WO2011043814A2 - Fluorescent proteins and uses thereof - Google Patents

Abstract

The present invention provides novel fluorescent proteins. In particular aspects of the invention, a novel fluorescent protein of the invention has an increase in quantum yield. In other particular aspects of the invention, a novel fluorescent protein of the invention has altered spectral properties such that, for example, the protein can be utilized in laser-based applications and methodologies.

Description

FLUORESCENT PROTEINS AND USES THEREOF

Cross-Reference to Related Application

This international application claims benefit of priority under 35 U.S.C. §1 19(e) of provisional U.S. Serial No., 61/249,712 filed October 8, 2009, now abandoned, the entirety of which is hereby incorporated by reference

Statement of Federally Sponsored Research and Development

This invention was made with government support under NIH Grant No. DK077140 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to luminescent proteins, nucleic acids encoding the same, compositions, complexes, and combinations comprising the proteins, and methods using the proteins, nucleic acids, complexes, compositions and combinations. More specifically, the present invention is relates to modified cerulean proteins containing amino acid mutations resulting in tertiary structure stabilization.

Description of the Related Art

The discovery of green fluorescent protein (GFP) in the early 1960s ultimately heralded a new era in cell biology by enabling investigators to apply molecular cloning methods. Fusing and otherwise using GFP and its color-shifted genetic derivatives in connection with a wide variety of protein and enzyme targets has enabled monitoring of cellular processes in living systems using microscopy and other methodologies. When coupled to technical advances in widefield fluorescence and confocal microscopy, including ultrafast low light level digital cameras and multitracking laser control systems, GFP and its color-shifted genetic derivatives have improved service in live-cell imaging experiments. As a result of GFP color-shifted genetic derivatives, a full complement of colors for genetically-encoded fluorescent proteins has nearly been achieved. However, the majority of these proteins are less than ideal probes for live cell microscopy because of problems with, for example, overall brightness when observed with existing imaging technology. Typically, fluorescent proteins have been plagued by, for example, low quantum yields, inefficient maturation, two-component fluorescence profiles, and suboptimal excitation by existing illumination sources. These limitations are particularly relevant for cyan fluorescent proteins because they are widely used as donors in Forster resonance energy transfer (FRET) assays and energy transfer efficiency is directly related to the donor quantum yield. Further limiting their utility, cyan fluorescent proteins are optimally excited at ~430 nm. This wavelength is positioned between conventional visible and ultraviolet optical ranges and matches poorly with widely used argon ion laser excitation sources (458 nm). Thus, current cyan fluorescent proteins are not ideally suited for laser-based applications such as confocal microscopy.

Thus, the prior art is deficient in fluorescent proteins that overcome the limitations of existing fluorescent proteins. The present invention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

A structure-based design approach was used to improve the fluorescence of Cerulean fluorescent protein thereby producing fluorescent proteins with improved fluorescence or functionality. mCerutean2 (SEQ ID NO: 6), one of the fluorescent proteins of the invention, has a quantum yield of 0.78 and permits detection of Forster resonance energy transfer (FRET) with 7.5-fold less protein than mCerulean. mNeptune (SEQ ID NO: 8), another fluorescent protein of the invention, has a red-shifted absorption spectra that is optimized for laser excitation, folds over twice as quickly as mCerulean, and is 1.75-fold brighter when expressed in cells. mCerulean3 (SEQ ID NO: 10), designed by incorporation of T65S into mCerulean2 successfully improved the fluorescence properties of mCerulean2 without changing the absorption or fluorescence emission spectra. mCerulean3 has a quantum yield of 0.87, and is ~25% brighter than mCerulean2. These characteristics of mCerulean2, mNeptune and mCerulean3 were unexpected (including, for example, the magnitude of increase in fluorescence).

The instant invention is drawn to mutant cerulean protein inclusive of, but not restricted to the ones described supra. The mutant cerulean proteins have at least one mutation of a tertiary structure stabilizing amino acid. The instant invention is also drawn to nucleic acid sequences encoding said mutant cerulean proteins. In certain embodiments of the instant invention, the amino acid mutation is at least one of T65S, S72A, Y145A, A206K, S147H, D148G, K166G, I167L, R168N, H169C, and T203I.

The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described herein, which form the subject matter of the invention. It should be appreciated by those skilled in the art that any conception and specific aspect or embodiment taught or disclosed herein may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that any description, figure, example, etc. is provided for the purpose of illustration and description only and is by no means intended to define the limits the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts the amino acid sequence of mCerulean2 (SEQ ID NO: 6) and nucleic acid sequence of mCerulean2 (SEQ ID NO: 7).

Figure 2 shows the amino acid sequence of mNeptune (SEQ ID NO: 8) and nucleic acid sequence of mNeptune (SEQ ID NO: 9).

Figure 3 depicts the amino acid sequence of wild type green fluorescent protein

(SEQ ID NO: 1) and nucleic acid sequence of wild type green fluorescent protein (SEQ ID NO: 2).

Figure 4 depicts the amino acid sequence of GFP with valine at position 2. (SEQ ID

NO: 3).

Figures 5A-5C show structure-guided optimization of cerulean. Figure 5A depicts

Cerulean (2q57.pdb), wherein residues S147 and D148 (red), and 166-169 (green) were targeted for mutagenesis to stabilize protein folding in this region and increase the quantum yield of the chromophore (cyan). Figure 5B shows Residue T203 that interacts with the ground state of the Cerulean chromophore, and was targeted for mutagenesis to red-shift the Cerulean spectra. Figure 5C shows Absorption (broken lines) and emission spectra (solid lines) for Cerulean (black), mCerulean2 (blue), and mNeptune (red). Spectra were normalized to the peak intensity.

Figure 6 shows an energy minimized structural model of mCerulean2 (red) overlaid on the original structure of Cerulean.

Figures 7A-7B depict advantages of mCerulean2 and mNeptune for Quantitative

Imaging. Figure 7A shows agarose beads were labeled with 0.13 mg/ml of recombinant mVenus, and increasing concentrations of mCerulean (black), mCerulean2 (blue), and mNeptune (red). FRET was detected by measuring fluorescence anisotropies in the cyan (cyan excitation, cyan emission; rCFP) and FRET (cyan excitation, yellow emission; rFRET) channel. Decreased anisotropies in the FRET channel indicate FRET. Error bars indicate s.e.m. Data was fit to a one phase exponential association. Figure 7B shows fluorescence of HEK 293 cells expressing mCerulean (black), mCerulean2 (blue), and mNeptune (red) was quantified >24 h post-transfection under equivalent imaging conditions. Bars indicate the mean cellular intensity, and error bars indicate the s.d. (n>100 cells). Statistical significance compared to mCerulean (p<0.001) as determined by a one-tailed ANOVA and Tukey multiple comparison test is indicated by ***.

Figure 8 shows a schematic representation of making and identifying novel fluorescent proteins.

Figure 9 depicts spectral properties of mutant cerulean proteins. Absorption (dashed lines) and emission spectra (solid lines) are shown for Cerulean (black), mCerulean2 (green), and mCerulean3 (blue). Spectra were normalized to the peak absorption or emission values.

Figures 10A-10F depict photostability of mutant cerulean proteins. Figure 10A shows COS7 cells transfected with plasmids encoding Cerulean and mCerulean3 and imaged at 37°C. Fluorescence intensity is represented using a pseudocolor lookup table for clarity. Scale bars indicate 10 μητι. Figure 10B shows normalized mean fluorescence decays of mutant cerulean proteins expressed in cells (n>7) illuminated as in Figure 10A. Figure 10C shows COS7 cells expressing Cerulean and mCerulean3 were bleached to 50% of their initial fluorescence, and monitored for reversible fluorescence recovery. Data indicates the mean % recovery after 15 min. Figure 10D depicts normalized mean fluorescence of agarose beads labeled with recombinant mutant cerulean proteins, imaged at 60s intervals under low power illumination (45 mW cm-2). At 5 minutes, the beads were continuously illuminated for 60 s (red bar). n=15 for mCerulean3, n=24 for Cerulean. Figure 10E depicts beads labeled with various mutant cerulean proteins treated as in Figure 10D. The mean fraction of irreversibly bleached fluorescence and reversible photoswitching 15 min post the 60s illumination period is expressed as % pre-bleach intensity. (n>15 for all groups). Figure 10F shows HEK293 cells transfected with the indicated fusion, and observed by fluorescence microscopy. The yellow/cyan FRET ratio of individual cells (circles) is shown (n=50). Bar indicates the mean.

Figure 11 shows fluorescence imaging of mCerulean3 fusion vectors recorded in laser scanning confocal fluorescence microscopy.

Figure 12 shows dependence of measured FRET ratios on illumination time. Agarose beads were labeled with equivalent concentrations of proteins containing modified Venus green fluorescent protein (mVenus) fused to a cyan donor protein as indicated. Beads were imaged consecutively using constant illumination intensity (455 nm LED, 600-2 W cm), but a varied illumination period. Cyan and yellow fluorescence were captured simultaneously using an Optical Insights Dual-View containing standard CFP/YFP filter sets. FRET ratios were normalized to the peak FRET ratio. Points indicate the mean and error bars indicate s.e.m. (n=10). mTFP1 fluorescence was irresolvable at exposure times less than 10 ms.

Figures 13A-13B depict fluorescence lifetime microscopy of mCerulean3.

Fluorescence lifetime images of mouse pituitary GHFT1 cells expressing mCerulean (Figure 13A, left), mCerulean3 (Figure 13A, right panel), mCerulean3-H2B and mCereulan3-H2B in concert with mVenus-H2B (Figure 13B, right panel). Images were obtained using the frequency domain method. The bottom panels show polar plot analyses of the lifetime distributions for each image using the first harmonic (20 MHz). The average lifetime was determined for each region of interest (red squares) and the scale bars indicate 10 pm.

Figures 14A-14D show measurement of secretory granule pH using a mVenus:mCerulean3 FRET probe. Figure 14A shows fluorescence emission spectra of the indicated fluorescent proteins were obtained at various pH. The plot indicates the mean of three integrated spectra normalized to values obtained at pH 9.5. Figure 14B shows calibration curve for live cell imaging was generated from images of beads labeled with recombinant FRET pH sensors. Images of beads in various pH buffers were taken under the same conditions used for imaging cells. Fluorescence from cyan and yellow channels was captured in a single image using excitation conditions for cyan illumination. Error bars indicate s.e.m. (n>10 beads), and the pH vs. fluorescence ratio relationship was well approximated by regression analysis. Figure 14C shows mVenus:mCerulean3 expressed in pancreatic bTC3 cells to measure luminal secretory granule pH in both starved cells (top) and cells stimulated with GLP1 (bottom, 3 min stimulation). Ratio images were psuedocolored to reflect the pH inside secretory granules. Figure 14D shows pH of secretory granules in untreated (n=400), and GLP1 treated cells (n=400), calculated from the cyan/yellow fluorescence ratios in bTC3 cells expressing VAMP2-mVenus:mCerulean3.

DETAILED DESCRIPTION OF THE INVENTION Unless otherwise noted, technical terms are used according to conventional usage.

Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar technical references.

As used herein, "a" or "an" may mean one or more. As used herein when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more than one. As used herein "another" may mean at least a second or more. Furthermore, unless otherwise required by context, singular terms include pluralities and plural terms include the singular.

As used herein, "about" refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term "about" generally refers to a range of numerical values (e.g., +/- 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term "about" may include numerical values rounded to the nearest significant figure.

As used herein, the amino acids, which occur in the various amino acid sequences appearing herein, are identified according to their well-known, three-letter or one-letter abbreviations. The nucleotides, which occur in the various polynucleotides appearing herein, are designated with the standard single-letter designations used routinely in the art.

The term "isolated" means altered "by the hand of man" from its natural state i.e. if it occurs in nature it has been changed or removed from its original environment, or both.

Therefore, the term contemplates a polynucleotide or protein removed from its natural environment, purified or separated, or substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical reactants, or other chemicals when chemically synthesized. Preferably, an isolated polynucleotide or protein is at least 60% free, more preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.

The term "nucleic acid" refers to any polyribonucleotide or polydeoxyribonucleotide and is intended to include modified or unmodified DNA, RNA, including mRNAs, DNAs, cDNAs, and genomic DNAs, or a mixed polymer, and can be either single-stranded, double- stranded or triple-stranded. For example, a polynucleotide may be a single-stranded or double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, or single-, double- and triple-stranded regions, single- and double-stranded RNA, RNA that may be single-stranded, or more typically, double-stranded, or triple-stranded, or a mixture of regions comprising RNA or DNA, or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The DNAs or RNAs may contain one or more modified bases. For example, the DNAs or RNAs may have backbones modified for stability or for other reasons. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name a few examples, are polynucleotides, as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful functions known to those skilled in the art. The term "polynucleotide" embraces such chemically, enzymatically or metabolically modified forms of nucleic acids, as well the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells. The term "polynucleotide" and in particular DNA or RNA, refers only to the primary and secondary structure and it does not limit it to any particular tertiary forms. The term also embraces short polynucleotides often referred to as oligonucleotides.

The term "protein" used herein generally refers to any polypeptide or peptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. The term refers to both short chains (i.e. peptides, oligopeptides and oligomers) and to longer chains. Proteins may contain amino acids other than the 20 gene encoded amino acids. Proteins include those modified by natural processes (e.g. processing and other post-translational modifications) and by chemical modification techniques. The same type of modification may be present in the same or varying degree at several sites in a given protein and a protein may contain many modifications. Modifications may occur in the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini. Examples of modifications include acetylation; acylation; ADP-ribosylation; amidation; covalent attachment of flavin, a heme moiety, a nucleotide or nucleotide derivative, a lipid or lipid derivative, or phosphotidylinositol; cross-linking; cyclization; disulfide bond formation; demethylation, formation of covalent cross-links; glycosylation; hydroxylation; iodination; methylation; myristoylation; oxidation; proteoytic processing; phosphorylation; S-nitrosation; racemization; lipid attachment; sulfation, gamma- carboxylation of glutamic acid residues; and hydroxylation [By way of example see Proteins-Structure and Molecular Properties 2.sup.nd Ed., Creighton, Freeman and Company, New York (1993), and Wold, P., Posttranslational Protein Modifications: Perspectives and Prospects, pages 1 -12 in Posttranslational Covalent Modification Of Proteins, B. C. Johnson, Ed. Academic Press, New (1983); Seifer et al., Meth. Enzymol 182:626 (1990); and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48 (1992)]. The polypeptides may be branched or cyclic, with or without branching.

"Variant(s)" as used herein refers to a polynucleotide or protein that differs from a reference polynucleotide or protein respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleotide sequence from another polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of an encoded polypeptide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the protein encoded by the reference sequence. A typical variant of a protein differs in amino acid sequence from another reference protein. Differences are generally limited so that the sequences of the reference protein and the variant are very similar overall and, in many regions, identical. A variant may differ in amino acid sequence by one or more substitutions, additions, and deletions in any combination. A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. Mutagenesis techniques, direct synthesis, and other recombinant methods known to skilled artisans may be used to produce variants of polynucleotides and proteins.

"Vector" (or plasmid) refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof Selection and use of such vehicles are well within the skill of the artisan. An expression vector includes vectors capable of expressing DNAs that are operationally associated with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA. Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome. Preferred plasmids for expression of proteins or chimeric proteins are those that are expressed in bacteria such as those described herein.

A "promoter element" or "promoter" refers to a segment of DNA or RNA that controls transcription of the DNA or RNA to which it is operationally associated. A promoter element includes specific sequences sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter element is referred to as the promoter. In addition, the promoter element includes sequences that modulate this recognition, binding and transcription initiation activity of RNA polymerase. The sequences may be cis acting or may be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, may be constitutive or regulated.

As used here, "derivative" or "mutant" (or other similar word or words having the same meaning) used in the context of an amino acid or nucleotide sequence is meant to encompass a subsequent amino acid or nucleotide sequence that has been derived from a previous amino acid or nucleotide sequence. For example, a derivative or mutant protein of GFP may have one or more amino acid substitutions, additions, or deletions of the wt GFP amino acid sequence or sequence of GFP with an added Val (Valine) at position 2. Cerulean is an example of a derivative or mutant protein of GFP with an added Val at position 2.

"Luminescent agent acceptor" refers to a substance that is capable of accepting energy from a luminescent protein or luminescent complex of the invention. In an embodiment, the luminescent agent acceptor is a chromoprotein or photoprotein. In a preferred embodiment, the luminescent agent acceptor is a green fluorescent protein.

As used herein, "detection method" means any of several methods known in the art to detect a molecular interaction event. The phrase "detectable signal", as used herein, is essentially equivalent to "detection method." Detection methods include detecting changes in mass (e.g., plasmin resonance), changes in fluorescence (e.g., fluorescent resonance energy transfer (FRET), lanthamide resonance energy transfer (LRET), FCCS, fluorescence quenching or increasing fluorescence, fluorescence polarization, flow cytometry), enzymatic activity (e.g., depletion of substrate or formation of a product, such as a detectable dye-NBT-BCIP system of alkaline phosphatase is an example), changes in chemiluminescence or scintillation (e.g., scintillation proximity assay, luminescence resonance energy transfer, bioluminescence resonance energy transfer and the like), and ground-state complex formation, excimer formation, colorimetric substance detection, phosphorescence, electro-chemical changes, and redox potential changes.

As described herein, the term 'structural defect' relates a specific site (for example, one or more amino acids) in the 3-D structure that if mutated would produce a fluorescent protein with improved fluorescence or functionality. A structural defect as described in the method can be, for example, a greater than 5 angstrom spacing between alpha carbons in adjacent and hydrogen-bonding in the beta strands of the fluorescent protein. A structural defect can also affect hydrogen-bonding between a secondary structure element and an adjacent amino acid with alpha-helical character, or between a secondary structure element and the chromophore. A structural defect can be a packing defect, a defective hydrophobic core, the presence of structure destabilizing amino acid interaction or amino acids that promote destabilization, or loss of secondary structure characteristics within the molecule, such as an beta strand or alpha helix. A structural defect can also be, for example, a site- specific mutation that may alter the energetics of the absorptive molecular orbital in the chromophore.

"Complementary," when referring to two nucleotide sequences, refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base- pairing. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules may be "partial", in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between the single stranded molecules. Two sequences of nucleotides may be considered complementary if they are capable of hybridizing, preferably with less than 25%, more preferably with less than 15%, even more preferably with less than 5%, most preferably with no mismatches between opposed nucleotides. Preferably the two molecules will hybridize under conditions of high stringency.

Appropriate stringency conditions which promote hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 -6.3.6. Stringency of hybridization in determining percentage mismatch may be as follows: 1 ) high stringency: O.l .times.SSPE, 0.1 % SDS, 65°C. 2) medium stringency: 0.2.times.SSPE, 0.1.% SDS, 50°C. 3) low stringency: LO.times.SSPE, 0.1 % SDS, 50°C. Equivalent stringencies may be achieved using alternative buffers, salts and temperatures.

The term "substantially" identical or homologous or similar varies with the context as understood by those skilled in the relevant art and generally means at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity.

Green fluorescent protein (GFP) is a protein composed of 238 amino acids (26.9kDa), which exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower green portion of the visible spectrum. GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm. In cell and molecular biology, GFP is frequently used as a reporter of expression. In modified forms, it has been used to make biosensors and many animals have been created that express GFP as a proof-of-concept that a gene can be expressed throughout a given organism. The GFP gene can be introduced into organisms and maintained in their genome through breeding, injection with a viral vector, or cell transformation. The GFP gene has been introduced and expressed in many bacteria, yeast and other fungi, fish (such as zebrafish), plant, fly, and mammalian cells, including human.

Since its cloning, GFP has been used to develop a number of derivatives with, for example, improved stability and changes in spectral properties. One of the first genetic engineering activities carried out using the wt sequence was optimization of its codon usage for mammalian expression. The primary amino acid sequence was kept intact, with the exception of an inserted valine at position 2 (Fig. 4). However, to avoid confusion with previously published work on GFP mutants, it is common for those of ordinary skill in the art to refer to the wt residue numbers. In certain aspects of the invention, mutations are made in the wt GFP sequence or the wt GFP sequence with an added valine at position 2. For example, mCerulean2 and mNeptune comprise mutations of the wt GFP sequence with an added valine at position 2. Therefore, for example, mNeptune actually has a mutation, among other mutations, at position 204.

Fluorescent GFP mutant proteins of the present invention are described in Table 1.

The Cerulean x-ray structure was utilized for structure-guided refinement of the fluorescence quantum yield of Cerulean (Malo, G. D. et al. Biochemistry 46, 9865-9873 (2007); and Rizzo, et al., Nat Biotechnol 22, 445-449 (2004)). Quenching of fluorescent proteins can be associated with structural instability of the tertiary structure (_-barrel) that surrounds the chromophore. In support of this, denaturation of fluorescent proteins completely and reversibly quenches fluorescence (Ward, W. W. & Bokman, S. H. Biochemistry 21 , 4535-4540 (1982)). Furthermore, engineered splits in the beta-barrel tertiary structure can be used to modulate fluorescence quenching in Ca2+ biosensors (Baird, et al., Proc Natl Acad Sci USA 96, 1 1241-11246 (1999); Nagai, et al., Proc Natl Acad Sci USA 98, 3197-3202 (2001 ); and Nakai, et al., K. Nat Biotechnol 19, 137-141 (2001)). In this invention, it was determined that there was a similar split in structure between strands 7 and 8 in the Cerulean tertiary structure beta-barrel (Figure 5a), suggesting that improved folding in this area may improve the quantum yield. Therefore to develop novel fluorescent proteins, the inventors concentrated on a mutagenesis strategy at this region of the protein. Table 1. Examples of GFP Mutant Proteins.

In certain embodiments of the invention teaching mutations (for example, mutations leading to novel fluorescent proteins of the invention), residues were mutated by replacing the wild-type amino acid with another natural or non-natural occurring amino acid (see, e.g., US Patent No. 6,783,946). Naturally occurring amino acids include, for example, alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V).

In certain embodiments of the present invention, the substitutions are conservative substitutions. In other embodiments, the substitutions are non-conservative substitutions. Conservative and non-conservative amino acid substitutions are known to those of ordinary skill in the art, for example, substituting an acidic amino acid for another acid amino acid may be considered a conservative substitution whereas substituting a basic amino acid for an acidic amino acid may be considered a non-conservative substitution; similarly, substituting a polar amino acid for another polar acid may be considered a conservative substitution whereas substituting a nonpolar amino acid for a polar amino acid may be considered a non-conservative substitution. Amino acids are generally grouped into the following categories (which can be used as a guide for determine whether or not a substitution is conservative or non-conservative): (1) polar/hydrophilic: N, Q, S, T, K, R, H,

D, E, C, and Y; (2) non-polar/hydrophobic: G, A, V, L, I, P, Y, F, W, M, and C; (3) acidic: D,

E, and C; (4) basic: K, R, and H; (5) aromatic: F, W, Y, and H; and (6) aliphatic: G, A, V, L, I, and P.

It will be appreciated by one of ordinary skill in the art that the amino acid mutations described herein can be substituted for other amino acid mutations at the specific residue or residues (or a nearby residue or residues) described herein that achieves the same effect as the novel fluorescent proteins described herein (e.g., increased fluorescence). It is well within the level of those of ordinary skill in the art to create fluorescent proteins based on the residues that the inventors have discovered and described herein to create additional fluorescent proteins aside from mCerulean2, mCerulean3 and mNeuptune. Therefore, additional fluorescent proteins containing an amino acid other than those described for mCerulean2, mCerulean3 and mNeptune are encompassed by the instant invention. For example, a novel fluorescent protein having an amino acid mutation at a position described for mCerulean2 and mNeptune in Table 2 wherein the position is the site for mutation but the amino acid may differ, is encompassed by the present invention. For example, the mutation at position 203 used to create mNeptune is not required to be isoleucine, but can be any naturally occurring or non-naturally occurring amino acid so long as the properties of the protein are altered or otherwise enhanced.

Table 2. Fluorescence Properties of Selected Fluorescent Proteins ( ^' Brightness¹^

Protein '(nm) _m ^b(nm) W) W) Φ (458 nm) pKa bleach^d (¾

Cerulean 434 474 43 J0OO 36,500 0.62 27(23) 4.7 90 s 0.67 mCerulean2 432 474 47 JD00 37,700 0.78 37(29) 4.8 97 s 0.90 mNeptune 440 485 49 P00 46,500 0.54 26(25) 5.3 72 s 1.54 mECFP 433 475 32500 - 0.40 13 4.7 ND ND

EGFP* 488 507 56000 - 0.60 34 6 247 s ND

"Peak absorbance wavelength. Peak emission wavelength, brightness calculated by the method of

Shaner et al. (Nat Methods 2, 905-909 (2005)). ^Time to bleach 50% of initial intensity with illumination

power set to 17/.W/cm². Refolding rate from denatured protein (Nagai, T. et al. Nat Biotechnol 20 , 87-90

(2002)). Values from Shaner et a I. ( at Methods 2, 905-909 (2005)).

The instant invention is drawn to mutant cerulean protein having at least one mutation of a tertiary structure stabilizing amino acid, and the nucleic acid encoding said mutant cerulean protein. In certain embodiments of the instant invention, the amino acid mutation is at least one of T65S, S72A, Y145A, A206K, S147H, D148G, K166G, I167L, R168N, H169C, and T203I or a combination thereof. In preferred embodiments, the mutant cerulean protein comprises amino acid mutations: S72A, Y145A, A206K, S147H, D148G, K166G, I167L, R168N, and H169C. In certain embodiments, the mutant cerulean protein comprises amino acid mutations S72A, Y145A, A206K, S147H, D148G, K166G, I167L, R168N, H169C and T203I. In yet other embodiments, the mutant cerulean protein comprises amino acid mutations T65S, S72A, Y145A, A206K, S147H, D148G, K166G, I167L, R168N, and H169C. In embodiments of the instant invention, the mutant cerulean protein has an amino acid sequence shown in SEQ ID NO:6, and is encoded by a nucleic acid sequence of SEQ ID NO: 7. In other embodiments, the mutant cerulean protein has an amino acid sequence shown in SEQ ID NO:8, encoded by a nucleic acid sequence of SEQ ID NO: 9. In yet other embodiments, the mutant cerulean protein has an amino acid sequence shown in SEQ ID NO: 10. Preferred embodiments of the instant invention encompass nucleic acid encoding a mutant cerulean protein described supra, said nucleic acid selected from the group consisting of: a) isolated nucleic acid which encodes the mutant cerulean protein; b) isolated nucleic acid which hybridizes to isolated DNA which encodes the mutant cerulean protein; and c) isolated nucleic acid differing from the isolated nucleic acid of a) and b) above in codon sequence due to the degeneracy of the genetic code, and which encodes the mutant cerulean protein.

Amino acid sequences of the invention encompass sequences comprising, consisting essentially of, and consisting of the amino acid sequences described supra. The invention also encompasses nucleotide sequences encoding the amino acid sequences of the present invention, the complementary nucleotide sequences, and mRNA sequences, which also include sequences comprising, consisting essentially of, and consisting of these polynucleotide sequences. In other certain aspects of the invention, the amino acid and nucleotide sequences of the invention include sequences that have a degree of sequence identity to the sequences of the invention. For example, the present invention includes amino acid sequences (and nucleotide sequences encoding the same) that share a specified degree of similarity with the sequence of mCerulean2 or mNeptune. In specific embodiments, similarity of sequences falling under the scope of the present invention include both amino acid and nucleotide sequences that have about 80% sequence identity, about 81 % sequence identity, about 82% sequence identity, about 83% sequence identity, about 84% sequence identity, about 85% sequence identity, about 86% sequence identity, about 87% sequence identity, about 88% sequence identity, about 89% sequence identity, about 90% sequence identity, about 91 % sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, and about 99% sequence identity.

In certain aspects, the invention is a chimeric protein comprising a mutant cerulean protein described supra, conjugated with a luminescent agent acceptor. A representative luminescent agent acceptor is, for example but not limited to, a yellow fluorescent protein. In certain preferred embodiments, it is a modified venus yellow fluorescent protein (mVenus).

In certain aspects of the invention, there is provided a kit suitable for use consisting of, consisting essentially of, or comprising a fluorescent protein of the invention (including, for example, mCerulean2, mCerulean 3 or mNeptune). In particular embodiments, the invention is drawn to a kit used for making and using a fluorescent protein of the invention in laboratory methods or other applicable uses (including, for example, to construct a fluorescent fusion protein comprising a fluorescent protein of the invention that can be expressed in living cells, tissues, and organisms). A kit may comprise a suitably aliquoted composition and/or additional agent compositions of the present invention. The components of the kit may be packaged in combination or alone in the same or in separate containers, depending on, for example, cross-reactivity or stability, and can also be supplied in solid, liquid, lyophilized, or other applicable form. A container means of the kits may generally include, for example, at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit can contain a second, third or other additional containers into which the additional components may be contained. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing the composition, additional agent, and any other reagent containers in close confinement for commercial sale. Such containers may include, for example, injection or blow molded plastic containers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. The compositions may also be formulated into a syringeable composition. In this case, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the contents of the container may be, for example, mixed with the other components of the kit.

However, in other embodiments the components of the kit may be provided as a dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the composition is placed, preferably, suitably allocated. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.

A fusion protein of the invention is constructed by fusing a fluorescent protein of the present invention with another 'protein of interest' (e.g. , fusing mCerulean2 to a mitochondrial protein to enable and facilitate monitoring of mitochondrial function or structure). The kinds of "protein of interest" to be fused with the fluorescent protein of the present invention is not limited. The methods for obtaining a fluorescent protein of the invention or a fusion protein of the present invention are also not limited. The means by which a fluorescent protein of the invention or a fusion protein of the present invention may be made is by chemical synthesis, semi-synthetic synthesis, or by recombinant synthesis (see, for example, U.S. Patent Nos. 7,566,774; 7,563,867; 7,504,480; 7,485,308; 7,351 ,566; 7,351 ,537; 7,256,010; 7,214,502 U.S. Application Pre-Grant Publication Nos. 20090247482; 20090247482; 20090246202; 20090226451 ; 20090226450; 20090226449; 20090226448; 20090220977; 20080248046; 20080241 155; 20080233134; 20080227110; 20070154918; 20070154912; 20070154910;and Recombinant DNA 2^nd Edition, published by Scientific American Books, 1992 (ISBN 0-7167-2282-8); all of which are incorporated by reference in their entirety). In the case of making a fusion protein, DNA encoding the desired fusion protein can be obtained by ligating those DNAs encoding a fluorescent protein of the invention and another protein of interest. A fusion protein of the present invention can be produced by introducing this DNA into a suitable expression system.

Fluorescent protein gene products can be introduced into mammalian and other cells using an appropriate vector (for example, a plasmid or virus) for transient or stable transfection. In transient, or temporary, gene transfer experiments (often referred to as transient transfection), plasmid or viral DNA introduced into the host organism does not necessarily integrate into the chromosomes, but can be expressed in the cytoplasm for a short period of time. Expression of gene fusion products, easily monitored by the observation of fluorescence emission using a filter set compatible with the fluorescent protein, usually takes place over a period of several hours after transfection and continues for hours after introduction of plasmid or viral DNA into mammalian cells. In many cases, the plasmid or viral DNA can be incorporated into the genome in a permanent state to form stably transformed cell lines. The choice of transient or stable transfection depends upon the target objectives of the investigation.

The basic plasmid vector configuration useful in fluorescent protein gene transfer experiments has several components. A plasmid for this purpose contains, for example, prokaryotic nucleotide sequences coding for a bacterial replication origin for DNA and an antibiotic resistance gene. These elements, often termed shuttle sequences, allow propagation and selection of the plasmid within a bacterial host to generate sufficient quantities of the vector for mammalian transfections. In addition, the plasmid contains, for example, one or more eukaryotic genetic elements that control the initiation of messenger RNA transcription, a mammalian polyadenylation signal, an intron (optional), and a gene for co-selection in mammalian cells. Transcription elements are necessary for the mammalian host to express the gene fusion product of interest, and the selection gene is usually an antibiotic that bestows resistance to cells containing the plasmid. These general features vary according to plasmid design, and many vectors have a wide spectrum of additional components suited for particular applications. EXAMPLE 1

Fluorescent Protein mCerulean2

Mutations were introduced in pairs into strands 7 and 8 of mCurelan, and screened for improved fluorescence. A total of 6 mutations (S147H / D148G / K166G / I167L / R168N / H169C) were introduced into the monomeric variant of Cerulean containing the A206K mutation (mCerulean), producing the novel protein mCerulean2 (Rizzo, et al. Microsc Microanal 12, 238-254 (2006); and Zacharias, et al. Science 296, 913-916 (2002)).

The properties of mCerulean2 fluorescence are shown in Table 2. mCerulean2 has a greatly improved quantum yield (0.78 vs. 0.62), and is almost 1.5-fold brighter than Cerulean. The energy minimized structural model of mCerulean2 (red) was overlaid on the original structure (Cerulean). Beta-strands 7 and 8 have moved closer together in the mCerulean2 model, and likely provide additional solvent exclusion and a higher fluorescence quantum yield (Figure 6). In addition, mCerulean2 has a measurable improvement in maturation time, and overall is 1.5 fold brighter when expressed in HEK 293 cells (Figure 7b). The magnitude in changes was an unexpected result of the invention.

The improved quantum yield of mCerulean2 made this fluorescent protein a preferential FRET donor for pairing with yellow fluorescent proteins. The Forster distance for pairing mCerulean2 with the mVenus yellow fluorescent protein was calculated to be 5.63 nm, compared with 5.40 nm for a mCerulean:mVenus pairing (Rizzo, et al. Microsc Microanal 12, 238-254 (2006)). The combination of improved efficiency and enhanced molecular brightness showed an unexpectedly greater increase in detecting intramolecular FRET (Figure 7a). To quantify the extent of this benefit, Ni chelating agarose beads were labeled with mVenus and increasing concentrations of mCerulean and mCerulean2 protein. FRET between mCerulean2 and mVenus was quantified by measuring fluorescence anisotropy for the donor cyan fluorescent protein and FRET (cyan excitation, yellow emission) channels (Rizzo, et al., Biophys J 88, L14-6 (2005)). Half-maximal FRET for the mCerulean2:mVenus pairing was detected with 7.5-fold less protein than for the mCerulean:mVenus pairing. Therefore, less protein was used for certain assays. This is critical for assessing, for example, interaction between proteins that are toxic to cells when produced in cells to overabundance. Without being bound by theory, both increased molecular brightness and increased Forster distance contribute to the improved FRET sensitivity observed for mCerulean2. EXAMPLE 2

Novel Fluorescent Protein mNeptune

Modern confocal microscopes generally use 440 nm or 458 nm lasers, which are red-shifted from the ~430 nm peak of cyan fluorescent proteins. Excitation of mCerulean2 at 458 nm is less than 80% efficient, and this wavelength is on the edge of its absorption peak. Thus, a small red shift in the absorption spectra could enhance the usefulness of cyan fluorescent proteins for laser-based applications without creating additional cross talk issues for multi-color imaging. To red shift the spectra, the inventors targeted residue T203 of Cerulean (Figure 5b). A total of 7 mutations (S147H / D148G / K166G / I167L / R168N / H169C /T203I) were introduced into the monomeric variant of Cerulean containing the A206K mutation (mCerulean), producing the novel protein mNeptune (Rizzo, et al. Microsc Microanal 12, 238-254 (2006); and Zacharias, et al. Science 296, 913-916 (2002)). Taking this approach, the inventors discovered the novel fluorescent protein mNeptune, which has a substitution (among other substitutions) at position 203 (T203I). The absorption peak of mNeptune was shifted by 8 nm (440 nm) and at 458 nm this fluorescent protein unexpectedly retained 95% of its peak absorption (Figure 5c, Table 2), making mNeptune an optimal match for laser based excitation sources. Although the molecular brightness of mNeptune was less than mCerulean2, maturation time was improved by 70%. Cells transfected with mNeptune were more brightly labeled compared to mCerulean (a 75% increase), and also mCerulean2 (Figure 7b), although FRET to mVenus was less efficient than mCerulean and mCerulean2 (Figure 7a). This was consistent with the reduced Forster distance (5.36 nm for the mNeptune:mVenus pairing) and reduced molecular brightness.

The T203I mutation reduced the quantum yield by approximately 30%. The faster maturation time for mNeptune suggested that protein folding is enhanced, rather than destabilized (Table 2). This is inconsistent with destabilized hydrogen bonding between beta-strands arising from T203I contributing to the reduced quantum yield. For other GFP derivatives, increased rotational freedom within the chromophore is associated with increased quenching (Megley, et al., J Phys Chem B 1 13, 302-308 (2009)). A similar mechanism is likely to occur in mNeptune, and is consistent with elimination of the hydrogen bonding associated with T203I mutation.

EXAMPLE 3

Method of Making Proteins with Improved Fluorescence

In the case of producing a recombinant protein, it was necessary to obtain the DNA encoding the protein. By utilizing the information of the amino acid sequences and nucleotide sequences provide herein, suitable primers were designed, and by using those to perform PCR using a DNA clone of various known fluorescent proteins, DNA fragments necessary for constructing DNA encoding fluorescent proteins of the present invention was prepared by, for example, site-directed mutagenesis. Further, DNA encoding a protein to be fused with a protein of the invention was also obtained in the same manner.

In the case of a fused fluorescent protein of the invention, it is obtained by fusing a fluorescent protein of the present invention with another protein in cells and monitoring the emitted fluorescence, temporal and spatial aspects of the fused protein in the cell were studied and analyzed. Thus, by observing a cell which was transformed or transfected with DNA encoding a fused fluorescent protein of the present invention by means of a fluorescent microscope, the localization and dynamic situation of the fused fluorescent protein in the cell were visualized and analyzed.

Mutants were introduced into (His)6mCerulean in the pQE9 vector7 by PCR using the Quikchange mutagenesis kit (Stratagene) and the degenerate primers (IDT). Mutant plasmids were transformed into XL10-Gold Ultracompetent cells (Stratagene) according the manufacturer's instructions. Transformed cells were plated on LB agar plates containing 100 mg/ml ampicillin, and incubated overnight at 37°C. For screening, 4- 5 colonies were transferred to a 25 mm filter (Nucleopore Track-Etch Membrane, Whatman). The filter was then placed colony side up on the bottom of a sterile 60 mm cell culture dish and screened for brightness (filter set ET436/20X exciter, T455LP beamsplitter, ET480-40m-2p (Chroma Technology Corp., Rockingham, VT) using an inverted Zeiss Axiovert 200M with a 1X, 0.025 NA objective lens. Dishes were heated to 50°C to improve contrast, and the brightest colonies were selected for additional screening. Colonies were grown in culture for 1 h at 37°C, and streaked on LB agar plates containing 100 mg/ml ampicillin. The following day, single colonies were transferred to a single filter for comparison, and the brightest colony was selected and cultured in 5 ml LB (100 mg/ml ampicillin) for DNA miniprep (QIAprep spin, Qiagen). Purified DNA was sequenced by the University of Maryland, Baltimore DNA sequencing facility, and also transformed into M15(pRep4) bacteria. Recombinant protein production was induced by addition of IPTG and cells were harvested and lysed as described (Rizzo, et al. Microsc Microanal 12, 238-254 (2006)). Recombinant proteins were purified using Ni loaded 1 ml HiTrap Chelating HP columns (GE Healthcare) according to the manufacturer's instructions. Protein concentration was determined using the advanced protein reagent, and SDS-PAGE as described (Id.). For bleaching measurements, HiTrap beads were labeled with fluorescent protein as described, and mounted in Prolong gold (Invitrogen). High-speed imaging of fluorescence decay under constant 455nm LED illumination was performed using a water-cooled Hamamatsu C9100-13 EM CCD was used to capture. The time to reach 50% initial intensity was reported. EXAMPLE 4

FRET Imaging

Beads were labeled as described in Youvan using 0.13 mg/mL mVenus, and recombinant cyan fluorescent proteins ranging from 0.0001 to 0.26 mg/ml (Youvan, et al., Biotechnology (1997)). FRET detection was performed on a Zeiss Axio Observer using described methodology for FRET detection by anisotropy (Piston, D. & Rizzo, M. 85, 415- 430 (2008)). Illumination was under 455 nm light emitting diode illumination filtered by a high efficiency 436/25 bandpass filter, a wire grid polarizer (VersaLight UV, Meadowlark Optics), and FT455 dichroic mirror. Observation was with a Plan-apochromat 40X/ 0.95 NA lens. Fluorescence polarization for cyan (BP480/40) and FRET fluorescence (BP525/50) was determined using the Optical Insights Dual-View to split parallel and perpendicular polarizations onto a single image captured by a Zeiss Axiocam HRm. Calculation of fluorescence anisotropies were performed as described (Piston, et al. 85, 415-430 (2008)). Statistical analysis and curve fitting were performed using Prism software (Graphpad).

For live cell imaging, mCerulean2 and mNeptune were cloned into the C1 vector using Nhel and BsrGI restriction sites as described (Rizzo, et al. Microsc Microanal 12, 238- 254 (2006)). HEK 293 cells were cultured in DMEM with 10%FBS and antibiotics. Cells were seeded on 35 mm glass bottom dishes with a No. 1.5 coverslip (Mattek) 1 day prior to transfection. Cells were transfected 1 .mu.g plasmid DNA using LipoD293 transfection reagent (SignaGen Laboratories), as per the manufacturer's instructions. Prior to imaging, media was replaced with phenol-red free Opti-MEM media (Invitrogen). Cells expressing pmCerulean-C1 , pmCerulean2-C1 , and pmNeptune-C1 were imaged >24 hours post- transfection using standard ECFP illumination and collection filters, with 455 LED illumination. Cells were maintained at 37°C during image collection.

EXAMPLE 5

Spectroscopic characterization

Absorption spectra were collected using a Shimadzu UV-mini absorbance spectrometer, and emission spectra were collected on PTI QM-3 fluorometer. Molar extinction coefficients and quantum yields were calculated as described using Cerulean as a reference standard for calibration (Rizzo, et al., Microsc Microanal 12, 238-254 (2006)).

Forster distances were calculated as described in Rizzo, and the pKa for fluorescence was measured and calculated also as described Rizzo (Rizzo, et al., Microsc Microanal 12, 238-

254 (2006); and Rizzo, et al., Nat Biotechnol 22, 445-449 (2004)). Renaturation assays were performed by the method of Nagai et al. (Nagai, T. et al. Nat Biotechnol 20, 87-90

(2002)). EXAMPLE 6

mCerulean3

An attempt to further optimize chromophore planarity was made by introducing the T65S mutation to reduce the bulkiness of the imidazolidinone ring. mCerulean3 (SEQ ID NO: 10), created by incorporation of T65S into mCerulean2 successfully improved the fluorescence properties of mCerulean2 without changing the absorption or fluorescence emission spectra (Fig. 9). mCerulean3 has a quantum yield of 0.87, and is ~25% brighter than mCerulean2 (Table 3).

mCerulean3 is exceptionally photostable compared to previously described CFPs. The bleaching t₀₅ for mCerulean3 is ~40-fold greater than mCerulean2 (Table 3). mCerulean3 also exhibited very little reversible photoswitching in vitro (1 %±0.4, n=15) (Fig.

10) and in COS-7 cells (2.5%±6.2, n=20). In addition, irreversible bleaching was <5% over the course of 200 images (Fig. 10A, Fig. 10B).

To test the suitability of mCerulean3 as a fusion protein, it was fused to a variety of different localization partners, including actin, myosin, and organelle-localized domains (Fig.

1 1) . Bright, successfully localized fusions were accomplished using both the N-terminus and C-terminus of mCerulean3, including those that require exceptional monomeric character, such as a-tubulin, intermediate filaments, connexin 43, histone H2B, and β-actin.

The improved quantum yield and photostability also makes mCerulean3 a preferential donor for FRET reactions with a yellow fluorescent protein (YFP). Consistent with the high quantum yield of mCerulean3, the Forster distance (R₀) for pairing mCerulean3 with the mVenus YFP was calculated to be 5.71 nm. This is 10% greater than the R₀ calculated for mCerulean and mVenus FRET (5.19 nm) and approximates that of FRET between mTurquoise and mVenus (/¾=5.70 nm).

More efficient coupling to mVenus (~40%) was observed in HeLa cells expressing a mCerulean3:mVenus fusion protein (FRET efficiency=0.33±0.05, n=15), compared to mCerulean:mVenus (0.24±0.04, n=15, P<0.0001 , ANOVA comparison with mTurquoise, mCerulean3, and mCerulean containing proteins) and mTFP1 :mVenus (0.25+0.03, n=15, P<0.0001 ). Consistent with the calculated Forster distances, FRET efficiency for between mTurquose and mVenus was not significantly different than with FRET between mCerulean3 and Venus (P>0.05), for either the mTurquoise:mVenus fusion (0.30±0.03, n=15), or the reversed mVenus: mTurquoise fusion (0.47+0.04, n=25, compared to 0.47+0.05, n=25 for mVenus:mCerulean3, P>0.05 for ANOVA with all the yellowxyan fusions).

Ratiometric imaging of mCerulean3:mVenus in HEK293 cells provided

CFP/YFP ratios with greatly reduced variance (Fig. 10). The s.d. of FRET ratios obtained for mVenus:mCerulean (±0.57, n=50), mVenus:mTurquoise (±0.37, n=50), and mVenus:mTFP1 (±0.41 , n=50) were at least 2.5— fold greater than the s.d. for mVenus:mCerulean3 (±0.15, n=50). Ratios for the mTFP1 were particularly high due to the ~20 nm shift in the fluorescence of mTFP1 poorly matched the conventional CFP/YFP filter sets used in these experiments.

The fluorescence instability of mCerulean, mTurquoise, and mTFP1 has a quantifiable effect on FRET ratio measurements. FRET ratios measurements of beads labeled with fused FRET partners containing these CFPs were 10-20% less at long illumination times (~1 s) compared with short ones (<1 ms) (Fig. 12). In contrast, FRET ratio measurements performed using mCerulean3 as a donor varied <2.5 % as exposure time was varied over 4 orders of magnitude. Thus, FRET ratio measurements performed using mCerulean3 as a donor protein are much more stable and accurate than those made using previous CFPs.

The long lifetime of mCerulean3 is also advantageous for FRET detection by fluorescence lifetime microscopy (Fig. 6). The fluorescence lifetimes for mCerulean (3.08±0.06 ns, r?=1 1 ) and mCerulean3 (3.94+0.04 ns, n=1 1 ) expressed in mouse pituitary GHFT1 cells were similar to in vitro measurements. Intramolecular FRET efficiencies determined from fluorescence lifetime measurements were also similar to those obtained by donor dequenching. The FRET efficiency for mCereulean3:mVenus (29%, mCerulean3 τ=2.77±0.11 ns, n=13) was 45% greater than mCerulean:mVenus (20% FRET efficiency, mCerulean τ=2.41 ±0.126 ns, A7=13). Fusion of mCerulean3 with histone H2B did not significantly affect the fluorescence lifetime of mCerulean3 (3.95±0.07 ns, n=1 1 , two-tailed t-test, p>0.05). Furthermore, we were able to detect FRET to histone H2B-mVenus in cells co-expressing H2B-mCerulean3 and H2B-mVenus. FRET efficiency (0.073) was 15% greater than observed with H2B-mCerulean as a donor (0.063), and the net reduction in mCerulean3 lifetime (-0.29 ns) was greater than twice the s.d. (±0.13 ns, n=10).

The fluorescence of mCerulean3 is very well suited for measurement of organelle pH because it is unusually stable at acidic pH compared to previous CFPs (Fig. 14A). mCerulean3 was incorporated into a FRET-based pH reporter by fusion with mVenus. mVenus has pH sensitive fluorescence (pKa=6) that steadily decreases from pH 9.5 to pH 4 (Fig. 14A). When coupled to mCerulean3, pH sensitive dequenching occurs as mVenus fluorescence decreases, producing a 3.2-fold increase in the cyan/yellow FRET ratio as the pH changes from 7 to 5 (Fig. 14B). This is a 70% increase in the dynamic range over the mVenus: mCerulean fusion (Fig. 14B), and is ~50% greater than the dynamic range of pHluorin probes. Expression of mVenus:mCerulean3 in cultured cells produced pH measurements for cytoplasm (7.2±0.06, n=10) and Golgi (6.1 +0.1 , n=10) that are consistent with previous measures.

Insulin secretory granule priming requires luminal acidification and it has been suggested that granule acidification contributes to hormonal enhancement of insulin secretion by glucagon-like peptide 1 (GLP1); however, the effect of GLP1 on the pH of individual granules is unknown. To quantify changes in the pH of secretory granules, the mVenus:mCerulean3 pH sensor was fused to the C-terminus of secretory granule protein VAMP2, and expressed it in βΤ03 insulinoma cells. The pH of individual secretory granules was explored by ratio imaging (Fig. 14C), and pH values were assigned by generation of a standard curve (Fig. 14B). The mean granule pH in glucose-starved cells was 5.9±0.4, whereas the mean granule pH in cells stimulated with GLP1 was slightly more acidic (5.8+0.4, P<0.001 , two-tailed t-test). Nonetheless, the number of matured granules with pH 5.5 was 30% greater (Fig. 14D). This is consistent with a GLP1 -stimulated increase in the primed vesicle population.

Development of mCerulean3 has achieved brighter, more reliable fluorescence by targeting residues that likely affect chromophore planarity. In addition to improving molecular brightness and photostability, the unusual acid stability of mCerulean3 is advantageous for quantifying the luminal pH of organelles. By providing a set of acid-bright probes with a physiologically useful pKa, mCerulean3 pH probes will enable mechanistic studies on organelle pH regulation. Finally, mCerulean3 is exceptionally useful as a FRET donor, and provides longer Forster distances and greatly improved measurement accuracy.

EXAMPLE 7

Making and Identifying Fluorescent Proteins with Increased Fluorescent Yield

The invention encompasses a method of making a novel fluorescent protein. The protein improved fluorescence or other spectral properties that increase the practical applicability of the fluorescent protein. For example, fluorescence can be increased so that less protein can be used for a given assay. This may be critical for assessing interaction between proteins that are toxic to cells when produced in cells to overabundance. Additionally, spectral properties can be improved to enable excitation and emission spectra that are compatible with current and future laboratory equipment and methodologies.

The general methodology for making a novel fluorescent protein is as follows (Figure 8): obtaining the 3-D structure of the fluorescent protein to be improved upon; identify structural defects in the fluorescent protein; design primers for site-directed mutagenesis in the region of the indentified structural defects; produce the mutated gene by PCR-based or other appropriate methods; transform bacterial with the gene product of (4); transfer colonies to black filter paper; heat black filter paper to 50°C and select brightest colony; recover plasmid and sequence; generate the recombinant protein; and determine the fluorescence properties of the mutant protein; repeat steps as necessary.

The 3-D structure of the fluorescent protein of the invention can be obtained by conventional means in the art (including, for example, X-ray crystallography). For example, of a fluorescent protein to be improved upon may be formed by a variety of different methods known in the art. For example, crystallizations may be performed by batch, dialysis, and vapor diffusion (sitting drop and hanging drop) methods. A detailed description of basic protein crystallization setups can be found in, for example, McRee, D. and David. P., Practical Protein Crystallography, 2nd Ed. (1999), Academic Press Inc. Further descriptions regarding performing crystallization experiments are provided in Stevens, et al. (2000) Curr. Opin. Struct. Biol.: 10(5):558-63, and U.S. Patent Nos. 7,319,016, 6,296,673, 5,419,278, and 5,096,676. Additionally, for example, crystallographic calculations are performed using the CCP4 program package (Collaborative Computational Project, N. The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50, 760- 763 (1994)). The X-ray crystallography methods that can be used in the invention are not restricted (see, for example, U.S. Patent Nos. 7,593,820; 7,585,656; 7,499,847; 7,491 ,731 ; 7,445,923; 7,235,367; and US Patent Application Publication Nos. 20090240475; 20090170128; 20090170117; 20090142822; 20090104679).

Identification of structural defects can be determined by a close study of the crystal structure. A structural defect is the determination of a specific site (for example, one or more amino acids) in the 3-D structure that if mutated would produce a fluorescent protein with improved fluorescence or functionality. A structural defect as described in the method can be, for example, a greater than 5 angstrom spacing between alpha carbons in adjacent and hydrogen-bonding in the beta strands of the fluorescent protein. A structural defect can also be, for example, a site-specific mutation that may alter the energetics of the absorptive molecular orbital in the chromophore.

Methods of carrying out the novel method of making a novel fluorescent protein described herein are generally known to those of ordinary skill in the field of molecular biology.

Although the present invention and its advantages have been described in detail in the Examples and other sections of the specification, it should be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of the invention. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, an invention described herein is intended to include within its scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which the invention pertains. All patents and publications herein are incorporated by reference to the same extent as if each individual publication was specifically and individually indicated as having been incorporated by reference in its entirety.

Claims

WHAT IS CLAIMED IS:

1. A mutant cerulean protein having at least one mutation of a tertiary structure stabilizing amino acid.

2. The mutant cerulean protein of claim 1 , wherein said amino acid mutation is at least one of: T65S, S72A, Y145A, A206K, S147H, D148G, K166G, I167L, R168N, H169C, and T203I.

3. The mutant cerulean protein of claim 2, wherein said protein contains amino acid mutations: S72A, Y145A, A206K, S147H, D148G, K166G, I167L, R168N, and H169C.

4. The mutant cerulean protein of claim 3, wherein said protein has an amino acid sequence shown in SEQ ID NO: 6.

5. The mutant cerulean protein of claim 3, having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 6.

6. The mutant cerulean protein of claim 3, having an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 6.

7. The mutant cerulean protein of claim 2, wherein said protein contains amino acid mutations: S72A, Y145A, A206K, S147H, D148G, K166G, I167L, R168N, H169C and T203I.

8. The mutant cerulean protein of claim 7, wherein said protein has an amino acid sequence shown in SEQ ID NO: 8.

9. The mutant cerulean protein of claim 7, having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 8.

10. The mutant cerulean protein of claim 7, having an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 8.

11. The mutant cerulean protein of claim 2, wherein said protein contains amino acid mutations: T65S, S72A, Y145A, A206K, S147H, D148G, K166G, I167L, R168N, and H169C.

12. The mutant cerulean protein of claim 1 1 , having an amino acid sequence shown in SEQ ID NO: 10.

13. The mutant cerulean protein of claim 1 1 , having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 10.

14. The mutant cerulean protein of claim 11 , having an amino acid sequence with at least 95% sequence identity to SEQ ID NO: 10.

15. A chimeric protein comprising the mutant cerulean protein of claim 1 , conjugated with a luminescent agent acceptor.

16. The chimeric protein of claim 15, wherein the luminescent agent acceptor is a green fluorescent protein, yellow fluorescent protein, orange fluorescent protein or red fluorescent protein.

17. The chimeric protein of claim 16, wherein the yellow fluorescent protein is a venus yellow fluorescent protein or citrine yellow fluorescent protein.

18. A chimeric protein comprising the mutant cerulean protein of claim 1 , conjugated with a luminescent agent acceptor.

19. A fusion protein comprising the mutant cerulean protein of claim 1 and a protein of interest.

20. The fusion protein of claim 19, wherein said protein of interest is a mitochondrial protein, golgi protein or membrane-localized protein.

21. A kit comprising: the mutant cerulean protein of claim 1 , and instructions for use.

22. A kit comprising: the mutant cerulean protein of claim 2, and instructions for use.

23. A mutant cerulean protein of sequence shown in SEQ ID NO.6, SEQ ID NO:8, SEQ ID NO: 10 or a conservative mutant thereof.

24. Nucleic acid encoding the mutant cerulean protein of claim 1 , said nucleic acid selected from the group consisting of:

a) isolated nucleic acid which encodes the mutant cerulean protein of claim 1 ; b) isolated nucleic acid which hybridizes to isolated DNA which encodes the mutant cerulean protein of claim 1 ; and

c) isolated nucleic acid differing from the isolated nucleic acid of a) and b) above in codon sequence due to the degeneracy of the genetic code, and which encodes the mutant cerulean protein of claim 1.

25. A vector capable of expressing the nucleic acid of claim 24, adapted for expression in a recombinant cell and regulatory elements necessary for expression of the nucleic acid in the cell.

26. A host cell transfected with the vector of claim 25, said vector expressing the mutant cerulean protein of claim 1.

27. The host cell of claim 26, wherein said cell is selected from the group consisting of bacterial cells, mammalian cells, plant cells, and insect cells.

28. Nucleic acid encoding the mutant cerulean protein of claim 2, said nucleic acid selected from the group consisting of:

a) isolated nucleic acid which encodes the mutant cerulean protein of claim 2; b) isolated nucleic acid which hybridizes to isolated DNA which encodes the mutant cerulean protein of claim 2; and

c) isolated nucleic acid differing from the isolated nucleic acid of a) and b) above in codon sequence due to the degeneracy of the genetic code, and which encodes the mutant cerulean protein of claim 2.

29. The Nucleic acid of claim 28, wherein said mutant cerulean protein has the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

30. A vector capable of expressing the nucleic acid of claim 28, adapted for expression in a recombinant cell and regulatory elements necessary for expression of the DNA in the cell.

31. The vector of claim 30, wherein said nucleic acid encodes the mutant cerulean protein having the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

32. A host cell transfected with the vector of claim 30, said vector expressing the mutant cerulean protein of claim 2.

33. The host cell of claim 32, wherein said cell is selected from the group consisting of bacterial cells, mammalian cells, plant cells, and insect cells.

34. Nucleic acid encoding the mutant cerulean protein of claim 3, said nucleic acid selected from the group consisting of:

a) isolated nucleic acid which encodes the mutant cerulean protein of claim 3; b) isolated nucleic acid which hybridizes to isolated DNA which encodes the mutant cerulean protein of claim 3; and

c) isolated nucleic acid differing from the isolated nucleic acid of a) and b) above in codon sequence due to the degeneracy of the genetic code, and which encodes the mutant cerulean protein of claim 3.

35. The Nucleic acid of claim 34, wherein said mutant cerulean protein has the amino acid sequence shown in SEQ ID NO: 6.

36. The Nucleic acid of claim 34, having a sequence shown in SEQ ID NO: 7.

37. Nucleic acid encoding the mutant cerulean protein of claim 7, said nucleic acid selected from the group consisting of:

a) isolated nucleic acid which encodes the mutant cerulean protein of claim 7; b) isolated nucleic acid which hybridizes to isolated DNA which encodes the mutant cerulean protein of claim 7; and

c) isolated nucleic acid differing from the isolated nucleic acid of a) and b) above in codon sequence due to the degeneracy of the genetic code, and which encodes the mutant cerulean protein of claim 7.

38. The nucleic acid of claim 37, wherein said mutant cerulean protein has the amino acid sequence shown in SEQ ID NO: 8.

39. The nucleic acid of claim 37, having a sequence shown in SEQ ID NO: 9.

40. Nucleic acid encoding the mutant cerulean protein of claim 1 1 , said nucleic acid selected from the group consisting of:

a) isolated nucleic acid which encodes the mutant cerulean protein of claim 11 ;

b) isolated nucleic acid which hybridizes to isolated DNA which encodes the mutant cerulean protein of claim 11 ; and

c) isolated nucleic acid differing from the isolated nucleic acid of a) and b) above in codon sequence due to the degeneracy of the genetic code, and which encodes the mutant cerulean protein of claim 1 1.

41. The nucleic acid of claim 40, wherein said mutant cerulean protein has the amino acid sequence shown in SEQ ID NO: 10.

42. A method of producing a fluorescent protein with increased fluorescence yield, comprising:

identifying structural defects in a fluorescent protein;

performing site-directed mutagenesis in one or more regions encoding the structural defects;

transforming bacteria with the mutated gene product;

selecting the bacteria colony exhibiting greatest increase in fluorescence; and recovering the plasmid and sequence to generate the recombinant protein with increased fluorescence yield.

43. The method of claim 42, wherein the structural defects are one or more of packing defects, a defective hydrophobic core, unfavorable side chain interactions, defects affecting hydrogen-bonding between secondary structure elements or between secondary structure elements and adjacent amino acids within the molecule, and defects that reduce secondary structure character within the molecule.