDsRed-I125R. If they were not boiled prior to SDS-PAGE, both tDsRed-I125R and DsRed-I125R migrated as dimers with an apparent molecular weight of approximately 50 kDa. DsRed-I125R, which was not boiled, also had a large component that appeared to be denatured, but a dimer (50 kDa) fluorescent band was clearly visible. tDsRed-I125R also had a denaturing component (65 kDa versus 50 kDa) that migrated more slowly than the complete fluorescent species. However, when boiled, tDsRed-I125R migrated with approximately the same molecular weight (65 kDa) as the two monomers, while DsRed-I125R migrated with a monomer molecular weight of 32 kDa.

  These results show that linking two DsRed monomers to form an intramolecularly bound tandem dimer prevents the formation of intermolecular oligomers without affecting the emission or excitation spectrum of the red fluorescent protein. Demonstrate.

Example 6
Red fluorescent protein with improved maturation efficiency
Wild-type DsRed and Q66M In an attempt to improve the rate and effect of maturation maturation of DsRed, DsRed's Q66 was converted to all other naturally occurring amino acids by site-directed mutagenesis. By picking up a fluorescent colony, almost all single mutations (F, N, G, T, H, E, K, D, R, L, C) at this position are harmful and loss of fluorescence or red It was confirmed that this led to the inability to mature into fluorescent species. However, only one substitution, Q66M, resulted in a protein with significantly improved properties.

  Initially, both wild-type DsRed and Q66M DsRed were produced in bacteria (E. coli) and rapidly purified and matured in cuvettes sealed with parafilm. Parafilm was chosen to allow diffusion of oxygen into the protein solution and to prevent water loss from the solution. Maturity was monitored by taking the time course of an absorption scan. FIG. 26 is a plot of maturation versus time as red chromophore peak absorption. Since proteins are not expressed immediately or precisely with the same efficiency, two different curves do not start with the same value and they reach slightly different endpoint values. For comparison purposes, the amplitudes of both curves were first normalized to the same final absorption. The curve was then fitted to a three-component kinetic model and the time reference for each scan was adjusted so that the curve crossed zero time.

Wild-type DsRed and Q66M DsRed fluorescence Whether the fluorescence emission and excitation spectra of wild-type DsRed and Q66M DsRed mutants use 558 nm excitation for DsRed or 583 nm emission and 566 nm excitation for Q66M DsRed Or it collected by monitoring luminescence at 590 nm. The results are shown in FIG. Of note is the red shift of the entire Q66M DsRed fluorescence spectrum, as well as a significant decrease in the excitation shoulder at 480 nm for Q66M DsRed compared to the wild type.

Q66M DsRed mature complete wild-type DsRed, Q66M DsRed, and other DsRed mutants, K83R DsRed, were boiled briefly in pH 1 HCl and then run on an SDS polyacrylamide gel. When the red chromophore is formed, the protein becomes acid-degradable at residue 66, so the relative amount of hydrolyzate relative to the full-length protein is an indicator of maturity integrity. The gel was Coomassie stained and imaged using a flatbed scanner, and then the relative intensities of all bands were quantified using the software NIH Image. After maturity completeness was normalized to molecular weight (because the Coomassie staining band density is related to mass, not the molar concentration of the protein in the band), the normalized intensity of the split fragment bands was normalized to the normal of all bands. It was calculated by dividing by the sum of the chemical strength. The results are shown in FIG. As expected, K83R DsRed contained only a trace amount of red protein and only full-length protein was seen. However, wild-type DsRed was roughly broken down by 2/3 into split fragments, and Q66M DsRed was almost completely degraded into split fragments.

  In conclusion, when Q66M DsRed is expressed in bacteria, the bacteria appear to be fluorescent slightly faster than bacteria expressing wild-type DsRed. Furthermore, the Q66M DsRed protein appears to be deeper pink than the wild-type DsRed, which is orange. Finally, the excitation spectrum of Q66M DsRed has a significantly smaller hump at 480 nm than wild-type DsRed, suggesting that species that absorb at 480 nm (the immature green form of the protein) are poor. Together, these data indicate that Q66M DsRed has significantly improved properties over the wild type DsRed protein.

Example 7
Preparation of a further improved variant of the monomer red fluorescent protein, mRFP1 The engineering of the monomer DsRed, mRFP1 (SEQ ID NO: 8) described in the previous examples is a genetically encoded red of unmodified DsRed. At least three problems associated with use as fluorescent fusion labels have been overcome. Specifically, (1) mRFP1 is a monomer, (2) it matures rapidly, and (3) it is not efficiently excited at a wavelength suitable for GFP imaging. However, mRFP1 is relatively dim (extinction coefficient (EC) 44,000 M ⁻¹ and quantum yield (QY) 0.25) and is therefore limited to certain applications. In studies to improve the brightness of mRFP1, the directed evolution strategy by randomizing specific residues has continued to hope to find variants with improved spectral properties.

In the first such library to improve the properties of mRFP1, the following codon substitutions were made in the mRFP1 cDNA:
N42Q to NNK (= 20 amino acids)
V44A to NNK (= 20 amino acids)
L46 to MTC (= I or L)
Q66 to NNK (= 20 amino acids in total)
K70 to ARG (= K or R)
V71A to GYC (= V or A) This library contains a genetic diversity of 262,144 cDNAs encoding 64,000 different amino acid sequences. This library was transformed into E. coli JM109 (DE3) and bacterial colonies were screened manually (approximately 50,000 independent colonies) as described in the preceding examples. Colonies that showed improved brightness or a different color were picked and the gene sequence was determined to confirm amino acid substitution. The top clones identified from this library were classified into several different categories as follows.

Red shift mutant:
Q66M + T147S; x588m610, EC-58,000M- ¹ , Q-0.25
Q66M; x588m610, EC ~ 52,000M- ¹ , QY ~ 0.25
Blue shift mutant:
Q66T + Q213L; x574m595, EC-34,000M- ¹ , QY-0.25
Q66T; x564m581, EC to 23,000M- ¹ , QY to 0.25
Q66S; x558m578, other interesting variants that are less dim than Q66T:
N42H + Q66G; x504m516, dim
V44M + Q66G; x504m516, dim
Q66L; absorption maximum at 502 nm, practically non-fluorescent The top mutant isolated from this library is mRFP1 + Q66M / T147S, which was named mRFP1.1. This mutant does not improve quantum yield compared to mRFP1, but mRFP1.1 is almost 30% brighter than mRFP1 due to improved EC. This improvement is due to the apparent increase in the fraction of proteins that form the mature red chromophore at the expense of non-fluorescent species that absorb at 502 nm (displaying the absorption and emission spectra of mRFP1.1). (See Figure 29). A favorable T147S mutant resulted from errors during PCR amplification of cDNA using Taq polymerase. The Q66M mutation has previously been shown to improve the fluorescence of the DsRed dimeric I125R mutant (Baird, GS (2001) Ph.D. Thesis, University of California, San Diego). The amino acid sequence of mRFP1.1 is shown in FIG. 30 (SEQ ID NO: 79), and the nucleotide sequence of mRFP1.1 is given in FIG. 31 (SEQ ID NO: 80).

  All publications, GenBank accession number sequence submissions, patents and published patent applications mentioned in the above specification are hereby incorporated by reference in their entirety. Various modifications and variations of the described compositions and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in connection with various specific embodiments, it should be understood that the invention as claimed should not be unduly limited by such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in protein chemistry or biological technology or related industries are intended to be within the scope of the following claims.

Claims (19)

A polynucleotide encoding a Discosoma red fluorescent protein (DsRed) variant comprising the amino acid sequence of SEQ ID NO: 8 .   The polynucleotide of claim 1 wherein the encoded DsRed variant has improved fluorescence maturation as compared to DsRed of SEQ ID NO: 1.   A polynucleotide encoding a tandem dimer comprising two DsRed protein variants encoded by the polynucleotide of claim 1 operably linked by a peptide linker. 4. The polynucleotide of claim 3 , wherein the peptide linker is 10-25 amino acids long. The polynucleotide of claim 4 wherein the peptide linker is 12-22 amino acids in length. 4. The polynucleotide of claim 3 , wherein the peptide linker is selected from the group consisting of GHGTGSTGSGSS (SEQ ID NO: 17), RMGTSTGSTKGQL (SEQ ID NO: 18), and RMGTSGSGKPGSGEGSTKGQL (SEQ ID NO: 19). The polynucleotide of claim 3 , wherein the tandem dimer is a homodimer. The polynucleotide of claim 3 , wherein the tandem dimer is a heterodimer. 9. A fusion protein comprising at least one DsRed protein variant encoded by the polynucleotide of any one of claims 1-8 operably linked to at least one polypeptide of interest. Polynucleotide. The polynucleotide of claim 9 wherein the fusion protein comprises a peptide tag. The polynucleotide of claim 10 , wherein the peptide tag is a polyhistidine peptide tag. A kit comprising at least one polynucleotide according to any one of claims 1-11 . 12. A kit comprising at least one polypeptide encoded by the polynucleotide of any one of claims 1-11 . A vector comprising a polynucleotide selected from the polynucleotide of any one of claims 1-11 . 15. A vector according to claim 14 , wherein the vector is an expression vector. A host cell comprising the vector according to claim 14 or 15 . A method for detecting transcriptional activity comprising:
(a) a host cell comprising a vector comprising a nucleotide sequence encoding a DsRed fluorescent protein variant according to any one of claims 1 to 11 operably linked to at least one expression control sequence; And providing a means for assaying the fluorescence of said fluorescent protein variant; and
(b) assaying the fluorescence of the fluorescent protein variant produced by the host cell, and using the fluorescence of the fluorescent protein variant as an indicator of transcription activity;
Said method. A polynucleotide encoding a polypeptide probe suitable for use in fluorescence resonance energy transfer (FRET), comprising at least one polynucleotide according to any one of claims 1-11 . A method for analyzing in vivo localization or transport of a polypeptide of interest comprising:
(a) providing the polynucleotide of claim 9 and a host cell or non-human tissue; and
(b) visualizing the fusion protein expressed in the host cell or tissue;
Said method.

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