JP2009131260A - Non-aggregating fluorescent protein and method for using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid composition encoding a non-aggregating chromo/fluorescent protein and its mutant and a protein encoded by the composition. <P>SOLUTION: Provided are polypeptides comprising non-aggregating colored and/or fluorescent proteins (the non-aggregating feature arises from the modulation of residues in the N-terminal of the protein and the chromo and/or fluorescent feature arises from the interaction of two or more residues of the protein), nucleic acids encoding the polypeptides, antibodies to the subject proteins, and transgenic cells and organisms are provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

This application is a continuation-in-part of Patent Application No. 10 / 006,922 filed on December 4, 2001, and is based on Patent Application No. 60 / 270,983 filed on February 21, 2001. Claiming priority, the disclosures of these applications are fully incorporated herein.

Introduction Field of the Invention The field of the invention is chromoproteins and fluorescent proteins.

Background of the Invention Labels are tools for marking a protein, cell, or organism of interest and play a prominent role in many biochemical, molecular biology, and medical diagnostic applications. A variety of different labels have been developed including radioactive labels, dye labels, fluorescent labels, chemiluminescent labels and the like. However, there is still interest in developing new labels. Of particular interest is the development of new protein labels, including chromoproteins and / or fluorescent protein labels.

  An important new class of fluorescent proteins recently developed is the reef-building coral fluorescent protein described in Matz, MV et al. (1999) Nature Biotechnol., 17: 969-973. Reef Coral Fluorescent Proteins). Although these fluorescent proteins show many advantages, certain types are prone to high molecular weight aggregation, which can cause problems and consequently limit their applicability.

  Therefore, the development of a non-aggregating form of this important new class of fluorescent proteins is of great interest. The present invention satisfies this need.

Related Literatures US Patents of Interest include: 6,066,476 (Patent Document 1); 6,020,192 (Patent Document 2); 5,985,577 (Patent Document 3); 5,976,796 (Patent Document 4); 5,968,750 ( Patent Document 5); 5,968,738 (Patent Document 6); 5,958,713 (Patent Document 7); 5,919,445 (Patent Document 8); 5,874,304 (Patent Document 9); and 5,491,084 (Patent Document 10) Is included. International patent applications of interest include WO 00/46233 (patent document 11); WO 99/49019 (patent document 12); and DE 197 18 640 A (patent document 13). It is. Anderluh et al., Biochemical and Biophysical Research Communications (1996) 220: 437-442 (Non-Patent Document 2); Dove et al., Biological Bulletin (1995) 189: 288-297 (Non-Patent Document 3); Fradkov et al., FEBS Lett. 2000) 479 (3): 127-30 (non-patent document 4); Gurskaya et al., FEBS Lett., (2001) 507 (1): 16-20 (non-patent document 5); Gurskaya et al., BMC Biochem. (2001) ) 2: 6 (Non-Patent Document 6); Lukyanov, K. et al. (2000) J Biol Chemistry 275 (34): 25879-25882 (Non-Patent Document 7); Macek et al., Eur. J. Biochem. (1995) 234: 329-335 (Non-Patent Document 8); Martynov et al., J Biol Chem. (2001) 276: 21012-6 (Non-Patent Document 9); Matz, MV et al. (1999) Nature Biotechnol., 17: 969-973 (Non-patent document 1); Terskikh et al., Science (2000) 290: 1585-8 (non-patent document 10); Tsien, Annual Rev. of Biochemistry (1998) 67: 509-544 (non-patent document 11); Tsien, Nat. Biotech. (1999) 17: 956-957 (Non-Patent Document 12); Ward et al., J. Biol. em. (1979) 254: 781-788 (Non-Patent Document 13); Wiedermann et al., Jarhrestagung der Deutschen Gesellschact fur Tropenokologie-gto.Ulm.17-19.02.1999.Poster P-4.20 (Non-Patent Document 14); Yanushevich et al. FEBS Lett (January 30, 2002) 511 (1-3): 11-4 (Non-Patent Document 15); and Yarbrough et al., Proc Natl Acad Sci USA (2001) 98: 462-7 (Non-Patent Document 16). ) Is also of interest.
U.S. Patent No. 6,066,476 US Patent 6,020,192 U.S. Pat.No. 5,985,577 U.S. Patent No. 5,976,796 U.S. Patent No. 5,968,750 U.S. Pat.No. 5,968,738 U.S. Patent No. 5,958,713 U.S. Pat.No. 5,919,445 U.S. Pat.No. 5,874,304 U.S. Pat.No. 5,491,084 International Publication No. 00/46233 International Publication No.99 / 49019 DE 197 18 640 A Matz, MV et al. (1999) Nature Biotechnol., 17: 969-973 Anderluh et al., Biochemical and Biophysical Research Communications (1996) 220: 437-442 Dove et al., Biological Bulletin (1995) 189: 288-297 Fradkov et al., FEBS Lett. (2000) 479 (3): 127-30 Gurskaya et al., FEBS Lett., (2001) 507 (1): 16-20 Gurskaya et al., BMC Biochem. (2001) 2: 6 Lukyanov, K. et al. (2000) J Biol Chemistry 275 (34): 25879-25882 Macek et al., Eur. J. Biochem. (1995) 234: 329-335 Martynov et al., J Biol Chem. (2001) 276: 21012-6 Terskikh et al., Science (2000) 290: 1585-8 Tsien, Annual Rev. of Biochemistry (1998) 67: 509-544 Tsien, Nat. Biotech. (1999) 17: 956-957 Ward et al., J. Biol. Chem. (1979) 254: 781-788 Wiedermann et al., Jarhrestagung der Deutschen Gesellschact fur Tropenokologie-gto.Ulm.17-19.02.1999.Poster P-4.20 Yanushevich et al., FEBS Lett (January 30, 2002) 511 (1-3): 11-4 Yarbrough et al., Proc Natl Acad Sci USA (2001) 98: 462-7

SUMMARY OF THE INVENTION Nucleic acid compositions encoding non-aggregating dye / fluorescent proteins and variants thereof, and proteins encoded by them are provided. The protein of interest is a non-aggregating colored and / or fluorescent protein (non-aggregating attributes are generated by adjusting the N-terminal residues of the protein, and the pigmented and / or fluorescent attributes are two of the protein Or a polypeptide generated by the interaction of more residues). Also provided are fragments of the nucleic acids of the invention, and peptides encoded thereby, as well as antibodies to the proteins of the invention, as well as transgenic cells and organisms. The protein and nucleic acid compositions of the present invention are useful in a variety of different applications. Finally, kits are provided for use in such applications, for example kits comprising the nucleic acid compositions of the invention.

Definitions In accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art can be utilized. Such techniques are explained fully in the literature. For example, Maniatis, Fritsch, and Sambrook, “Molecular Cloning: A Laboratory Manual” (1982); “DNA Cloning: A Practical Approach”, Volumes I and II (DNGlover 1985); “Oligonucleotide Synthesis” (MJGait 1984) "Nucleic Acid Hybridization" (BDHames and SJHiggins (1985)); "Transcription and Translation" (BDHames and SJHiggins (1984)); "Animal Cell Culture" (RIFreshney (1986)); "Immobilized Cells and Enzymes" (IRL Press, (1986)); see B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

  A “vector” is a replicon, such as a plasmid, phage, or cosmid, to which another DNA segment can be added such that replication of that segment occurs.

  “DNA molecule” refers to a polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in either a single stranded form or a double stranded helix. The term refers only to the primary and secondary structure of the molecule and is not intended to limit the molecular structure to any particular tertiary form. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (eg, restriction fragments), viruses, plasmids, and chromosomes.

  A DNA “coding sequence” is a DNA sequence that is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA derived from eukaryotic mRNA, genomic DNA sequences derived from eukaryotic (eg, mammalian) DNA, and synthetic DNA sequences. . A polyadenylation signal and transcription termination sequence may be located 3 'to the coding sequence.

  As used herein, the term “hybridization” refers to two nucleic acids that form an antiparallel duplex stabilized by hydrogen bonding between residues of opposite nucleic acid strands. This refers to the process of chain association.

  The term “oligonucleotide” refers to a short (less than 100 bases long) nucleic acid molecule.

  “DNA regulatory sequences” as used herein are transcriptional and translational, such as promoters, enhancers, polyadenylation signals, terminators, etc., that provide and / or control the expression of a coding sequence in a host cell. Regulatory sequence.

  A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3 ′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence contains the minimum number of bases or elements required to initiate transcription at a detectable level above the background, with the transcription start site as the 3 'end boundary. As shown, it spreads upstream (5 'direction). It is considered that not only a transcription initiation site but also a protein binding domain responsible for binding of RNA polymerase is found in the promoter sequence. Eukaryotic promoters will often, but not always, contain a “TATA” box and a “CAT” box. A variety of promoters, including inducible promoters, can be used to drive the various vectors of the present invention.

  As used herein, the terms “restriction endonuclease” and “restriction enzyme” refer to bacterial enzymes that each cleave double-stranded DNA at or near a specific nucleotide sequence.

  When exogenous or heterologous DNA has been introduced into the cell, the cell has been “transformed” or “transfected” by such DNA. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. For example, in prokaryotes, yeast, and mammalian cells, transforming DNA can be maintained on episomal elements such as plasmids. With respect to eukaryotic cells, a stably transformed cell is one in which the transformed DNA is integrated into the chromosome so that it can be inherited by daughter cells through chromosome replication. This stability is evidenced by the ability of eukaryotic cells to establish cell lines or clones composed of a population of daughter cells containing transforming DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that can be stably grown in vitro for many generations.

  A “heterologous” region of a DNA construct is a segment of identifiable DNA in a larger molecule that is not found in nature in association with the larger DNA molecule. Thus, if the heterologous region encodes a mammalian gene, the gene is usually considered to be adjacent to DNA that is not adjacent to the mammalian genomic DNA in the genome of the source organism. In another example, the heterologous DNA includes a coding sequence in a construct in which portions of genes from two different sources are collected to create a fusion protein product. Allelic changes or naturally occurring mutational events do not generate a heterologous region of DNA as defined herein.

  As used herein, the term “reporter gene” refers to a heterologous promoter or enhancer element that allows the product to be easily and quantitatively assayed when the construct is introduced into a tissue or cell. Refers to the encoded sequence.

The amino acids described herein are preferably “L” isomers. The amino acid sequence is represented by a single letter (A: alanine; C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine; G: glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M: Methionine; N: asparagine; P: proline; Q: glutamine; R: arginine; S: serine; T: threonine; V: valine; W: tryptophan; Y: tyrosine; X: any residue). NH ₂ refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In line with standard polypeptide nomenclature, J Biol. Chem., 243 (1969), 3552-59 is used.

  The term “immunologically active” induces a specific immune response in a suitable animal or cell of a natural, recombinant or synthetic dye / fluorescent protein or any oligopeptide thereof. Define the ability and ability to bind to a specific antibody. As used herein, “antigenic amino acid sequence” means an amino acid sequence that can elicit an antibody response in a mammal, either alone or in association with a carrier molecule. In connection with the binding of an antibody to an antigen, the term “specific binding” is a well-understood term in the art, where an antibody binds to the antigen that produced the antibody and is otherwise unrelated. It does not bind to any antigen.

  As used herein, the term “isolated” refers to a polynucleotide, polypeptide, polypeptide, polypeptide, antibody, or polynucleotide, polypeptide that is present in an environment different from that in which the host cell naturally occurs. , Antibodies, or host cells are intended to be described.

  Bioluminescence (BL) is defined as the emission of light by an organism that is sufficiently visible in the dark and affects the visual behavior of animals (eg, Harvey, EN (1952). Bioluminescence. New York) : Academic Press; Cell Physiology (N. Speralakis) pp. 651-681, Hastings, JW (1995). Bioluminescence. New York: Academic Press .; Wilson, T. and Hastings, JW (1998). Bioluminescence. Annu Rev Cell Dev Biol 14,197-230). For bioluminescence, so-called very low light radiation (Murphy, ME and Sies, H. (1990), Visible-range low-level), which can be detected in virtually all biological structures using a sensitive illuminometer. Chemiluminescence in biological systems. Meth. Enzymol. 186,595-610; Radiotic, K, Radenovic, C, Jeremic, M. (1998) Spontaneous ultra-weak bioluminescence in plants: origin, mechanisms and properties. Gen Physiol Biophys 17,289-308), And bamboo growth cone luminescence (Totsune, H., Nakano, M., Inaba, H. (1993). Chemiluminescence from bamboo shoot cut. Biochem. Biophys. Res Comm. 194, 1025-1029), or fertilization of animal eggs Radiation of light (Klebanoff, SJ, Froeder, CA, Eddy, EM, Shapiro, BM (1979). Metabolic similarities between fertilization and phagocytosis. Conservation of peroxidatic mechanism. J. Exp. Med. 149, 938-953; Schomer, B And Epel, D. (1998). Redox changes during fertilization and maturation of marine invertebrate eggs. Dev Biol 203,1-11 Such as, perhaps weak light emission which does not play any ecological role is not included.

DESCRIPTION OF SPECIFIC EMBODIMENTS Nucleic acid compositions encoding non-aggregating dye / fluorescent proteins and variants thereof, and the proteins they encode are provided. The protein of interest is a non-aggregating protein that is colored and / or fluorescent (non-aggregating characteristics arise from the adjustment of the N-terminal residue of the protein, and the pigmented and / or fluorescent characteristics are Generated by the interaction of two or more residues). Also of interest are proteins that are substantially similar to the specific protein or variants of the specific protein. Nucleic acid fragments and peptides encoded by them are also provided, as well as transgenic cells and organisms comprising antibodies against the proteins of the invention, and nucleic acid / protein compositions of the invention. The protein and nucleic acid compositions of the present invention are useful in a variety of different applications. Finally, kits are provided for use in such applications, for example kits comprising the nucleic acid compositions of the invention.

  Before further describing the present invention, the present invention is not limited to the specific embodiments of the invention described below, and variations of the specific embodiments may be made and are also within the scope of the appended claims. Please understand that. It should also be understood that the terminology used is for describing particular embodiments and is not intended to limit the invention. Instead, the scope of the present invention is established by the appended claims.

  In this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. included. 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.

  Where a range of values is provided, each value that falls between the upper and lower limits of the range (up to one tenth of the lower limit unless otherwise specified), and others in the stated range It is understood that any stated value or value in between is encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also encompassed by the present invention, provided that any limits are specifically excluded from the stated ranges. Where the stated range includes one or both of the limits, ranges excluding either or both of the included limits are also included in the invention.

  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. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. Is done.

  All publications mentioned in this specification describe cell lines, vectors, methodologies, and other inventive components described in the publications that can be used in connection with the invention described herein. And for the purposes of disclosure, incorporated herein by reference.

  In a further description of the invention, the nucleic acid composition of the invention is first described, followed by discussion of the protein composition, antibody composition, and transgenic cell / organism of the invention. The following provides an overview of representative methods in which the proteins of the present invention are beneficial.

Nucleic Acid Compositions As summarized above, the present invention provides nucleic acid compositions that encode non-aggregating chromoproteins and fluorescent proteins and variants thereof, as well as fragments and homologues of these proteins.

  As summarized above, the proteins encoded by the nucleic acids of the invention are non-aggregating chromoproteins and / or fluorescent proteins. Non-aggregating means that the proteins do not aggregate, that is, the mutual complex does not form a high molecular weight aggregate. As used herein, an “aggregate” refers to a higher order molecular complex, eg, a complex comprising two or more protein tetramers. The molecular weight of such aggregates is typically greater than about 100 kDa, and more typically greater than about 150 kDa. Aggregates are distinguished from multimers (the term “multimer” refers to oligomers such as dimers, trimers, and tetramers). Non-aggregating polypeptides of the present invention include polypeptides that exhibit reduced in vitro and / or in vivo aggregation compared to corresponding aggregated analogs, eg, corresponding wild type proteins.

  In certain embodiments, the polypeptides of the invention exhibit reduced in vitro aggregation compared to corresponding aggregating analogs, such as the corresponding wild type protein. “Reduction of aggregation in vitro” refers to the reduction of aggregation in a cell-free system or solution. In some embodiments, the non-aggregating polypeptide is less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 60% of the aggregation exhibited by the corresponding aggregated analog under the same in vitro conditions. Less than 50%, less than about 40%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%, for example, a book present in a sample Less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5% of the polypeptides of the invention aggregate. In vitro conditions suitable for comparing a polypeptide of the invention to the corresponding aggregating analog are conditions that do not prevent aggregation of the aggregating analog, eg, standard physiological conditions. Any of a wide variety of buffer systems used in the art to study physiological phenomena can be used for in vitro comparisons. Non-limiting examples of such conditions include a salt concentration in the range of about 0.01 mM to about 0.1 mM; a temperature in the range of about 19 ° C. to about 25 ° C .; and a pH in the range of about 6.5 to about 8.0. However, these are not limited. Buffers suitable for comparison of aggregation include, but are not limited to, any physiological buffer; Tris-Cl, phosphate buffered saline; Tris buffered saline; borate buffered saline, etc. Not done. An example is 1 × Tris-Cl buffer, pH 8.8, 0.1% SDS, room temperature. An exemplary assay for determining whether the DsRed variants of the present invention form aggregates is as described in the Examples. Briefly, a standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protocol is used to isolate DsRed mutant proteins produced recombinantly in bacterial cells such as E. coli. The sample is not boiled before loading on the gel. Standard conditions for SDS-PAGE are described in Short Protocols in Molecular Biology, 4th edition, 1999, edited by FM Ausubel et al., John Wiley & Sons, Inc. Typically, the sample is electrophoresed in the presence of about 0.1% SDS in 1 × Tris-Cl buffer (pH about 8.8).

  In some embodiments, non-aggregating polypeptides of the invention exhibit reduced aggregation in vivo. “Reduction of aggregation in vivo” refers to the reduction of aggregation within a cell. In some embodiments, the non-aggregating polypeptide is a eukaryotic cell or cell population of the same cell type, in another eukaryotic cell from the same cell line, in the same prokaryotic cell, or in the same cell type. Less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, about 25% of the aggregation exhibited by the corresponding aggregating analog Less than, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. Generally, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10% of the non-aggregating polypeptides of the invention present in a cell or cell population, Or less than about 5% aggregate. Methods for measuring the degree of aggregation are known in the art; a given variant can be compared to the corresponding aggregated analog, eg, compared to the corresponding aggregated wild-type polypeptide. Any known method can be used to determine whether it exhibits a decrease. Such methods include “pseudo-native” protein gel electrophoresis as described in the Examples; gel filtration; ultracentrifugation; circular dichroism; These are not limited. Aggregation can be measured by light scattering, as described in the Examples. For unaggregated proteins, the ratio of absorbance at shorter wavelengths to absorbance at longer wavelengths is close to zero. In some embodiments, the ratio of absorbance at 400 nm to absorbance at 566 nm of the non-aggregating polypeptide is about 0.01 to about 0.1, about 0.015 to about 0.09, about 0.02 to about 0.08, about 0.025 to about 0.07, or about 0.03. Within the range of ~ 0.06.

  In many embodiments, the non-aggregating polypeptides of the present invention are in a manner sufficient to create a non-aggregating variant of a naturally occurring protein or an aggregated variant thereof, such as reversing or moderate charge. For the sake of summation, it has an amino acid sequence that differs from the corresponding wild-type sequence due to an N-terminal mutation that adjusts the charge appearing on the side chain group of the N-terminal residue. More specifically, a basic residue located near the N-terminus of the protein is substituted, for example, the Lys residue and Arg residue near the N-terminus are negatively charged or neutral residues Is replaced by N-terminus means within about 50 residues from the N-terminus, often within about 25 residues at the N-terminus, more often within about 15 residues at the N-terminus, and in many embodiments Residue modifications occur within about 10 residues of the N-terminus. In many embodiments, the specific residues of interest include 2, 3, 4, 5, 6, 7, 8, 9, and 10.

  As mentioned above, in addition to the non-aggregating properties of the polypeptides of the invention encoded by the nucleic acids of the invention, the polypeptides of the invention are also characterized by being colored and / or fluorescent. Dye and / or fluorescent protein means a protein that is colored, i.e. colored, the protein may be fluorescent or non-fluorescent, e.g. Depending on the irradiation, low, medium or high fluorescence may be exhibited. In any case, the protein of the present invention of interest has a colored characteristic, i.e. a pigmented and / or fluorescent characteristic, that is a single residue of the protein, more specifically a single single residue. A protein that arises from the interaction of two or more residues of a protein rather than from its side chains. Therefore, the fluorescent proteins of the present invention do not include proteins that exhibit only fluorescence from residues that themselves act as intrinsic fluors, ie, tryptophan, tyrosine, and phenylalanine. Therefore, the fluorescent protein of the present invention is fluorescent such that the fluorescence is generated from some structure in the protein other than the single residue, for example, from the interaction of two or more residues. It is a protein.

  In many embodiments, the polypeptide encoded by the nucleic acid of the invention is a naturally occurring protein, often a variant of a protein that is present in a cnidarian species, for example, a class of nematode species. In certain embodiments, the nucleic acid further comprises (1) a non-bioluminescent species, often a non-bioluminescent cnidarian species, such as a non-bioluminescent flower moth species; or (2) It is characterized in that it encodes a non-aggregating variant of a wild-type protein (or variant thereof) that is not from a (Pennatulacean. Therefore, the nucleic acids of these embodiments encode non-aggregating variants of proteins derived from bioluminescent flower worm species unless they are of the order Umella, such as Renillan or Ptilosarcan. obtain. Of particular interest in certain embodiments are non-aggregating variants of the following specific wild type proteins (or variants thereof): (1) disclosed in patent application No. 10 / 006,922 amFP485, cFP484, zFP506, zFP540, drFP585, dsFP484, asFP600, dgFP512, dmFP592 (the disclosure of which is incorporated herein by reference); (2) hcFP640 disclosed in patent application No. 09 / 976,673 (the disclosure is (3) CgCP disclosed in patent application 60 / 255,533 (the disclosure of which is incorporated herein by reference); and (4) patent application 60 / 332,980. HcriGFP, zoanRFP, scubGFP1, scubGFP2, rfloRFP, rfloGFP, mcavRFP, mcavGFP, cgigGFP, afraGFP, rfloGFP2, mcavGFP2, mannFP (this disclosure is incorporated herein by reference). Specific non-aggregating fluorescent polypeptides of interest include FP1-NA; FP3-NA; FP4-NA; FP6-NA; E5-NA; 6 / 9Q-NA; 7A-NA; mutM35-5 dimer- NA and the like are included, but are not limited to, and these specific non-aggregating variants are further described below.

  In some embodiments, the nucleic acids of the invention encode a polypeptide that exhibits an increased isoelectric point (pI) relative to a reference protein, eg, a corresponding wild-type protein, in addition to the features described above. The isoelectric point can be determined using any method known in the art. In some embodiments, pI is the theoretical pI calculated as described in the Examples. In some embodiments, the muteins of the invention have a pI in the range of about 5.50 to about 7.00, about 5.75 to about 6.75, about 6.00 to about 6.50, or about 6.10 to about 6.40.

  The fluorescence intensity of a particular fluorescent protein is determined by the quantum yield multiplied by the maximum extinction coefficient. The brightness of a chromoprotein can be represented by a maximum extinction coefficient. In some embodiments, a polypeptide encoded by a nucleic acid of the invention exhibits substantially the same or greater brightness in the cell as compared to a reference protein, eg, the corresponding wild type protein, eg, a variant Is at least about 10%, at least about 20%, at least about 30% than the reference protein. At least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100% (or 2 times), at least about 150%, at least about 3 times, Or at least about 4 times, or more, high brightness in the cells. A “cell” can be a prokaryotic cell or a eukaryotic cell. Methods for measuring luminance are known in the art. Luminance is measured using any known method including, but not limited to, visual screening, spectrophotometry, spectrofluorimetry, fluorescence microscopy, fluorescence activated cell sorting (FACS) machine, etc. obtain. In some examples, the brightness of a mutant protein of the invention in a cell can be visually compared to the brightness of a reference protein in a cell of the same cell type, or in another cell of the same cell line.

  A nucleic acid composition is an open reading frame encoding a non-aggregating dye / fluorescent polypeptide of the present invention, ie, a dye / fluorescent protein coding sequence, and under appropriate conditions, the non-aggregating according to the present invention It means a composition comprising a sequence of DNA that can be expressed as a sex dye / fluorescent protein. The term also encompasses nucleic acids that are homologous, substantially similar, or identical to the nucleic acids of the invention. Thus, the present invention provides not only coding sequences that encode the proteins of the present invention, but also homologs thereof.

  In addition to the specific nucleic acid composition described above, homologues of the above sequences are also of interest. In certain embodiments, the sequence similarity between homologues is at least about 20%, sometimes at least about 25%, 30%, 35%, 40%, 50%, 60%, 70%, or more, And may include 75%, 80%, 85%, 90%, and 95%, or more. Sequence similarity is calculated based on a reference sequence that may be a subset of a larger sequence, such as a conserved motif, coding region, flanking region, etc. The reference sequence is usually at least about 18 nt long, more usually at least 30 nt long, and may extend to the complete sequence being compared. Algorithms for sequence analysis, such as BLAST described in Altschul et al. (1990), J. Mol. Bilo. 215: 403-10 (using default settings, ie parameters W = 4 and T = 17), Known in the art. Of particular interest in certain embodiments are nucleic acids of substantially the same length as the nucleic acids identified as SEQ ID NOs: 13; 14; 15; 17; 19; 21; and 23 (substantially The same length means that the difference in length does not exceed about 20%, usually does not exceed about 10%, more usually does not exceed about 5%); And has at least about 90%, usually at least about 95%, more usually at least about 99% sequence identity over any length of the nucleic acid to any of these sequences. It is. In many embodiments, the nucleic acid has a sequence that is substantially similar (ie, the same) or identical to the sequences of SEQ ID NOs: 14; 15; 17; 19; 21; and 23 ing. Substantially similar means that the sequence identity is generally at least about 60%, usually at least about 75%, often at least about 80%, 85%, 90%, or 95% It means that there is even thought to be.

  Nucleic acids that encode proteins encoded by the nucleic acids but differ in sequence from the nucleic acids due to the degeneracy of the genetic code are also provided.

  Nucleic acids that hybridize to the nucleic acids under stringent conditions are also provided. An example of stringent hybridization conditions is hybridization at 50 ° C. or higher and 0.1 × SSC (15 mM sodium chloride / 1.5 mM sodium citrate). Another example of stringent hybridization conditions is: solution: 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10 Overnight incubation at 42 ° C. in% dextran sulfate and 20 μg / ml denatured sheared salmon sperm DNA, followed by washing of the filter at 0.1 × SSC at about 65 ° C. Stringent hybridization conditions are hybridization conditions that are at least as stringent as the representative conditions described above, and are at least about 80% stringent compared to the specific stringent conditions described above, A condition is considered at least as equally stringent if it is typically at least about 90% stringent. Other stringent hybridization conditions are known in the art and can also be utilized to identify the nucleic acids of this particular embodiment of the invention.

  Nucleic acids encoding variants of the non-aggregating proteins of the invention are also provided. Mutant nucleic acids can be generated by random mutagenesis or targeted mutagenesis using well-known techniques that are routine in the art. In some embodiments, the chromoprotein or fluorescent protein encoded by the nucleic acid encoding the homologue or variant has the same fluorescent properties as the wild-type fluorescent protein. In other embodiments, the homologue or mutant nucleic acid encodes a chromoprotein or fluorescent protein with altered spectral properties, as described in more detail herein.

  As described in more detail below, the nucleic acids of the invention can be present in a suitable vector for extrachromosomal maintenance or for integration into the host genome.

  The nucleic acid composition of the present invention may encode all or part of the protein of the present invention. Double-stranded or single-stranded fragments can be obtained from the DNA sequence by chemical synthesis of oligonucleotides, restriction enzyme digestion, PCR amplification and the like by conventional methods. Usually, a DNA fragment will be at least about 15 nt, usually at least about 18 nt or about 25 nt, but may be at least about 50 nt. In some embodiments, the nucleic acid molecules of the invention can be about 100 nt, about 200 nt, about 300 nt, about 400 nt, about 500 nt, about 600 nt, about 700 nt, or about 720 nt in length. The nucleic acids of the invention can encode fragments or full-length proteins of the invention, for example, the nucleic acids of the invention can be about 25 aa, about 50 aa, about 75 aa, about 100 aa, about 125 aa, about 150 aa, about 150 aa Polypeptides of 200aa, about 210aa, about 220aa, about 230aa, or about 240aa, up to the length of the entire protein can be encoded.

  The polynucleotides and constructs thereof are provided. These molecules can be generated synthetically by a number of different protocols known to those skilled in the art. Suitable polynucleotide constructs are described using standard recombinant DNA techniques described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY. And current regulations in the Guidelines for Recombinant DNA Research of the United States Dept. of HHS and the National Institute of Health (NIH) And purified.

  Nucleic acids encoding a fusion protein of a protein of the invention or fragment thereof fused to a second protein, such as a degradation sequence, a signal peptide, a protein of interest (ie, the protein being studied), etc. are also provided. Fusion proteins include polypeptides of the invention or fragments thereof, and non-aggregating polypeptides ("fusion partners") fused in-frame to the N-terminus and / or C-terminus of the polypeptides of the invention. Can be included. A fusion partner can be a polypeptide (eg, an epitope tag) capable of binding to an antibody specific for that fusion partner; an antibody or binding fragment thereof; provide a catalytic function or induce a cellular response Polypeptides; including but not limited to ligands or receptors or mimetics thereof. In such fusion proteins, the fusion partner is generally not naturally associated with the non-aggregating protein portion of the invention of the fusion protein, and in certain embodiments, a cnidarian protein or derivative / fragment thereof. But not found in cnidarian species.

  Also provided are constructs comprising a nucleic acid of the invention inserted into a vector that can be used for a number of different applications, including amplification, protein production, and the like. Viral and non-viral vectors containing plasmids can be prepared and used. The choice of vector will depend on the type of cell in which amplification is desired and the purpose of the amplification. Certain vectors are useful for amplifying and making large quantities of desired DNA sequences. There are also vectors suitable for expression in cultured cells. In addition, there are vectors that are suitable for transfer and expression in cells in the complete animal or human body. The selection of an appropriate vector is well within the skill of the art. Many such vectors are commercially available. To prepare the construct, a partial or full length polynucleotide is inserted into the vector, typically by addition with a DNA ligase to a cleaved restriction enzyme site in the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination in vivo. Typically this is achieved by adding homologous regions on either side of the desired nucleotide sequence of the vector. The homologous region is added, for example, by ligation of oligonucleotides or by polymerase chain reaction using primers that contain both the homologous region and a portion of the desired nucleotide sequence.

  Among applications, there are also provided expression cassettes or expression systems that are particularly beneficial in the synthesis of the proteins of the invention. For expression, the gene product encoded by the polynucleotide of the invention is expressed in any convenient expression system, including, for example, bacterial, yeast, insect, amphibian, and mammalian systems. Suitable vectors and host cells are described in US Pat. No. 5,654,173. In the expression vector, the polynucleotide of the present invention is appropriately linked to a control sequence in order to obtain desired expression characteristics. These control sequences can include promoters (added to the 5 ′ end of the sense strand or the 3 ′ end of the antisense strand), enhancers, terminators, operators, repressors, and inducers. The promoter may be regulated or constitutive. In some situations, it may be desirable to use a conditionally active promoter, such as a tissue specific promoter or a developmental stage specific promoter. These are ligated to the desired nucleotide sequence using the techniques described above for ligation with vectors. Any technique known in the art can be used. In other words, the expression vector is inducible or may be constitutive, a transcription and translation initiation region (the coding region is operably linked under transcriptional control of the transcription initiation region), As well as the termination region of transcription and translation. These regulatory regions may be native to the species of the invention from which the nucleic acids of the invention are obtained, or may be derived from exogenous sources.

  Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. There may be selectable markers that function in the expression host. Expression vectors can be used inter alia for the production of said fusion protein.

  An expression cassette can be prepared that includes a transcription initiation region, gene or fragment thereof, and a transcription termination region. Of particular interest is a functional epitope, usually at least 8 amino acids long, more usually at least about 15 amino acids long to about 25 amino acids long, up to the length of the complete open reading frame of the gene. Or the use of sequences that allow the expression of the domain. After introduction of the DNA, the cells containing the construct can be selected with a selectable marker, the cells can be amplified and then used for expression.

  The expression system can be utilized with prokaryotes or eukaryotes according to conventional methods, depending on the purpose of expression. For mass production of proteins, unicellular organisms such as E. coli, B. subtilis, S. cerevisiae, insect cells combined with baculovirus vectors, or higher such as vertebrates Biological cells such as COS7 cells, HEK293, CHO, Xenopus oocytes and the like can be used as expression host cells. In some situations, it is desirable for the expressed protein to express a gene in eukaryotic cells that would benefit from conventional folding and post-translational modifications. Small peptides can also be synthesized in the laboratory. Polypeptides that are a subset of the complete protein sequence can be used to identify and examine portions of the protein that are important for function.

  Particular expression systems of interest include expression systems derived from bacteria, yeast, insect cells, and mammalian cells. Representative lines from each of these categories are listed below.

Bacteria Bacterial expression systems include Chang et al., Nature (1978) 275: 615; Goeddel et al., Nature (1979) 281: 544; Goeddel et al., Nucleic Acids Res. (1980) 8: 4057; European Patent No. 0 036,776. U.S. Pat. No. 4,551,433; DeBoer et al., Proc. Natl. Acad. Sci. (USA) (1983) 80: 21-25; and Siebenlist et al., Cell (1980) 20: 269.

Yeast Expression systems in yeast include Hinnnen et al., Proc. Natl. Acad. Sci. (USA) (1978) 75: 1929; Ito et al., J. Bacteriol. (1983) 153: 163; Kurtz et al., Mol. Cell. Biol. (1986) 6: 142; Kunze et al., J. Basic Microbiol. (1985) 25: 141; Gleeson et al., J. Gen. Microbiol. (1986) 132: 3459; Roggenkamp et al., Mol. Gen. Genet. 1986) 202: 302; Das et al., J. Bacteriol. (1984) 158: 1165; De Louvencourt et al., J. Bacteriol. (1983) 154: 737; Van den Berg et al., Bio / Technology (1990) 8: 135; Kunze et al., J. Basic Microbiol. (1985) 25: 141; Cregg et al., Mol. Cell. Biol. (1985) 5: 3376; US Pat. Nos. 4,837,148 and 4,929,555; Beach and Nurse, Nature (1981) 300 David et al., Curr. Genet. (1985) 10: 380; Gaillardin et al., Curr. Genet. (1985) 10:49; Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112: 284-289. Tilburn et al., Gene (1983) 26: 205-221; Yelton et al., Proc. Natl. Acad. Sci. (USA) (1984) 81: 1470-1474; Kelly and Hynes, EMBO J. (198 5) 4: 475479; European Patent No. 0 244,234; and WO 91/00357.

Insect cells Expression of heterologous genes in insects is described in US Patent No. 4,745,051; The Molecular Biology Of Baculoviruses (1986) (ed. By W. Doerfler), Friesen et al., The Regulation of Baculovirus Gene Expression; Patent 0 155,476; and Vlak et al., J. Gen. Virol. (1988) 69: 765-776; Miller et al., Ann. Rev. Microbiol. (1988) 42: 177; Carbonbone et al., Gene (1988) 73: Maeda et al., Nature (1985) 315: 592-594; Lebacq-Verheyden et al., Mol. Cell. Biol. (1988) 8: 3129; Smith et al., Proc. Natl. Acad. Sci. (USA) (1985) 82: 8844; Miyajima et al., Gene (1987) 58: 273; and Martin et al., DNA (1988) 7:99. Numerous baculovirus strains and variants, and the corresponding permissive insect host cells, are described by Luckow et al., Bio / Technology (1988) 6: 47-55, Miller et al., Generic Engineering (1986) 8: 277-279, and Maeda et al. Nature (1985) 315: 592-594.

Mammalian cells Mammalian expression is described in Dijkema et al., EMBO J. (1985) 4: 761, Gorman et al., Proc. Natl. Acad. Sci. (USA) (1982) 79: 6777, Boshart et al., Cell (1985) 41. : 521, and US Pat. No. 4,399,216. Other characteristics of mammalian expression include Ham and Wallace, Meth. Enz. (1979) 58:44, Barnes and Sato, Anal. Biochem. (1980) 102: 255, U.S. Pat. Nos. 4,767,704, 4,657,866, Promoted as described in 4,927,762, 4,560,655, WO 90/103430, WO 87/00195, US Reissue Patent 30,985.

  When any of the above host cells, or any other suitable host cell or host organism, is used to replicate and / or express a polynucleotide or nucleic acid of the invention, the resulting replicated nucleic acid, RNA, An expressed protein or polypeptide is included within the scope of the present invention as a product of a host cell or host organism. The product is recovered by any suitable means known in the art.

  The nucleic acids of the present invention may be produced in various ways known in the art to generate targeted changes in the sequence of the encoded protein, the properties of the encoded protein including the fluorescent properties of the encoded protein, etc. Can be mutated. A DNA sequence or protein product so mutated is usually considered substantially similar to the sequences provided herein, e.g., at least one nucleotide or amino acid is considered to be different. At least 2, but up to about 10 nucleotides or amino acids may be different. The sequence changes can be substitutions, insertions, deletions, or combinations thereof. Deletions include more than domain or exon deletions, such as deletions ranging from 10aa, 20aa, 50aa, 75aa, 100aa, 150aa, or more It can further include large changes. Techniques for in vitro mutagenesis of cloned genes are known. Examples of protocols for site-directed mutagenesis are Gustin et al. (1993), Biotechniques 14:22; Barany (1985), Gene 37: 111-23; Colicelli et al. (1985), Mol. Gen. Genet. 199. : 537-9; and Prentki et al. (1984), Gene 29: 303-13. Methods for site-directed mutagenesis are described by Sambrook et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp. 15.3-15.108; Weiner et al. (1993), Gene 126: 35-41; Sayers et al. (1992). ), Biotechniques 13: 592-6; Jones and Winistorfer (1992), Biotechniques 12: 528-30; Barton et al. (1990), Nucleic Acids Res 18: 7349-55; Marotti and Tomich (1989), Gene Anal. Tech. 6: 67-70; and Zhu (1989), Anal Biochem 177: 120-4. Nucleic acid derivatives so mutated can be used to study the structure-function relationship of a particular dye / fluorescent protein, or to alter the properties of a protein that affect its function or control.

  Humanized forms of the nucleic acids of the invention are also of interest. As used herein, the term “humanization” refers to changes made to nucleic acid sequences to optimize codons for expression of proteins in human cells (Yang et al., Nucleic Acids Research 24 (1996), 4592-4593). See also US Pat. No. 5,795,737, which describes the humanization of proteins, the disclosure of which is incorporated herein by reference.

Protein / polypeptide compositions Non-aggregating chromoproteins and / or fluorescent proteins encoded by the nucleic acids of the invention and variants thereof, and related polypeptide compositions are also provided by the invention. As used herein, the term polypeptide composition refers to both full-length proteins and parts or fragments thereof. This term includes variants of naturally-occurring proteins, and variants of naturally-occurring proteins that are homologous or substantially similar to naturally-occurring proteins, described in more detail later. Is also included.

  In many embodiments, the proteins of the invention have an absorption maximum in the range of about 300 nm to 700 nm, usually about 350 nm to 650 nm, more usually about 400 nm to 600 nm. When the protein of the present invention is a fluorescent protein, i.e., when excited by light of one wavelength and then can emit light at another wavelength, the emission spectrum of the protein of the present invention is typically While the range of about 400 nm to 800 nm, usually about 425 nm to 775 nm, and more usually about 450 nm to 750 nm, the excitation spectrum of the proteins of the invention is typically about 300 nm to 700 nm, usually about 350 nm to 650 nm. More usually in the range of about 400 nm to 600 nm. The proteins of the invention generally have a maximum extinction coefficient in the range of about 10,000 to 50,000, usually about 15,000 to 45,000. The proteins of the present invention are typically about 150-300 amino acid residues long, generally about 200-300 amino acid residues long, generally about 15 kDa-35 kDa, usually about 17.5 kDa-32.5 It has a molecular weight in the range of kDa.

  In certain embodiments, the proteins of the present invention are bright (bright means that chromoproteins and their proteins are obtained by conventional methods (eg visual screening, spectrophotometry, spectrofluorimetry, fluorescence microscopy, FACS machine, etc.). Meaning that fluorescent variants can be detected). The fluorescence intensity of a particular fluorescent protein is determined by the quantum yield multiplied by the maximum extinction coefficient. The brightness of a chromoprotein can be represented by its maximum extinction coefficient.

  In certain embodiments, the proteins of the invention are rapidly folded after expression in a host cell. Rapid folding means that the protein quickly reaches a tertiary structure that produces pigmentary or fluorescent properties. In these embodiments, the protein is generally folded for a period not exceeding about 3 days, usually not exceeding about 2 days, more usually not exceeding about 1 day.

  The specific proteins of interest are the following specific protozoan species: Anemonia majano, Clavularia species, Zoanthus species, Zoanthus species, Discosoma striata ), Discosoma sp. "Red", Anemonia sulcata, Discosoma sp. "Green", Discosoma sp. "Magenta" pigments / fluorescent proteins (and their variants) Non-aggregating mutant polypeptide or variant. Specific non-aggregating fluorescent polypeptides of interest include FP1-NA; FP3-NA; FP4-NA; FP6-NA; E5-NA; 6 / 9Q-NA; 7A-NA, etc. These are not limited.

  Also provided are homologues or proteins (or fragments thereof) that differ in sequence from the amino acid sequence of a particular non-aggregating polypeptide provided above. Homologues are DGHiggins and PMSharp, “Fast and Sensitive multiple Sequence Alignments on a Microcomputer” (1989) CABIOS, 5: 151-153 DNAstar (1998) clustered algorithm MegAlign (used Are determined using ktuple 1, gap penalty 3, window 5, and diagonals saved 5) and at least About 10%, usually at least about 20%, more usually at least about 30%, and in many embodiments at least about 35%, usually at least about 40%, more usually at least about 60% amino acids It means a protein having sequence identity. In many embodiments, the homologue of interest has a much higher sequence identity, eg, 65%, 70%, 75%, 80%, 85%, 90%, or more.

  Also provided is a protein that is substantially identical to the sequence of the specific protein provided above (substantially identical means that the protein is at least about 60% of one of the specific proteins provided above, Usually means having an amino acid sequence identity of at least about 65%, more usually at least about 70%, and in some instances the identity is much higher, eg, 75%, 80%, 85%, 90%, 95%, or more).

  In many embodiments, homologues of the present invention have structural features found in the specific sequences provided above, including β-can folding.

  Also provided are proteins that are variants of the proteins specifically described above. A variant may retain the biological properties of a wild-type (eg, naturally occurring) protein or may have biological properties that are different from those of a wild-type protein. The term “biological property” of a protein of the invention includes extinction maxima, emission maxima, max extinction coefficient, brightness (eg wild type protein or another fluorescent protein such as green fluorescent protein from A. victoria). Spectral characteristics such as compared to a reference protein); in vivo and / or in vitro stability (eg, half-life), etc., but are not limited thereto. Variants include single amino acid changes, deletion of one or more amino acids, N-terminal truncations, C-terminal truncations, insertions, and the like.

  Variants can be generated using standard techniques of molecular biology, such as random mutagenesis and targeted mutagenesis. Several variants are described herein. Given that guidance is provided in the examples, using standard techniques, one of skill in the art can readily produce a wide variety of additional variants to determine whether the biological properties have been altered. Can be tested. For example, fluorescence intensity can be measured using a spectrophotometer at various excitation wavelengths.

  Also provided are variants of the proteins specifically provided above. In general, such polypeptides include full-length proteins, as well as fragments thereof, in particular fragments corresponding to biologically active fragments and / or functional domains; other proteins of the polypeptides of the invention or their It includes the amino acid sequence encoded by the open reading frame (ORF) of the gene encoding the wild type protein of the present invention, including a fusion with a portion. Fragments of interest are typically considered to be at least about 10 aa long, usually at least about 50 aa long, may be 300 aa or longer, but are generally considered not to exceed about 1000 aa . Here, the fragment will have the same amino acid range as a protein of the invention that is at least about 10 aa long, usually at least 15 aa long, and in many embodiments at least 50 aa long. In some embodiments, a polypeptide of the invention has a length of about 25aa, about 50aa, about 75aa, about 100aa, about 125aa, about 150aa, about 200aa, about 210aa, about 220aa, about It is 230aa long, or about 240aa long and up to the entire protein. In some embodiments, the protein fragment retains all or substantially all of the biological properties of the wild-type protein.

  The proteins and polypeptides of the present invention can be expressed using any convenient protocol, for example, as described above, by expressing the recombinant gene or nucleic acid coding sequence encoding the protein of interest in a suitable host, as described above. It can be made synthetically. Any convenient protein purification technique may be utilized, and suitable protein purification methodologies are described in Guide to Protein Purification, (Deuthser ed.) (Academic Press, 1990). For example, lysates can be prepared from the original source and purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, and the like.

Antibody Compositions Antibodies that specifically bind to the non-aggregating fluorescent protein of the present invention are also provided. Suitable antibodies can be obtained by immunizing a host animal with a peptide comprising all or part of the protein of the present invention. Suitable host animals include mice, rats, sheep, goats, hamsters, rabbits and the like. The immunogen can include the complete protein, or fragments and derivatives thereof.

The first step for the preparation of polyclonal antibodies is the immunization of the host animal with the target protein, preferably considered to be present in substantially pure form, containing less than about 1% of contaminants. The immunogen can include the complete target protein, fragments or derivatives thereof. In order to increase the host animal's immune response, the target protein may be combined with an adjuvant, which includes an alum, dextran, sulfate, a polymeric anion, an oil-water emulsion such as Freund's adjuvant, Freund's complete adjuvant and the like are included. The target protein may be conjugated to a synthetic carrier protein or a synthetic antigen. A variety of hosts can be immunized to produce polyclonal antibodies. Such hosts include rabbits, guinea pigs, rodents such as mice, rats, sheep, goats and the like. The target protein is usually administered an initial dose intradermally, followed by one or more additional doses, usually at least two. After immunization, blood is collected from the host, followed by separation of serum from the blood cells. Ig present in the resulting antiserum can be further fractionated using known methods such as ammonium salt fractionation, DEAE chromatography and the like.

  Monoclonal antibodies are produced by conventional techniques. In general, the spleen and / or lymph nodes of the immunized host animal provide the origin of the plasma cells. Plasma cells are immortalized by fusion with myeloma cells to produce hybridoma cells. To confirm that the antibodies produced have the desired specificity, culture supernatants from individual hybridomas are screened using standard techniques. Animals suitable for the production of monoclonal antibodies against human proteins include mice, rats, hamsters and the like. In general, hamsters, guinea pigs, rabbits and the like can be considered as animals for producing antibodies against mouse proteins. Antibodies can be purified from hybridoma cell supernatants or ascites by conventional techniques, such as affinity chromatography using proteins coupled to insoluble supports, protein A sepharose, and the like.

  An antibody may be produced as a single chain rather than a normal multimeric structure. Single chain antibodies are described in Jost et al. (1994) J.B.C.269: 26267-73, and others. DNA sequences encoding the variable region of the heavy chain and the variable region of the light chain are linked to a spacer encoding at least about 4 amino acids of small neutral amino acids, including glycine and / or serine. The protein encoded by this fusion allows assembly of a functional variable region that retains the specificity and affinity of the original antibody.

  In certain embodiments, humanized antibodies are also of interest. Methods for humanizing antibodies are known in the art. A humanized antibody can be the product of an animal having a transgenic human immunoglobulin constant region gene (see, eg, International Patent Applications, WO 90/10077 and 90/04036). Alternatively, the antibody of interest may be designed by recombinant DNA technology to replace the CH1, CH2, CH3, hinge domain, and / or framework domain with the corresponding human sequence (WO (See 92/02190).

  The use of Ig cDNA for the construction of chimeric immunoglobulin genes is known in the art (Liu et al. (1987) P.N.A.S.84: 3439 and (1987) J. Immunol. 139: 3521). mRNA is isolated from antibody-producing hybridomas or other cells and used to generate cDNA. The cDNA of interest can be amplified by polymerase chain reaction using specific primers (US Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library is created and screened to isolate the sequence of interest. The DNA sequence encoding the variable region of the antibody is then fused to a human constant region sequence. The sequence of the human constant region gene can be found in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, N.I.H. Publication 91-3242. The human C region gene is readily available from known clones. The choice of isotype is thought to be driven by the desired effector function such as complement binding or activity in antibody-dependent cytotoxicity. Preferred isotypes are IgG1, IgG3, and IgG4. Either the human light chain constant region, kappa or lambda, can be used. The chimeric humanized antibody is then expressed by conventional methods.

Antibody fragments such as Fv, F (ab ′) ₂ and Fab can be prepared by complete protein cleavage, for example by protease or chemical cleavage. Alternatively, a truncated gene is designed. For example, a chimeric gene encoding a portion of the F (ab ') ₂ fragment may contain a translation stop codon to generate a truncated molecule after the DNA sequence encoding the CH1 and hinge domains of the heavy chain. It is done.

  In order to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent linking of the V region segment to the human C region segment, A consensus sequence can be used. C region cDNA can be modified by site-directed mutagenesis to place a restriction site at a similar position in the human sequence.

  Expression vectors include plasmids, retroviruses, YACs, EBV-derived episomes, and the like. Convenient vectors are those that encode a functionally complete human CH or CL immunoglobulin sequence with appropriate restriction sites designed to allow any VH or VL sequence to be easily inserted and expressed. . In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site located in front of the human C region and is present in the human CH exon. It also occurs in the splice region. Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding region. The resulting chimeric antibodies are retroviral LTRs such as the SV-40 early promoter (Okayama et al. (1983) Mol. Cell. Bio. 3: 280), Rous sarcoma virus LTR (Gorman et al. (1982) PNAS 79: 6777), And Moloney murine leukemia virus LTR (Grosschedl et al. (1985) Cell 41: 885); can be connected to any strong promoter, including the native Ig promoter and the like.

Transgenic The nucleic acids of the invention can be used to generate site-specific genetic modifications in transgenic non-human plants or animals, or cell lines. The transgenic cells of the invention contain one or more nucleic acids according to the invention present as transgenes (this definition includes parental cells transformed to contain the transgene and their progeny) . In many embodiments, a transgenic cell is a cell that does not normally carry or contain a nucleic acid according to the present invention. In embodiments where the transgenic cell naturally contains a nucleic acid of the invention, the nucleic acid will be present in a non-natural location within the cell, i.e., at a non-natural location within the cell's genomic material. Will be incorporated. Transgenic animals with altered endogenous loci can be generated via homologous recombination. Alternatively, the nucleic acid construct is randomly integrated into the genome. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs and the like.

  Transgenic organisms of the invention include cells and multicellular organisms that are endogenous knockouts, such as plants and animals, in which expression of the endogenous gene is at least reduced if not eliminated. In the transgenic organism of interest, the protein or variant thereof is expressed in a cell or tissue where it is not normally expressed and / or expressed at a level not normally present in such a cell or tissue, Also included are cells and multicellular organisms such as plants and animals.

  A DNA construct for homologous recombination has the desired (one or more) genetic modifications and is believed to contain at least a portion of the gene of the invention, including a region homologous to the target locus. It is done. A DNA construct for random integration need not contain a homologous region to mediate recombination. Conveniently includes markers for positive selection and negative selection. Methods for producing cells with altered target genes via homologous recombination are known in the art. See Keown et al. (1990), Meth. Enzymol. 185: 527-537 for various techniques for transfecting mammalian cells.

For embryonic stem (ES) cells, ES cell lines may be used, or embryonic cells may be obtained fresh from a host, such as a mouse, rat, guinea pig or the like. Such cells are grown on a suitable fibroblast feeder layer or grown in the presence of leukemia inhibitory factor (LIF). If ES or embryonic cells are transformed, they can be used to create transgenic animals. After transformation, the cells are seeded on the feeder layer in an appropriate medium. Cells containing the construct can be detected by utilizing a selective medium. After sufficient time for the colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct. Those colonies that are positive can then be used for embryo manipulation and blastocyst injection. Blastocysts are obtained from 4-6 week old superovulated females. ES cells are trypsinized and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocyst is returned to each uterine horn of the pseudopregnant female. The females are then allowed to reach maturity and the resulting offspring are screened for the construct. By providing blastocysts of different expression types and genetically modified cells, chimeric progeny can be easily detected.

  Chimeric animals are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous offspring. If the genetic modification causes lethality at some point during development, the tissue or organ can be maintained as an allogeneic or allogenic graft or transplant, or in in vitro culture. The transgenic animal can be any non-human mammal such as a laboratory animal, domestic animal. Transgenic animals can be used in functional studies, drug screening, and the like. Representative examples of the use of transgenic animals include:

  Transgenic plants can be created in a similar manner. Methods for preparing transgenic plant cells and transgenic plants are described in US Pat. Nos. 5,767,367; 5,750,870; 5,739,409; 5,689,049; 5,689,045; 5,674,731; No. 5,629,470; No. 5,595,896; No. 5,576,198; No. 5,538,879; No. 5,484,956, the disclosures of which are incorporated herein by reference. Methods for producing transgenic plants are also reviewed in Plant Biochemistry and Molecular Biology (Edited by Lea and Leegood, John Wiley & Sons) (1993) pp.275-295. Briefly, appropriate plant cells or tissues are harvested depending on the nature of the plant species. Thus, in certain instances, protoplasts will be isolated, and such protoplasts can be isolated from a variety of different plant tissues, such as leaves, hypocotyls, roots, and the like. For protoplast isolation, harvested cells are incubated in the presence of cellulase to remove cell walls (where the exact incubation conditions depend on the type of plant and / or tissue from which the cells were derived). Fluctuate). The resulting protoplasts are then separated from the resulting cell debris by sieving and centrifugation. As an alternative to using protoplasts, embryogenic explants containing somatic cells may be used for the preparation of transgenic hosts. Following cell or tissue harvest, the exogenous DNA of interest is introduced into the plant cell, and a variety of different techniques are available for such introduction. Incubating the protoplast with naked DNA, eg, a plasmid, containing the exogenous coding sequence of interest in the presence of a multivalent cation, eg, PEG or PLO, by obtaining an isolated protoplast; and the exogenous of interest Opportunities arise through DNA-mediated gene transfer protocols, including electroporation of protoplasts in the presence of naked DNA containing sex sequences. Protoplasts that have successfully incorporated exogenous DNA are then selected, grown into callus, and finally grown into transgenic plants through contact with appropriate amounts and ratios of stimulatory factors such as auxin and cytokinin . When using embryogenic explants, a convenient way to introduce exogenous DNA into target somatic cells is through the use of particle acceleration protocols or “gene gun” protocols. The resulting explants are then grown into chimeric plants and crossed to obtain transgenic offspring. As an alternative to the naked DNA approach described above, another convenient method for generating transgenic plants is Agrobacterium-mediated transformation. For Agrobacterium-mediated transformation, a co-integrative or binary vector containing exogenous DNA is prepared and then transferred to an appropriate Agrobacterium strain, such as A. tumefaciens. And introduce. The resulting bacteria are then incubated with prepared protoplasts or tissue explants, such as leaf discs, to produce callus. The callus is then grown under selective conditions, selected and subjected to a growth medium to induce root and shoot growth, ultimately producing a transgenic plant.

Utility The non-aggregating chromoproteins and fluorescent variants thereof of the present invention are beneficial in a variety of different applications, but application is necessarily dependent on whether the protein is a chromoprotein or a fluorescent protein Different. The typical uses of each of these protein types are described below, but the uses described below are merely representative and do not limit the use of the protein of the present invention to the following. .

Chromoprotein The chromoprotein of the present invention is beneficial in a variety of different applications. One application of interest is the use of the protein of the invention as a colorant that can impart color or pigment to the particular composition in question. Of particular interest in certain embodiments are non-toxic chromoproteins. The chromoproteins of the invention can be incorporated into a variety of different compositions of interest (typical compositions of interest include food compositions, pharmaceuticals, cosmetics, organisms such as animals and plants, etc.). When used as a colorant or pigment, an amount of chromoprotein sufficient to impart the desired color or pigment is incorporated into the composition in question. The chromoprotein may be incorporated into the composition in question using any convenient protocol, and the particular protocol utilized is at least in part, the nature of the composition in question to be colored. Inevitably depends on. Protocols that can be utilized include, but are not limited to, mixing, diffusion, friction, spraying, pouring, and tattooing.

  A chromoprotein may also be useful as a label in an analyte detection assay, such as an assay for a biological analyte of interest. For example, the chromoprotein is incorporated into an adduct comprising an analyte-specific antibody or binding fragment thereof and then described in US Pat. No. 4,302,536, the disclosure of which is incorporated herein by reference. As such, it can be utilized in immunoassays for analytes of interest in complex samples. Instead of antibodies or binding fragments thereof, the chromoproteins of the invention or chromogenic fragments thereof may be ligands that specifically bind to the analyte of interest, or others, as will be readily apparent to those skilled in the art. It may be conjugated with a moiety, growth factor, hormone or the like.

  In still other embodiments, the chromoproteins of the invention can be used as selectable markers in recombinant DNA applications such as the generation of transgenic cells and organisms as described above. Thus, it is possible to design specific transgenic production protocols to utilize the expression of the chromoproteins of the invention as a selectable marker for either successful or unsuccessful protocols. Thus, the appearance of the color of the chromoprotein of the present invention in the expression form of a transgenic organism produced by a particular process is often incorporated in a manner in which the particular organism provides transgene expression in the organism. Can be used to indicate successful possession of the transgene of interest. When used as a selectable marker, nucleic acids encoding chromoproteins of the invention can be utilized in the transgenic production process described in more detail above. Particular transgenic organisms of interest in which the proteins of the invention may be utilized as selectable markers include transgenic plants, transgenic animals, transgenic bacteria, transgenic fungi and the like.

In still other embodiments, the chromoproteins (and fluorescent proteins) of the present invention are useful as sunscreens, selective filters, etc., in a manner similar to the protein uses described in WO 00/46233. is there.

Fluorescent Proteins The fluorescent proteins of the present invention (and other components of the present invention described above) are useful in a variety of different applications, including but not limited to the following. The first application of interest is the use of the proteins of the invention in fluorescence resonance energy transfer (FRET) applications. In these applications, the protein of the present invention is a second fluorescent protein or dye, such as a fluorescent protein such as that described in Matz et al., Nature Biotechnology (October 1999) 17: 969-973, such as US Patents. No. 6,066,476; No. 6,020,192; No. 5,985,577; No. 5,976,796; No. 5,968,750; No. 5,968,738; No. 5,958,713; No. 5,919,445; No. 5,874,304 (the disclosures of which are incorporated herein by reference) Aequoria victoria-derived green fluorescent protein or fluorescent variants thereof, as described, other fluorescent dyes such as coumarin and its derivatives such as 7-amino-4-methylcoumarin, aminocoumarin, body pea ( Bodipy dyes such as Bodipy FL, cascade blue, fluorescein and its derivatives, such as fluorescein Sothiocyanate, and Oregon green, rhodamine dyes such as Texas Red, tetramethylrhodamine, eosin, and erythrosine, cyanine dyes such as Cy3 and Cy5, macrocyclic chelates of lanthanide ions such as quantum dyes Chemiluminescent dyes, such as US Pat. Nos. 5,843,746; 5,700,673; 5,674,713; 5,618,722; 5,418,722; 5,418,155; 5,330,906; 5,229,285; 5,221,623; 5,182,202 (the disclosures of which are hereby incorporated by reference) In combination with luciferases including those described in (incorporated herein by reference) to function as donors and / or acceptors. Specific examples in which FRET assays utilizing the fluorescent proteins of the present invention may be used include protein-protein interactions, such as mammalian-to-hybrid systems, transcription factors, as biosensors for many different events. Detection of merization, multimerization of membrane proteins, complex formation of multiple proteins, etc. (in this case, the peptide or protein is covalently linked and linked to the FRET fluorescent combination comprising the fluorescent protein of the invention) Peptides or proteins that are protease-specific substrates, such as for caspase-mediated cleavage, or signals that increase or decrease FRET, such as PKA regulatory domains (cAMP sensors), phosphorylation (eg, phosphorylation sites within the linker) Or the linker is linked to the phosphorylation / dephosphorylation domain of another protein Including, but not limited to, binding specificity or linkers that undergo conformational changes upon receipt (if the linker has a Ca ²⁺ binding domain). Representative fluorescent resonance energy transfer or FRET applications in which the proteins of the invention are useful include US Pat. Nos. 6,008,373; 5,998,146; 5,981,200; 5,945,526; 5,945,283; 5,911,952; No. 5,866,336; No. 5,863,727; No. 5,728,528; No. 5,707,804; No. 5,688,648; No. 5,439,797 (the disclosures of which are incorporated herein by reference) There are no restrictions.

  Another application where the fluorescent proteins of the present invention are beneficial is BRET (Bioluminescence Resonance Energy Transfer). BRET is a protein-protein interaction assay based on energy transfer from a bioluminescent donor to a fluorescent acceptor protein. The BRET signal is measured by the amount of light emitted by the acceptor relative to the amount of light emitted by the donor. The ratio of these two values increases as the two proteins approach. BRET assay methods are well documented in the literature. See, for example, US Pat. Nos. 6,020,192; 5,968,750; and 5,874,304; and Xu et al. (1999) Proc. Natl. Acad. Sci. USA 96: 151-156. The BRET assay method genetically fuses a bioluminescent donor protein and a fluorescent acceptor protein independently with two different biological partners, partner A-bioluminescent donor fusion and partner B-fluorescent This can be done by creating a receptor fusion. Changes in the interaction between the partner portions of the fusion protein, eg, modulated by a ligand or test compound, can be monitored by changes in the ratio of light emitted by the bioluminescent and fluorescent portions of the fusion protein. In this application, the protein of the invention functions as a donor protein and / or an acceptor protein. The BRET assay can be used in many of the aforementioned assays as well as FRET.

The fluorescent protein of the present invention is used as a biosensor in prokaryotic cells and eukaryotic cells, for example, as a Ca ²⁺ ion indicator; as a pH indicator, as a phosphorylation indicator, other ions such as magnesium ion, sodium ion, potassium • It is also useful as an indicator for ions, chlorine ions, and halogen ions. For example, for the detection of Ca ions, it is known that proteins containing the EF-hand motif move from the cytosol to the membrane upon binding to Ca ²⁺ . These proteins contain myristoyl groups that are embedded in the molecule by hydrophobic interactions with other regions of the protein. Binding of Ca ²⁺ induces a conformational change that exposes the myristoyl group, which is then available for insertion into the lipid bilayer (referred to as the “Ca ²⁺ -myristoyl switch”). Such an EF-hand containing protein fusion with fluorescent protein (FP) is an indicator of intracellular Ca ²⁺ by monitoring translocation from the cytosol to the plasma membrane by confocal microscopy. Can be. EF-hand proteins suitable for use in this system include recoverin (1-3), calcineurin B, troponin C, vicinin, neurocalcin, calmodulin, parvalbumin, etc. Are included, but are not limited to these. For pH, a system based on hisactophilins can be used. Hisactophilin is a myristoylated histidine-rich protein known to exist in Dictyostelium. Their binding to actin and acidic lipids is clearly pH-dependent within the range of cytoplasmic pH fluctuations. In living cells, membrane binding appears to prevail over the interaction of hisctophilin and actin filaments. At pH 6.5 they migrate to the plasma membrane and nucleus. In contrast, at pH 7.5 they are evenly distributed throughout the cytoplasmic space. This change in distribution is reversible and is attributed to histidine clusters exposed in loops on the molecular surface. The reversion of subcellular distribution within the cytoplasmic pH variation is consistent with a pK of 6.5 for histidine residues. Cell distribution is independent of protein myristoylation. By fusing FP (fluorescent protein) with histophyllin, the intracellular distribution of the fusion protein can be followed by laser scanning confocal microscopy, or standard fluorescence microscopy. Quantitative fluorescence analysis performs cell line scans (laser scanning confocal microscopy) or other electronic data analysis (eg, using metamorph software (Universal Imaging)), This can be done by averaging the data collected in the cell population. Substantial pH-dependent redistribution of hisactophyrin-FP from the cytosol to the plasma membrane occurs within 1 to 2 minutes and reaches a steady state level after 5 to 10 minutes. The reverse reaction occurs on a similar time scale. As such, a histophyllin-fluorescent protein fusion protein that works in a similar manner can be used to monitor real-time cytosolic pH changes in living mammalian cells. Such methods can be used to measure pH changes as a result of high throughput applications such as growth factor receptor activation (eg, epidermal growth factor or platelet derived growth factor), chemotaxis stimuli / cell motility. In the detection of intracellular pH change as a second messenger, it is useful for monitoring intracellular pH in pH manipulation experiments and the like. For the detection of PKC activity, the reporter system takes advantage of the fact that a molecule called MARCKS (myristoylated alanine-rich C kinase substrate) is a PKC substrate. It is linked to the plasma membrane by a region of positively charged amino acids (ED-domain) that binds by myristoylation and electrostatic interactions with negatively charged plasma membranes. Upon activation of PKC, the ED-domain is phosphorylated by PKC, thereby being negatively charged, and MARCKS moves from the plasma membrane to the cytoplasm as a result of electrostatic repulsion (the “myristoyl-electrostatic switch”). be called). The fusion of the N-terminus of MARCKS, which extends from the myristoylated motif of MARCKS to the ED-domain, with the fluorescent protein of the present invention provides a detection system for PKC activity. When phosphorylated by PKC, the fusion protein moves from the plasma membrane to the cytosol. This transfer can be accomplished by standard fluorescence microscopy or confocal microscopy, eg Cellomics technology, or other High Content Screening systems (eg Universal Imaging / Becton Dickinson). Tracked using. The above reporter systems are used as indicators of PKC activity in high content screening applications, such as screening for PKC inhibitors, and in many screening scenarios involving reagents that may interfere with this signaling pathway. Has application. Methods of using fluorescent proteins as biosensors include those described in US Pat. Nos. 972,638; 5,824,485, and 5,650,135 (and references cited therein), The disclosure is incorporated herein by reference.

  The fluorescent proteins of the present invention are also useful in applications including automated screening of arrays of cells expressing fluorescent reporting groups by using microscopic imaging and electronic analysis. Screening can be used for drug discovery and in the field of functional genomics: For example, the proteins of the present invention may change in multicellular reorganization and migration, such as the formation of multicellular tubules by endothelial cells (angiogenesis). Can be used as a marker for whole cells to detect cell migration, wound healing, neurite outgrowth, etc. via the Fluoroblok Insert System (Becton Dickinson) Cellular activity, stimulated kinase and transcription factor movement (such as protein kinase C, protein kinase A, transcription factor NFkB, and NFAT); cell cycle proteins such as cyclin A, cyclin B1, and cyclin E, of cleaved substrates Fingers for signal transduction such as protease cleavage and phospholipid with subsequent movement High content markers of intracellular structure such as endoplasmic reticulum, Golgi apparatus, mitochondria, peroxisome, nucleus, nucleolus, plasma membrane, histone, endosome, lysosome, microtubule, actin・ Detection used as a tool for screening (co-localization of other fluorescent fusion proteins and their localization markers is used as an indicator of the movement of intracellular fluorescent fusion proteins or as a marker alone), etc. Used as markers fused to proteins (eg targeting sequences) and proteins). Examples of applications involving automated screening of cell arrays in which the fluorescent proteins of the invention are beneficial include US Pat. No. 5,989,835; and International Publication Nos. 0017624; 00/26408; 00/17643; No. 00/03246, the disclosures of which are incorporated herein by reference.

  The fluorescent proteins of the present invention are also useful in high throughput screening assays. The fluorescent protein of the present invention is a stable protein having a half-life of more than 24 hours. Also provided are destabilized forms of the fluorescent proteins of the invention having a shorter half-life that can be used as transcription reporters for drug discovery. For example, the protein according to the present invention is fused with a putative proteolytic signal sequence derived from a protein having a shorter half-life, such as a PEST sequence derived from a mouse ornithine decarboxylase gene, a mouse cyclin B1 disruption box, and ubiquitin. Can be. See US Pat. No. 6,130,313 (the disclosure of which is incorporated herein by reference) for a description of destabilizing proteins and vectors that can be utilized to make them. Signal transduction pathways such as AP1, NFAT, NFkB, Smad, STAT, p53, E2F, Rb, myc, CRE, ER, GR, and TRE promoters are unstable of the fluorescent proteins of the present invention for drug screening Can be detected using a chemical form.

  The protein of the present invention can be used as a second messenger detection agent, for example, by fusing the protein of the present invention with a specific domain such as a PKCγCa binding domain, PKCγDAG binding domain, SH2 domain, and SH3 domain.

  The secreted form of the protein of the invention can be prepared, for example, by fusing a secretory leader sequence with the protein of the invention and constructing the secreted form of the protein of the invention, and used in a variety of different applications. Can be done.

  The proteins of the present invention are also useful in fluorescence activated cell sorting applications. In such applications, the fluorescent protein of the present invention is used as a label to mark a cell population, and the resulting labeled cell population is then a fluorescently labeled cell known in the art. Sorted by a sorter. The FACS method is described in US Pat. Nos. 5,968,738 and 5,804,387, the disclosures of which are incorporated herein by reference.

  The proteins of the invention are also useful as in vivo markers in animals (eg, transgenic animals). For example, the expression of the protein of the invention can be driven by a tissue-specific promoter, and such methods are useful in gene therapy studies, for example, in testing the efficiency of transgene expression, among other applications. A representative application of fluorescent proteins in transgenic animals, exemplifying this class of application of the proteins of the invention, is found in WO 00/02997, the disclosure of which is incorporated herein by reference. It is.

  Further applications of the proteins of the invention include application as markers in calibration for quantitative measurements (fluorescence and protein) after injection into cells or animals; oxygen biosensor instrument for monitoring cell viability Application as a marker or reporter; application as a marker or label for animals, pets, toys, food, etc.

  The fluorescent proteins of the present invention are also useful in protease cleavage assays. For example, cleavage inactivated fluorescence assays can be developed using the proteins of the present invention. In this case, the protein of the invention is remodeled to include a protease-specific cleavage sequence without destroying the fluorescence of the protein. When the fluorescent protein is cleaved by the activated protease, the fluorescence will decrease sharply due to the destruction of the functional chromophore. Alternatively, fluorescence activated by cleavage can be developed using the proteins of the invention. In this case, the protein of the invention is designed to contain an additional spacer sequence in the vicinity / inside of the chromophore. Since the functional chromophore portion is cleaved by the spacer, this variant fluorescence activity will be significantly reduced. The spacer will be constituted by two identical protease-specific cleavage sites. Degradation by activated proteases excises the spacer and the two remaining “subunits” of the fluorescent protein can be reassembled to produce a functional fluorescent protein. Both of the above types of applications can be developed in assays for a variety of different types of proteases, such as caspases.

  The proteins of the invention can also be used in assays to determine phospholipid composition in biological membranes. Enables binding to specific phospholipids to identify / visualize patterns of phospholipid distribution within biological membranes, for example, allowing co-localization of membrane proteins in specific phospholipid rafts A fusion protein of the protein of the present invention (or any other type of covalent or non-covalent modification of the protein of the present invention) can be achieved using the protein of the present invention. For example, the PH domain of GRP1 has a high affinity for phosphatidylinositol triphosphate (PIP3), but no affinity for PIP2. Therefore, a fusion protein between the GRP1 PH domain and the protein of the present invention can be constructed to specifically label PIP3-rich areas in biological membranes.

  Yet another application of the protein of the present invention is that a switch from one fluorescent color to another (eg, from green to red) associated with the aging of a fluorescent protein may affect gene expression, eg, developmental genes. Application as a fluorescence timer used to determine activation / inactivation of expression, cell cycle dependent gene expression, circadian rhythm specific gene expression, etc.

  The non-aggregating fluorescent proteins of the present invention can also be used in cell labeling applications as described in US Patent Application No. 60 / 261,448, the disclosure of which is incorporated herein by reference. For example, non-aggregating proteins of the invention and members of specific binding partners that bind to cell surface molecules (eg, ligands that bind to cell surface receptors; antibodies that bind to cell surface proteins; reverses that bind to cell surface proteins) Fusion proteins comprising a receptor (such as a counterreceptor) can be used to identify and / or fractionate and / or isolate one or more cell populations from a mixture of cells. In some embodiments, the fusion protein multimerizes, and in some of these embodiments, cell labeling applications exploit the multimerization attributes. Accordingly, in some embodiments, the present invention provides intrinsic fluorescent multimeric non-aggregating fusion protein complexes comprising the polypeptides of the invention, and methods for use of the fusion protein complexes in cell labeling methods. .

  In one non-limiting example, a fusion protein comprising an antigen binding portion of an MHC molecule and a polypeptide of the present invention can be produced using standard molecular biology techniques. Such fusion proteins are expected to multimerize but not aggregate. The multimerization properties of the non-aggregating components of the fusion protein will function to bring together the MHC portions of the fusion protein so that a multimeric fusion protein complex is formed. Multimeric fusion protein complexes can also be loaded with peptide antigens. Such fusion proteins loaded with peptide antigens can be used to identify T lymphocytes that retain T cell receptors specific for the same peptide antigen on their surface. Because the peptide antigen and MHC peptide presentation domain of the fusion protein complex can both vary, the intrinsic fluorescent multimeric complex is generally almost infinite based on the antigen (and MHC) specificity of the antigen receptor. A variety of T lymphocytes can be used to fluorescently label.

  Accordingly, it is another aspect of the present invention to provide a method for using the proteins of the present invention to detectably label (stain) T lymphocytes based on the specificity of the antigen receptor. .

  In a first embodiment, the method comprises subjecting the T lymphocytes to be labeled with a polypeptide of the invention for a time and under conditions sufficient to allow detectable binding of the complex to T lymphocytes. Comprising contacting with an intrinsic fluorescent multimeric non-aggregating fusion protein complex comprising a peptide antigen specific for the antigen receptor of T lymphocytes and an MHC peptide presentation domain.

  In this regard, the complex's ability to give the complex as a whole binding activity to T lymphocytes sufficient to achieve detectable binding to the antigen receptor (TCR) on the lymphocyte surface. A T lymphocyte is said to be specific for a peptide antigen if the affinity of the antigen receptor (TCR) for the peptide antigen in the MHC context is sufficiently high.

  According to this definition, a single T lymphocyte having a single or multiple TCR species on its surface is said to be specific for multiple (typically closely related) peptide antigens. It is not impossible. In such cases, the TCR will typically have a greater affinity for one of the peptides than for the other. In many cases, the peptide antigen (s) for which T lymphocytes are said to be specific by this definition will also be able to stimulate cytokine expression by T lymphocytes; however, the MHC tetramer Such a functional response is not required since the availability of the multimeric complex of the present invention for is not limited to the labeling and identification of functionally responsive T lymphocytes.

  The reasonable conditions and times for such labeling can be conveniently adapted from those used in the art to stain T lymphocytes with MHC tetramers or MHC / Ig fusions. .

For example, Altman et al., Science 274: 94-96 (1996), published 200,000 cells using MHC tetramer by incubation at 4 ° C. for 1 hour at a concentration of approximately 0.5 mg / ml tetramer. NIAID Tetramer facility (http://www.niaid.nih.gov/reposit/tetramer/genguide.htm) is currently used for high temperatures To reduce the incubation time and optimize the signal-to-noise ratio, it is recommended to stain at 4 ° C, room temperature, and 37 ° C for 15-60 minutes, respectively. Greten et al., Proc. Natl. Acad. Sci. USA 95: 7568-7573 (1998) use 1 × 10 ⁶ peripheral blood mononuclear cells at 4 ° with 3 μg of MHC class I MHC / Ig chimera. It is dyed.

Thus, T lymphocytes are conveniently about 10 ⁴ , 10 ⁵ , 10 ⁶ , or even using 15 to 60 minutes incubation at temperatures between 4 ° C. and 37 ° C. At least about 0.1 μg, typically at least about 0.25 μg, more typically at least about 1 μg, 2 μg, 3 μg, 4 μg, or even at least 5 μg to label 10 ⁷ peripheral blood mononuclear cells Multimers can be used and labeled with intrinsic fluorescent multimeric complexes in the methods of the invention.

  Starting with these extensive exemplary guidelines, those who are skilled in labeling T lymphocytes using MHC tetramers, MHC / Ig fusions, and fluorophore-conjugated antibodies can easily and optimally Specific labeling conditions could be determined. Variables that affect the amount of multimeric reagent to be used and the temperature and duration of staining include those associated with T lymphocytes (specific for complex peptide antigens (and MHC) of T lymphocytes in the sample). Number, total number of cells in the sample, type of cell sample (eg whole blood, whole blood after erythrocyte lysis, Ficoll purified peripheral blood mononuclear cell (PBMC) fraction)), and stoichiometry of the labeled complex and Included are those related to multimeric complexes, including molecular weight, identity of the peptide antigen, and selection of MHC alleles contained in the complex.

  To optimize the labeling conditions, a single species of multimeric complex should be used and labeling conditions (eg, temperature, time length, complex concentration, cell number, cell concentration, cell purity) should be varied and labeled Labeling reactions using parallel aliquots of cell samples can be easily performed. Negative controls include labeling reactions that do not use multimers, labeling reactions that use multimers lacking peptides, and / or peptide antigens that may not be recognized by MHC peptide-presenting domains and / or T lymphocytes in cell samples A labeling reaction using a multimer containing can be included. The labeling efficiency for each aliquot can be readily determined by counting by flow cytometry of T lymphocytes in the sample that bind to the fluorescent complex. As is well known in the flow cytometry art, labeled cells can be washed prior to flow cytometry to remove unbound complexes from the cells and media. All of these techniques and means are conventional in the flow cytometry field and are routinely performed by technicians.

  Typically, the T lymphocytes to be labeled are present in a heterogeneous cell sample, the goal of labeling is to detect antigen-specific T lymphocytes within this population, and in many cases, It is to count.

  Accordingly, in another aspect, the present invention provides a method for detecting T lymphocytes specific for a selected antigen in a cell sample. The method involves subjecting a sample to a complex under sufficient time and conditions to allow for detectable binding of the complex to T lymphocytes in the sample specific for the selected antigen and MHC. Intrinsic fluorescent multimeric complex according to the invention (the peptide antigen of the complex is the selected antigen and the MHC presentation domain of the complex is considered to be constrained by the T lymphocytes that it is desired to detect And then detecting specific T lymphocytes in the sample by fluorescence of the complexes bound to them.

  Detection of fluorescence associated with the cells is typically accomplished with a flow cytometer such as a FACSVantage ™, FACSVantage ™ SE, or FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). Executed using. The laser selected for excitation is determined by the absorption spectrum of the multimeric complex and any additional fluorophores that are desired to be detected simultaneously in the sample. For example, if the multimeric complex has endogenous DsRed spectral characteristics (eg, a homotetramer in which all fusion protein subunits have an endogenous DsRed GFP-like chromophore), A standard argon ion laser having a 488 nm series can be used for excitation. For detection, the type of filter set and detector will be selected according to the multimeric complex desired to be detected in the sample and the emission spectrum of any additional fluorophore. For example, if the multimeric complex has an intrinsic DsRed spectral feature with an emission maximum at about 583 nm, fluorescent emission from the complex can be detected in the FL2 channel using a PE setup.

  Alternatively, the fluorescence of the complex bound to the cells can be detected using a microfluorometer such as IMAGN2000 (Becton Dickinson Immunocytometry Systems, San Jose, Calif., USA). The application of microfluorometers for characterization of blood cells, and the conditions therefor, are particularly described in Seghatchian et al., Transfus. Sci. 22 (1-2): 77-9 (2000); Glencross et al., Clin. Lab. Haematol. 21 (6): 391-5 (1999); and Read et al., J. Hematother. 6 (4): 291-301 (1997).

  Alternatively, fluorescence of the complex bound to the cells can be detected using a laser scanning cytometer (Compucyte Corp., Cambridge, MA, USA).

  The fluorescence of multimeric complexes bound to cells is described in Skinner et al., “Cutting edge: In situ tetramer staining of antigen-specific T cells in tissues”, J. Immunol. 165 (2): 613-7 (2000). Detected directly on the microscope slide, whether from a touch prep, a cytospin prep, or from a tissue sample, using essentially similar conditions to obtain.

  T lymphocyte-containing samples are typically whole blood samples collected directly into anticoagulation collection tubes (eg, EDTA-containing or heparin-containing Vacutainer ™ tubes, Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA) Can be a peripheral venous blood specimen.

  Advantageously, the T lymphocyte-containing sample is a whole blood sample that has been processed prior to detection with a red blood cell (RBC) lysing agent, particularly as described in Chang et al. US Pat. Nos. 4,902,613 and 4,654,312. (Solubilizers are well known in the art and are available from a number of suppliers (FACS ™ Lysing Solution, Becton Dickinson Immunocytometry Systems, San Jose, CA, USA; Cal-Lyse ™ Lysing Solution, Caltag Labs, Burlingame, CA, USA; No-Wash Lysing Solution, Beckman Coulter, Inc., Fullerton, CA). The sample may optionally be washed after RBC lysis and before detection.

  The sample from which T lymphocytes are desired to be detected by the method of the present invention may be a peripheral blood fraction, advantageously a mononuclear cell (PBMC) fraction. PBMCs were centrifuged by Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and cell preparation blood collection tubes (Vacutainer ™ CPT ™ Cell Preparation Tube, Becton Dickinson, Franklin Lakes , NJ, USA) and can be prepared according to well-known art-recognized techniques, including direct centrifugation.

  The sample is advantageously a sample enriched in T lymphocytes, such as cultured lymphocytes (such as clonal cell lines or multiple clonal cultures), lymphs extracted or eluted from tissues containing lymphocyte infiltrates. Spheres (eg, tumor infiltrating lymphocytes extracted from tumor biopsy material and selectively amplified in culture), lymphocytes collected from lymph or thymus, or after the first fluorescently labeled cell sorting The obtained lymphocyte may be sufficient.

  In another embodiment, the method further comprises counting antigen-specific T lymphocytes so detected. Counts conveniently include total cell count, percentage of antigen-specific T lymphocytes in assayed cells (total cells, mononuclear cells, or T lymphocytes), or antigens in T cell subsets. It can be expressed in the form of a percentage of specific lymphocytes.

  For some of these metrics, it is further necessary to quantify the total number of T lymphocytes in the whole sample, or the total number of T lymphocytes in a particular subset in the sample.

  Accordingly, in other embodiments, the methods of the invention contact a sample with at least one fluorophore-conjugated antibody (the antibody is selected from the group consisting of a pan-T antibody and a T-cell subsetting antibody). And then simultaneously detecting fluorescence from the multimeric fluorescent complex and the fluorophore-conjugated antibody.

  Antibodies usefully used in this embodiment include antibodies specific for CD3, CD4, CD8, CD45RO, CD45RA, and CD27. As will be appreciated, the antibody is typically specific for a marker as expressed by the taxonomic species from which T lymphocytes are detected (human, mouse, rat, etc.), or It will be cross-reactive with them.

  As is well known in the flow cytometry field, pan-T and / or T lymphocyte sub-setting antibodies can either trigger data acquisition, live gating, or pre-gating data. Or may be used for all.

  In many samples, it is often advantageous to obtain a large number of events in the method of the present invention because of the small number of antigen-specific T lymphocytes present in many samples. In addition, it may be possible to improve the signal-to-noise ratio for detecting antigen-specific T lymphocytes by triggering or gating on fluorescence from antibodies specific for T lymphocyte-activated antigens. .

  Thus, in another embodiment, the method comprises contacting a sample with at least one fluorophore conjugated antibody specific for a T cell activating antigen, and then from the multimeric fluorescent complex and the fluorophore conjugated antibody. And simultaneously detecting the fluorescence of. The antibody is usefully specific for an activating antigen selected from the group consisting of CD69, CD25, CD71, and MHC class II (HLA-DR for human T lymphocyte labeling). obtain.

  Advantageously, the antibodies used in the methods of the invention comprise a fluorophore, typically the emission of which is an intrinsic fluorescent multimeric T cell labeling complex, and other simultaneously used in the methods. From the fluorophore emission, it will be directly conjugated with a fluorophore that is distinguishable by flow cytometry. Fluorophores usefully include fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridinin chlorophyll protein (PerCP), allophycocyanin (APC), Texas Red, Alexa Fluor 488 (Molecular Probes, Inc. ., Eugene Or.), And tandem fluorophores PerCP-CY5.5, PE-Cy5, PE-Cy7, and APC-Cy7. The antibody may usefully be conjugated with biotin, allowing second stage detection using streptavidin labeled with a fluorophore.

The method of the invention for detecting and counting antigen-specific T lymphocytes includes flow cytometry of limiting dilution assay, ELISPOT, and intracellular cytokine expression as well as the use of MHC tetramers and MHC / Ig chimeras. It can be used for the same purposes as prior art methods, including other functional assays such as detection (Waldrop et al., J. Clin. Invest. 99 (7): 1739-50 (1997)). Thus, the method can be used to assess the response of CD4 ⁺ and CD8 ⁺ T cells, for example, against infection, against vaccines, and in autoimmunity.

  Detection of antigen-specific T lymphocytes is linked directly or indirectly to sorting depending on the equipment used, and thus, in another aspect of the invention, T lymphocytes specific for the selected antigen. A method is provided for concentrating or reducing a sample with respect to a sphere.

  In general, the method involves subjecting a sample under a time and under conditions sufficient to allow detectable binding of the complex to T lymphocytes in the sample specific for the selected antigen and MHC. The intrinsic fluorescent multimeric complex of the present invention (the peptide antigen of the complex is the selected antigen, and the MHC presentation domain of the complex is considered to be constrained by T lymphocytes that are desired to be enriched or reduced In contact with a). After binding, the labeled T lymphocytes are enriched or reduced based on the fluorescence of the complex bound to them.

  Such a method is conveniently performed using a fluorescence activated cell sorter (sorting based at least in part on fluorescence from the multimeric complex of the present invention is Reduce the sample removed and concentrate the aliquot on which the cells are placed).

  However, it is also possible to use the multimers of the present invention to enrich or reduce cells using means other than fluorescence activated cell sorting.

  For example, by further conjugation of the TCR binding complex to superparamagnetic particles, T lymphocytes that are specifically stained with multimers can be separated magnetically rather than fluorometrically. This can be done, for example, using an antibody specific for the epitope of the fusion protein (eg, if DsRed contributes to the GFP-like chromophore and / or multimerization domain, the antibody can be Clontech Labs, Palo Alto, CA A DsRed specific antibody commercially available from USA).

  As another example, by further conjugating the TCR binding complex with biotin, followed by the use of an avidin affinity substrate, biotin / avidin affinity interaction rather than fluorescence can be used specifically in multimers. Stained T lymphocytes can be isolated. Further labeling of this multimeric complex can be done indirectly using a biotin-conjugated antibody specific for the epitope of the fusion protein. Alternatively, the multimer itself may be pre-conjugated with biotin either chemically or enzymatically when designing a BirA substrate peptide into a complex (typically a fusion protein).

  Samples enriched for antigen-specific T cells according to the method of the present invention are specific interactions of antigen-specific T cells with antigen-presenting cells, cytotoxic targets, B cells, or other cellular components of the immune system. Can be used in vitro for this study. Samples enriched for antigen-specific T cells according to the methods of the invention may be used in vitro to modify such interactions. See, for example, Dal Porto et al., Proc. Natl. Acad. Sci. USA 90: 6671-6675 (1993).

  Samples enriched for antigen-specific T cells according to the methods of the invention can also be used for in vivo therapeutic interventions such as tumor immunotherapy. See, for example, Oelke et al., Clin. Cancer Res. 6 (5): 1997-2005 (2000).

  The antibodies of the invention described above are also useful in a number of applications, including differentiation of the proteins of the invention from other fluorescent proteins.

Kits for use in the practice of one or more of the above applications, typically comprising a construct comprising an element for making a protein of the invention, eg, a vector comprising a coding region of the protein of the invention Kits are also provided by the present invention. The kit components of the present invention are typically present in a suitable container and in a suitable storage medium, such as a buffer solution. Antibodies to the provided proteins can also be present in the kits of the invention. In certain embodiments, the kit comprises a plurality of different vectors, each encoding a protein of the invention (in which case the vectors are for expression in different environments and / or under different conditions, eg, constitutive expression). Such as promoterless vectors that are designed and contain strong promoters for expression in mammalian cells), custom insertion of promoters and multiple cloning sites for tailored expression, etc. Including.

  In addition to the above components, the kit of the present invention will further include instructions for performing the method of the present invention. These instructions can be present in the kit of the present invention in a variety of forms, and one or more forms may be present in the kit. One form in which these descriptions may exist is information printed on a suitable medium or substrate, such as in a kit packaging, in a package insert, etc. (eg, one or more sheets of information printed). As. Yet another means would be a computer readable medium having information recorded thereon, such as a disc, CD, or the like. Yet another possible means is a website address that can be used over the Internet to access information at a remote location. Any convenient means can be present in the kit.

  The following examples are presented for purposes of illustration and not limitation.

Experiment
I. Sequence of Parent Aggregating Wild-type Flowerworm Class Proteins and Variants thereof The following table summarizes the properties of nine specific wild-type flowerworm class proteins of the present invention.

(Table 1)

II. Mutagenesis Site-directed mutagenesis was performed by PCR with primers containing the appropriate target substitution. All mutants were cloned between the BamHI and HindIII restriction sites of the pQE30 vector (Qiagen). The recombinant protein was 6 × histidine tagged to contain the sequence “MRHHHHHHGS” instead of the first Met. After overnight expression in E. coli, the fluorescent protein was purified using TALON Metal Affinity Resin (CLONTECH). SDS-PAGE analysis revealed that the protein was at least 95% pure.

  More specifically, mutagenesis is performed by the overlap extension method (Ho, SN, Hunt, HD, Horton, RM, Pullen, JK, Pease, LR Site-directed mutagenesis by overlap extension using the polymerase chain reaction. (Gene 1989 Briefly, two overlapping fragments of the FP coding region were amplified: a “forward cloning” primer and a “reverse mutagenesis” primer were Used for amplification, “forward mutagenesis” and “reverse cloning” primers were used for 3 ′ end fragment amplification PCR was performed using Advantage® 2 polymerase mix ( Polymerase Mix) (CLONTECH) was performed in 25 μl (final volume) of 1 × manufacturer's buffer supplemented with 100 μM of each dNTP, 0.2 μM of each primer, and 1 ng of plasmid DNA. The parameters were set at 95 ° C for 10 seconds, 65 ° C for 30 seconds, 72 ° C for 30 seconds, and 20 cycles were completed using a PTC-200 MJ Research thermocycler. To remove, the 5 'and 3' fragments were excised from a 2% low melting point agarose gel in 1x TAE buffer, and the gel pieces were subjected to 3 freeze-thaw cycles to drain the DNA solution. The 5 ′ and 3 ′ fragments were combined to obtain full-length cDNA as follows: equal amounts of 5 ′ fragment solution, 3 ′ fragment solution, and Advantage® 2 polymerase mix, buffer , And 3 × PCR mix containing dNTPs were mixed together and subjected to 2-3 cycles of 95 ° C. 20 seconds, 65 ° C. 30 minutes, 72 ° C. 30 seconds. 1 μl of diluted sample, using forward cloning primer and reverse claw As a template for PCR with the amplification primers (for amplification of the 5 'and 3' fragments described above), resulting in a full-length coding region with one or more target substitutions A ready-for-cloning fragment was produced.

  Mutant cDNA was digested with BamHI and HindIII (cloning primers contain sites for these endonucleases) and then cloned into pQE30 (Qiagen) digested with BamHI and HindIII. The recombinant protein contained a 6 × His tag at the N-terminus.

  Selected E. coli clones were grown at 37 ° C. in 50 ml until the optical density (OD) was 0.6. At that time, recombinant FP expression was induced with 0.2 mM IPTG. The culture was then incubated overnight. The next day, cells were collected by centrifugation, resuspended in buffer (20 mM Tris-HCl, pH 8.0; 100 mM NaCl) and disrupted by sonication. The fluorescent protein was purified from the soluble fraction using Talon Metal Affinity Resin (CLONTECH). The protein was at least 95% pure by SDS-PAGE.

III. Manufactured Variants Table 2 provides details on representative non-aggregating variants produced using the above protocol.

(Table 2)

  FIG. 14 provides an alignment of certain non-aggregating mutants as described above.

IV. Characterization of representative non-aggregating mutants
A. Material and Method Theory pI
Theoretical pI is created by appending "http: //" followed by ".html" to "expasy.pku.edu.cn/tools/protoparam", Appel RD, Bairoch A., Hochstrasser DF A new Generation of information retrieval tools for biologists: calculated using the ProtParam program available on the website described in the example of the ExPASy WWW server. Trends Biochem. Sci. 1994, 19: 258-260.

Methods for assessing protein aggregation
1． “Pseudo-native” protein electrophoresis. For a rapid assessment of the aggregation properties of the mutated protein, we used a simple method termed “pseudo-native” protein electrophoresis. This method is based on the application of non-boiled protein samples to conventional sodium dodecyl sulfate polyacrylamide gels (SDS-PAG). Under these conditions, FP remains fluorescent. Furthermore, these conditions maintain the supramolecular structure of the applied protein. High molecular weight aggregated protein remains on top of the gel, and tetrameric protein migrates as a> 100 kD band.

2． Light scattering. This method is based on light scattering in solution by aggregated protein particles. The larger the size and amount of such particles, the greater the light scattering. Since light scattering depends on the wavelength of light (scattering is much more pronounced for short waves), aggregation results in a general slope of the absorption spectrum. In general, assessing aggregation by the ratio of absorbance at a shorter wavelength (minimum absorbance of unaggregated protein) to absorbance at a longer wavelength (maximum absorbance of unaggregated protein) preferable. For example, in the case of E57, aggregation can be evaluated by measuring the ratio of absorbance such as absorbance (400 nm) / absorption (566 nm). In non-aggregated protein samples, this ratio should be close to zero.

  Mutants were tested at a concentration of 1 mg / ml under the same buffer conditions that did not prevent aggregation.

3． Luminance in mammalian cell lines. Certain mutants were transiently transfected into the mammalian cell line Phoenix using the C1 vector (CLONTECH). Aggregation was assessed by visual inspection of the number of cells expressing FP using a fluorescence microscope.

Four. This process is ambiguous because it is difficult to measure the exact kinetics of in vivo aggregation in living cells. Presumably aggregation is dependent on FP concentration, as brighter cells generally show more pronounced FP aggregates. Nevertheless, as soon as the signal becomes visible, aggregates can be observed even with low-fluorescence cells. This indicates that the threshold of FP concentration sufficient for aggregation is very low.

  Aggregation of purified FP is further observed in vitro. For example, almost all flower pods FP partially precipitate from solution (PBS) without loss of color or fluorescence. To visualize the aggregation of purified FP, we used “pseudo-native” protein electrophoresis based on discontinuous SDS-PAGE of unheated protein samples (Baird, GS, Zacharias, DA and Tsien , RY (2000) Proc. Natl. Acad. Sci. USA 97, 11984-11989). Under these conditions, FP retains not only fluorescence properties but also supramolecular structure: high molecular weight aggregated proteins remain on top of the gel and oligomers migrate as high molecular weight bands.

B. Results Red fluorescent drFP583 was the first protein subjected to mutagenesis. An improved variant of DsRed (a commercially available form of drFP583 with modified codon usage optimized for expression in mammalian cells), named E57, was used as the parent gene ( Table 2). A variant of E57 was produced containing the substitutions R2A, K5E, K9T (different combinations). After expression and purification in E. coli, these proteins were analyzed by pseudo-native PAGE (see above). All mutants showed a low level of aggregation compared to the parent E57, and the R2A substitution appeared to have the strongest effect on this result (not shown). Contains all three of these substitutions (R2A, K5E, and K9T), showing no aggregation (Figure 15) and very similar to E57 with respect to excitation-radiation maximum, fluorescence intensity, and maturation rate A variant called E57-NA was selected as the optimal protein.

  E57-NA showed an excellent fluorescence image in mammalian cells (FIG. 16). In most cells, nuclei and nucleoli were clearly visible, and cell boundaries and processes were clear. In contrast, the fluorescence of E57 expressing cells was ambiguous and there was no visible intracellular structure. The boundaries of the fluorescent signal often did not coincide with the cell boundaries and processes, so the cells appeared round. Thus, both in vivo and in vitro studies confirmed that the E57-NA protein is hypoaggregating. Importantly, preliminary studies of E57-NA in other laboratories have shown that cell lines (B. Angres, Clontech, Palo-Alto, CA, USA, personal communication), Xenopus embryos (A. Zaraisky) IBCH, Moscow, Russia, personal communications) and plants (A.Touraev, IMG, Vienna Biocenter, Vienna, Austria, personal communications) and this protein compared to both DsRed and E57 during expression Greatly reduced toxicity. Currently, E57-NA is commercially available from Clontech as DsRed2.

  Recently, a variant of DsRed of interest, named “Timer” to change color over time, has been described. In order to produce a non-aggregating form of this protein, we utilized the three substitutions described above. A novel mutant Timer-NA was produced (Table 2) that possessed virtually the same maturation characteristics as the parent Timer but did not form aggregates on pseudo-native PAGE (Figure 15). In mammalian cells, the difference between Timer and Timer-NA was similar to the difference between E57 and E57-NA (Figure 16).

  The target FP was ds / drFP616 (6 / 9Q), which shows fluorescence shifted to red with a peak at 616 nm. This protein was produced by random mutagenesis after shuffling of two red FPs (dsFP593 and drFP583). However, ds / drFP616 showed very high aggregation. Similar to E57 and Timer (Figure 14, Table 2), mutations in the two Lys residues at positions 5 and 9 in the N-terminal region of ds / drFP616 are significant in the amount of aggregated protein on pseudo-native PAGE. Although resulting in a decrease, remaining aggregation was detected in the Lys mutant ds / drFP616-K5E / K9T (not shown). After screening of protein-producing E. coli clones, those that showed a complete lack of aggregation on pseudo-native PAGE were selected (FIG. 15). Sequence analysis revealed that this clone contained two additional mutations (Ser-2 and Cys-3 accidentally deleted during the cloning procedure) in the N-terminal region of the protein. . When expressed in eukaryotic cells, the variant named ds / drFP616-NA (6 / 9QNA) showed a significant improvement in fluorescence image, similar to E57-NA and Timer-NA. In contrast to the bright but not fully structured blot-like image of the parent ds / drFP616, ds / drFP616-NA was more uniformly distributed in the nucleus and cytoplasm (FIG. 16).

  Subsequently, based on the improved mutants previously produced by random mutagenesis (Table 2), the mutagenesis strategy described above was performed using four different FPs: green zFP506 protein, yellow zFP538 protein, red Of asFP595 protein, and greenish blue (cyan) amFP486 protein. When expressed in E. coli, the mutant protein showed greater brightness and faster and more complete protein folding compared to the corresponding wild-type protein (unpublished data). However, the introduced substitutions did not affect the aggregation properties of these FPs. In an attempt to reduce the tendency to aggregate, all lysines near the N-terminus of the protein were mutated (Figure 14, Table 2). In vitro analysis of the resulting secondary protein variants did not confirm aggregation (FIG. 15). In addition, four non-aggregating mutants (zFP506-N66M-NA, zFP538-M129V-NA, amFP486-K68M-NA, and asFP595-M35-5-NA) are E57-NA, Timer-NA, and ds. It showed a clear improvement in fluorescence image in mammalian cells similar to / drFP616-NA (Figure 16, images for amFP486 and asFP595 not shown).

C. CONCLUSION In summary, it can be concluded that the basic residues near the N-terminus of the class FP are playing a prominent role in the formation of protein aggregates. Many examples of significant effects of single amino acid substitutions on protein aggregation have been demonstrated. Similarly, substitution of 1-3 residues within FP resulted in a significant increase in protein solubility.

V. Additional characterization

^aこの方法は、非煮沸タンパク質試料のSDS-PAGEへの適用に基づく。これらの条件では、FPは蛍光性であり、電気泳動の条件は超分子構造を保存する。
^b比率：（吸光（400nm）−吸光（650nm）/（吸光（566nm）−吸光（650nm）） Table 3 Characteristics of E57-based mutants with altered pI

^a This method is based on the application of non-boiled protein samples to SDS-PAGE. Under these conditions, FP is fluorescent and electrophoresis conditions preserve the supramolecular structure.
^b Ratio: (Absorption (400nm)-Absorption (650nm) / (Absorption (566nm)-Absorption (650nm)))

  From the above considerations and results, it is clear that the present invention provides important novel mutant fluorescent proteins and nucleic acids encoding them (when the mutants of the present invention are compared to a reference chromogenic / fluorescent protein) The improved properties, i.e. non-aggregating, and the proteins and nucleic acids of the invention are beneficial in a variety of different applications). As such, the present invention is a significant contribution to the art.

  All publications and patent applications cited in this specification are herein incorporated by reference as if each particular publication or patent application was specifically and individually indicated to be incorporated by reference. Incorporated. The citation of any publication is for disclosure prior to the filing date and should not be construed as an admission that the invention is entitled to antedate such publication due to prior invention.

  The present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, but certain types of techniques may be considered without departing from the spirit or scope of the appended claims in view of the teachings of the invention. It will be readily apparent to those skilled in the art that changes and modifications can be made to the present invention.

The nucleotide and amino acid sequences of wild type amFP486 (NFP-1) are provided (SEQ ID NOs: 01 and 02). The nucleotide and amino acid sequences (SEQ ID NOs: 03 and 04) of wild type zFP506 (NFP-3) are provided. The nucleotide and amino acid sequences (SEQ ID NOs: 05 and 06) of wild type zFP538 (NFP-4) are provided. The nucleotide and amino acid sequences of wild type drFP583 (NFP-6) (SEQ ID NOs: 07 and 08), and alternative nucleotide and amino acid sequences thereof are provided. The nucleotide and amino acid sequences (SEQ ID NOs: 09 and 10) of wild type asFP600 (NFP-7) are provided. The nucleotide sequence and amino acid sequence (SEQ ID NO: 11 and 12) of the 6 / 9Q hybrid protein are provided. The nucleotide sequence (SEQ ID NO: 13) of mutant E57-NA (DsRED2) is provided. The nucleotide sequence (SEQ ID NO: 14) of mutant E5-NA (Timer NA) is provided. The nucleotide and amino acid sequences of FP3-NA (SEQ ID NOs: 15 and 16) are provided. The nucleotide and amino acid sequences of NFP4-NA (SEQ ID NOs: 17 and 18) are provided. The nucleotide sequence and amino acid sequence of mut32-NA (SEQ ID NOs: 19 and 20) are provided. The nucleotide sequence and amino acid sequence (SEQ ID NO: 21 and 22) of mutant FP7-NA are provided. The nucleotide sequence and amino acid sequence (SEQ ID NO: 23 and 24) of the mutant FP7-NA dimer are provided. Multiple alignment of the N-terminal region of fluorescent proteins. The arrow above the sequence represents the first β-sheet in the protein based on the structure of GFP and drFP583. Amino acid residues substituted in non-aggregating mutants are shaded. Pseudo-non-denaturing gel electrophoresis of parent fluorescent proteins (odd lanes) and their non-aggregating mutants (even lanes). The photo was obtained under UV illumination. Aggregated protein is observed in the gel for concentration. Oligomeric proteins migrate in the separation gel as high molecular weight bands (expected MW for FP monomers and tetramers is about 27 and 108 kDa). Molecular weight standards are shown on the left side of the gel. Lanes: 1-DsRed mutant E57; 2-E57-NA; 3-DsRed mutant Timer; 4-Timer-NA; 5-ds / drFP616; 6-ds / drFP616-NA; 7-zFP506 mutant N66M; 8 -ZFP506-N66M-NA; 9-zFP538 mutant M129V; 10-ZFP538-M129V-NA; 11-amFP486 mutant K68M; 12-amFP486-K68M-NA; 13-asFP595 mutant M35-5; 14-M35- 5-NA; 15-EGFP. Fluorescent image of cells expressing the parent fluorescent protein (left column) and the corresponding non-aggregating fluorescent protein (right column) (see Table 2 for details). The protein name is shown on the left. EGFP expressing cells are shown for comparison as well-known non-aggregating fluorescent proteins (bottom).

Claims (20)

  A nucleic acid encoding a non-aggregating pigment variant or a fluorescent variant of an agglutinating chromoprotein or fluorescent protein of a cnidarian or a variant thereof.   2. The nucleic acid of claim 1, wherein the cnidarian chromoprotein or fluorescent protein is derived from a non-bioluminescent cnidarian species.   3. The nucleic acid according to claim 2, wherein the non-bioluminescent cnidarian species is a class of flower worms.   2. The nucleic acid of claim 1, having a sequence of residues that is substantially the same or identical to the nucleotide sequence of at least 10 residues in length of SEQ ID NOs: 14; 15; 17; 19; 21; and 23. .   2. A fragment of the selected nucleic acid according to claim 1.   A construct comprising a vector and the nucleic acid of claim 1. (A) a transcription initiation region functional in the expression host;
(B) a nucleic acid according to claim 1; and (c) an expression cassette comprising a transcription termination region functional in the expression host.   8. A cell comprising an expression cassette according to claim 7, or a progeny thereof, which is present as part of an extrachromosomal element or integrated into the genome of the host cell as a result of the introduction of the expression cassette into the host cell. A method for producing a chromoprotein and / or a fluorescent protein comprising:
9. A method comprising the steps of growing the cells of claim 8 expressing said protein; and isolating said protein substantially free of other proteins.   A protein encoded by the nucleic acid according to claim 1 or a fragment thereof.   11. An antibody that specifically binds to the protein of claim 10.   A transgenic cell or a progeny thereof comprising a transgene that is the nucleic acid of claim 1.   A transgenic organism comprising a transgene that is the nucleic acid of claim 1.   11. An improved method comprising the step of utilizing the protein of claim 10 in applications utilizing chromoproteins or fluorescent proteins.   An improved method comprising the step of using a nucleic acid according to claim 1 in an application using a nucleic acid encoding a chromoprotein or a fluorescent protein.   A kit comprising the nucleic acid according to claim 1.   A method for producing a nucleic acid according to claim 1, wherein at least one N-terminal residue codon of a sequence encoding a clinging animal aggregating chromoprotein and / or fluorescent protein is adjusted to produce the nucleic acid. A method comprising the steps of making.   18. The method of claim 17, wherein at least one residue is a basic residue.   19. The method of claim 18, wherein the adjustment is a substitution of a basic residue with a neutral residue.   19. The method of claim 18, wherein the basic residue is lys or arg.

Images