# Patent

## Title
Teal fluorescent proteins (mTFP1)

## Abstract

The present invention provides an isolated nucleic acid sequence encoding a non-oligomerizing Clavularia teal fluorescent protein (mTFP) variant having a tyrosine-derived chromophore, as well as fragments and derivatives thereof. Also provided is a method for engineering the nucleic acid sequence, a vector comprising the nucleic acid sequence, a host cell comprising the vector, and use of the vector in a method for expressing the nucleic acid sequence. The present invention further provides an isolated nucleic acid, or mimetic or complement thereof, that hybridizes under stringent conditions to the nucleic acid sequence. Additionally, the present invention provides a non-oligomerizing mTFP variant encoded by the nucleic acid sequence, as well as derivatives, fragments, and homologues thereof. Also provided is an antibody that specifically binds to the mTFP variant. The present invention further provides a tandem dimer comprising two mTFP dimers, operatively linked by a peptide linker.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims priority benefit of U.S. Provisional Patent Application No. 60/682,848 filed on May 20, 2005, the contents of which are incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to fluorescent proteins and, in particular, monomeric teal fluorescent proteins.
BACKGROUND
The Aequorea Victoria green fluorescent protein (GFP), along with its various homologues and mutants (Shaner, 2005; Shimomura, 1979), has enabled live-cell fluorescence imaging of recombinant fusion proteins to become a popular and widely-accessible technique in cell-biology research (Tsien, 1998; Zhang, 2002). The defining feature of Aequorea GFP is its ability to autonomously generate a green fluorophore within the confines of its distinctive β-barrel structure (Shimomura, 1979; Yang, 1996; Ormo, 1996). The chromophore of GFP is post-translationally and autonomously generated, through a stepwise process that involves a main-chain cyclization (Gly67 N to Ser65 C), a dehydration (Ser65 C-N), and an oxidation (Tyr66 Ca-C) that effectively conjugates the phenolic side chain of tyrosine 66 to a five-membered ring heterocycle formed from the main-chain atoms of serine 65, tyrosine 66, and glycine 67 (FIG. 1). In the ground state of the wild-type GFP, the chromophore exists as a mixture of neutral phenol (maximum absorbance at 395 nm) and anionic phenolate forms (maximum absorbance at 475 nm). In the excited state, the neutral phenol form deprotonates to form the phenolic anion; therefore, only a single fluorescence emission peak (maximum fluorescence at 504 nm) is observed.
The steric, electrostatic, and hydrogen-bonding environment imposed upon the chromophore by the surrounding residues strongly influences the fluorescence properties. The GFP chromophore has proven remarkably amenable to genetic modification of both its covalent structure and its local environment, and this tolerance has been exploited for the creation of wavelength-shifted variants (Tsien, 1998). Aequorea GFP variants (Shaner, 2005) have been engineered with altered colors, brightness, photostability, ion-sensitivity (Hanson, 2002), and photoswitching properties (Lukyanov, 2005). Amino-acid substitutions at position 65 and at several other residues in the immediate vicinity of the chromophore (e.g., position 203) have resulted in GFP variants (i.e., enhanced GFP (EGFP) with maximum fluorescence at 510 nm. A particularly important class of useful variants that have resulted from such efforts are the yellow fluorescent proteins (YFPs) that are defined by the Thr203Tyr mutation (Ormo, 1996) and an emission peak that is  ̃25 nm red-shifted from the wild type emission peak of  ̃504-509 nm. However, at present, there is no known report of an Aequorea GFP mutant with a tyrosine-derived chromophore and fluorescence that is blue-shifted relative to the wild-type protein (i.e., it has a maximum fluorescence that is less than 504 nm).
The term “cyan fluorescent protein”, or “CFP”, is generally reserved for any GFP homologue with maximum fluorescence emission between approximately 470 nm and 495 nm. To date, substitutions of tyrosine 66 to other aromatic amino acids have proved to be the only approach for blue-shifting the fluorescence emission relative to the wild-type protein, in order to produce a CFP. For example, the widely used Aequorea GFP-derived CFP known as avCFP (also commonly known as ECFP or CFP) was engineered by replacing Tyr66 of Aequorea GFP with a tryptophan, to give an indole-containing chromophore (FIG. 1) (Heim, 1994) that had an emission peak in the cyan region ( ̃480 nm) of the visible spectrum. Although the original Tyr66Trp mutant of Aequorea GFP was only weakly fluorescent, efforts to improve the brightness yielded the widely used variant ECFP (Heim, 1994; Miyawaki, 1997) and more recently-Cerulean (Rizzo, 2004) and CyPet (Nguyen, 2005). While avCFP has been proven as a useful fluorophore in multicolor labeling applications, and as the preferred Forster resonance energy transfer (FRET) donor to a YFP acceptor, its spectral properties limit its utility in some applications. Specifically, avCFP is relatively dim, has broad excitation and emission peaks (FIG. 2), and has a multi-exponential fluorescence lifetime. The multi-exponential fluorescence lifetime of avCFP complicates the use of this protein in fluorescence lifetime imaging (FLIM) applications. Some limitations have been partially addressed in the newer variants; Cerulean is twofold brighter and has a more homogenous fluorescence lifetime (Rizzo, 2004), while CyPet exhibits high FRET to the YFP variant YPet (Nguyen, 2005). However, despite these improvements, Cerulean and CyPet remain limited by fluorescent brightness that is less than 50% of the popular YFP variant Citrine (Shaner, 2005) and that is inferior to EGFP, and by fluorescence lifetimes that are poorly fit as single-exponentials, and a very broad fluorescence emission relative to other popular variants (FIG. 2) (Rizzo, 2004).
Thus, there is a need in the art for a fluorescent protein which mitigates the difficulties of the prior art.
SUMMARY OF THE INVENTION
In view of the problems associated with known fluorescent proteins, as described above, the inventors have engineered a novel non-oligomerizing CFP with a tyrosine-derived chromophore that has unexpectedly superior fluorescence properties, and have adopted the name ‘teal fluorescent proteins (TFP)’ with a preceding ‘d’ for dimeric or ‘m’ for monomeric, and a succeeding numerical identifier, to identify variants of the present invention.
Accordingly, in one aspect, the present invention provides an isolated nucleic acid sequence encoding a non-oligomerizing Clavularia teal fluorescent protein (TFP) variant having a tyrosine-derived chromophore. In certain embodiments, the nucleic-acid-sequence may be compatible with mammalian (e.g., human) codon usage. In one embodiment, the nucleic acid sequence has at least about 60% homology with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 6. In another embodiment, the nucleic acid sequence has at least about 75% homology with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 6. In still another embodiment, the nucleic acid sequence is substantially the same as, or identical to, the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 6. In another embodiment, the nucleic acid sequence is one that encodes the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 7. Also provided is a fragment or derivative of the nucleic acid sequence of the invention. The present invention further provides an isolated nucleic acid, or mimetic or complement thereof, which hybridizes under stringent conditions to the nucleic acid sequence of the invention.
In another aspect, the present invention provides a method of engineering an isolated nucleic acid sequence encoding a non-oligomerizing Clavularia teal fluorescent protein (TFP) variant having a tyrosine-derived chromophore, by screening a fully-synthetic gene library. In one embodiment, the gene library comprises the nucleotide sequence of SEQ ID NO: 1.
In still another aspect, the present invention provides a vector that includes a nucleic acid sequence encoding a non-oligomerizing teal fluorescent protein (TFP) variant having a tyrosine-derived chromophore. In one embodiment, the vector is a plasmid. In another embodiment, the nucleic acid sequence of the vector is cDNA. Also provided is a host cell comprising the vector. The present invention further provides use of the vector in a method for expressing the nucleic acid sequence in mammalian cells. In one embodiment, the nucleic acid sequence is expressed as a tandem genetic fusion to another protein.
In a further aspect, the present invention provides a non-oligomerizing Clavularia teal fluorescent protein (TFP) variant that includes a tyrosine-derived chromophore, as well as any derivative, fragment, or homologue thereof. The Clavularia TFP variant may be a monomer or dimer. In one embodiment, the chromophore comprises the amino acid sequence tyrosine-glycine (YG). For example, the chromophore may comprise the amino acid sequence glutamine-tyrosine-glycine (QYG); the chromophore may also comprise the amino acid sequence alanine-tyrosine-glycine (AYG), or cysteine-tyrosine-glycine (CYG), or glycine-tyrosine-glycine (GYG).
In another embodiment, the TFP variant comprises at least one or more of the following mutations: histidine 42 replaced with asparagine; leucine 44 replaced with valine or isoleucine; serine 62 replaced with threonine; asparagine 63 replaced with threonine; glutamine 66 replaced with cysteine or glycine or alanine; leucine 72 replaced with phenylalanine; alanine 80 replaced with proline; aspartate 81 replaced with asparagine; arginine 123 replaced with histidine; phenylalanine 124 replaced with leucine; aspartate 125 replaced with lysine; methionine 127 replaced with lysine or glutamate; leucine 141 replaced with threonine; lysine 142 replaced with glycine; glutamate 144 replaced with aspartate; proline 145 replaced with alanine; isoleucine 149 replaced with arginine; leucine 1-50 replaced with methionine; valine 158 replaced with lysine; isoleucine 161 replaced with valine; serine 162 replaced with lysine; serine 164 replaced with lysine; tyrosine 173 replaced with histidine; cysteine 175 replaced with valine; serine 179 replaced with threonine; lysine 182 replaced with arginine; valine 186 replaced with alanine; leucine 213 replaced with valine; asparagines 216 replaced with serine; tyrosine 221 replaced with asparagine; glycine 224 replaced with aspartate; or serine 226 replaced with proline.
The TFP variant of the present invention may comprise an amino acid sequence having at least about 60% homology with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 7. In one embodiment, the TFP variant comprises an amino acid sequence having at least about 75% homology with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 7. In another embodiment, the TFP variant comprises an amino acid sequence which is substantially the same as, or identical to, the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 7.
In one embodiment, the TFP variant of the present invention may have a fluorescence emission that is blue-shifted relative to wild-type cFP484 protein. In one embodiment, the TFP variant has a wavelength of maximum fluorescence emission that is less than about 504 nm. The TFP variant may also have an excitation spectrum ranging from about 350 to 500 nm and an emission spectrum ranging from about 450 to 600 nm. In one embodiment, the TFP variant has an excitation maximum ranging from about 450 to 460 nm and an emission maximum ranging from about 485 to 495 nm.
In yet another aspect, the present invention provides an antibody that specifically binds to the TFP variant of the invention. In one embodiment, the antibody is a polyclonal antibody; in another embodiment, the antibody is a monoclonal antibody.
In a further aspect, the present invention provides a tandem dimer comprising two TFP dimers, operatively linked by a peptide linker. In one embodiment, the tandem dimer comprises the amino acid sequence of SEQ ID NO: 5.
Additional aspects and advantages of the present invention will be apparent in view of the description, which follows. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings in which:
FIG. 1 (prior art) illustrates that, in wild-type avGFP, the green fluorescent chromophore arises from the post-translational modification of Ser65, Tyr66, and Gly67. The resulting chromophore contains either a neutral phenol or an anionic phenolate moiety. In avCFP and Cerulean, Tyr66 has been mutated to a tryptophan; thus, the resulting chromophore contains an indole moiety that is chemically distinct from either a phenol or phenolate group. TFP, a protein of the present invention, has a tyrosine-derived chromophore that differs from that of avGFP by the identity of the side chain at position 65, and the identity of the groups in near-spatial proximity to the fluorophore.
FIG. 2 (prior art) depicts the fluorescent emission spectra of avCFP, Cerulean, avGFP, and Citrine (a YFP). Note that both avCFP and Cerulean have strikingly broad emission peaks. A broad emission peak is undesirable when bandpass filters, which are typically used in fluorescence microscopy, are used to collect the fluorescence emission.
FIG. 3: Protein sequence alignments of mTFP1, coral CFPs, and relevant homologues. The aligned sequences are the designed synthetic gene library (SEQ ID NO: 8), mTFP1 (SEQ ID NO: 7), cFP484 from Clavularia sp. (Genbank accession AAF03374) (SEQ ID NO: 9), dsFP483 from Discosoma striata (Genbank accession AAF03370) (SEQ ID NO: 10), amFP486 from Anemonia majano (Genbank accession Q9U6Y6) (SEQ ID NO: 11), mCherry derived from Discosoma striata dsFP583 (Genbank accession AAV52164) (SEQ ID NO: 12) and EGFP derived from Aequorea GFP (SEQ ID NO: 13) (from Clontech vector pEGFP) (Shaner, 2004; Matz, 1999). Residues shaded cyan are changes or point mutations that were beneficial for mTFP1. Residues 66, 67, and 68 (enclosed in black box) are the precursors of the chromophore. Of the 227 structurally aligned residues of the 3 coral CFPs, there are 78 residues that are conserved in all 3 and an additional 91 residues that are conserved in 2 of the 3. Considering only those positions of two-thirds conservation, the inventors determined that cFP484 is the variant at 19%, dsFP483 at 34%, and amFP486 at 47% of the positions, and, cFP484 is the closest to the consensus sequence. The rationale behind the design of the gene library is summarized in Table 1. Numbering is consistent with DsRed and its monomeric variants that have no internal insertions or deletions relative to cFP484.
FIG. 4: Characterization of mTFP1 by Gel Filtration Chromatography. Gel filtration chromatography elution profile with detection at either 450 nm or 550 nm. The upper profile is a co-injection of dTFP0.2 and mCherry (Shaner, 2004) (a red monomer), the middle profile is dTomato (Shaner, 2004) (a red dimer) and mTFP1, and the lower profile is mTFP1 alone. The small 450 nm peak at the dimer elution volume is due to the weak absorbance of dTomato at this wavelength.
FIG. 5: pH-Dependence of the fluorescence emission of mECFP, mCerulean, and mTFP1. Each protein (stock solution of 1 mg/ml in 5 nm Tris pH 7) was diluted 1:100 in a 96-well plate containing 0.1 ml buffer (100 mM) at the indicated pH. Complete emission spectra at each pH were acquired in a plate reader equipped with monochromators.
FIG. 6: Relative rates of photo-bleaching of mECFP, mCerulean, and mTFP1. Photo-bleaching experiments were done directly on live colonies of E. coli on agar plates using the LED-based illumination system as described in the examples herein. Each data point represents the averaged mean fluorescence of 5 individual bacterial colonies expressing the indicated protein.
FIG. 7: Confocal fluorescence image of a Hela cell expressing mTFP1-actin (scale bar=10 μm). (A) HeLa cell expressing mTFP1-β-actin (scale bar=10 μm). (B) HeLa cell expressing mTFP1-α-tubulin (scale bar=10 μm).
FIG. 8: In vitro FRET responses for Cameleon (Miyawaki, 1997) constructs containing (A) mECFP, (B) mCerulean, and (C) mTFP1. For each protein the 3 spectra represent no Ca2+ (red), 10 mM Ca2+ (black), and no FRET (green). To obtain the ‘no FRET’ spectra, the linkers between the two fluorescent proteins were digested with trypsin under conditions where the fluorescent proteins themselves remain intact. The FRET efficiencies (E) in Table 1 were calculated using the formula E=1−(fluorescence at 490 nm before trypsin/fluorescence at 490 nm after trypsin).
FIG. 9: Fluorescence lifetime decay data for (A) mECFP, (B) mCerulean, and (C) mTFP1. Shown in each panel is the experimental data for the lifetime decay (open diamonds) and the instrument response function (IRF) for a scattering solution (open circles). The lifetime decay, convolved with the IRF, has been fitted with both single (green) and double (red) exponential decay functions. The inset is an expanded version of the region from 56 to 62 nm. For each protein, the residuals of the best fits are also shown. Excitation wavelength (nitrogen dye laser at 440 nm), emission wavelength (490 nm), and slits (1 nm) were the same for all experiments. Repeating the experiment under ‘magic angle’ conditions had no effect on the observed lifetimes.
FIG. 10A sets forth the complete DNA sequence of mTFP0.86 (SEQ ID NO:2) and 10B sets forth the complete DNA sequence of mTFP1 (or SEQ ID NO: 6).
FIG. 11 sets forth the complete DNA sequence of the synthetic gene library (SEQ ID NO:1). The synthetic gene library was digested with XbaI and BamHI, and ligated into pUC18 digested with the same enzymes. Transformation of E. coli with the ligated product gave at least 106 transformants. Plasmid DNA was prepared from the transformed E. coli, and digested with XhoI and EcoRI; the 736-bp fragment was then purified. This fragment was inserted into suitably-digested pBAD/HisB (Invitrogen) to create the final library suitable for expression and screening in colonies of E. coli strain LMG194.
FIG. 12A sets forth the complete protein sequence of mTFP0.86 (SEQ ID NO: 3) and 12B sets forth the complete protein sequence of mTFP1 (SEQ ID NO: 7).
FIG. 13 sets forth the complete DNA sequence of a tandem dimer of TFP, abbreviated as tdTFP0.3 (SEQ ID NO: 4).
FIG. 14 sets forth the complete protein sequence of tdTFP0.3 (SEQ ID NO: 5).
FIG. 15 shows the structure-based protein sequence alignment of the designed synthetic library (SEQ ID NO: 8), TFP (ClavCFP) (SEQ ID NO: 9), amFP486 (SEQ ID NO: 11), dsFP483 (SEQ ID NO: 10), dsFP583 (DsRed) (SEQ ID NO: 14), mRFP1 (SEQ ID NO: 15), and Aequorea GFP (avGFP) (SEQ ID NO: 16).
FIG. 16A illustrates degenerate amino acid positions in the synthetic library of FIG. 15, mapped onto the structure of DsRed, PDB ID 1G7K (Yarbrough, 2001). External positions are colored in green, and internal positions are colored in red. Position 66 is part of the chromophore and colored cyan. FIG. 16B shows residues at which degenerate codons were introduced in the designed library.
FIG. 17: Absorbance and fluorescence emission spectra of dTFP0.2, mTFP1, and selected fluorescence proteins. (A) Absorbance (open symbols) and fluorescence emission (filled symbols) spectra of dTFP0.2 (◯, ●) and mTFP1 (□, ▪). (B) Fluorescence emission spectra of mCerulean (◯) (Rizzo, 2004 & 2005), mTFP1 (□), EGFP (Δ), Citrine (●) (Griesbeck, 2001), mOrange (▪), and mCherry (▴)(Shaner, 2004).
FIG. 18 provides an overview of the library screening and the subsequent directed evolution process. Only 0.25% of the gene library encoded functional fluorescent proteins; of these, approximately 50% were green fluorescent and 50% were cyan fluorescent.
FIG. 19 illustrates fluorescence excitation and emission for Cerulean, dTFP0.2, and mTFP0.7. Note that mTFP0.7 is slightly red-shifted from dTFP0.2, but both proteins have a narrow fluorescence emission compared to Cerulean. The narrow fluorescence emission can be attributed to the different chromophore structure.
FIG. 20 depicts a demonstration of mTFP0.6 as a Forster (or fluorescence) resonance energy transfer (FRET) donor to Citrine (a YFP) in a ratiometric calcium sensor. The results of this experiment are summarized in Table 5 of the Examples.
FIG. 21 presents the fluorescence lifetime decay for Cerulean, dTFP0.2, and mTFP0.6.
FIG. 22 presents the fluorescent emission spectra of mTFP, avGFP, and Citrine (a YFP).



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
When describing the present invention, all terms not defined herein have their common art-recognized meanings. To the extent that the following description is of a specific embodiment or a particular use of the invention, it is intended to be illustrative only, and not limiting of the claimed invention. The following description is intended to cover all alternatives, modifications and equivalents that are included in the spirit and scope of the invention, as defined in the appended claims.
Aspects of the present invention relate to novel proteins from a naturally occurring protein that fluoresces at cyan wavelength to create a variant that is optimized for use in fluorescence imaging. Three naturally occurring homologues of Aequorea GFP (cFP484 from Clavularia sp., amFP486 from Anemonia majano, and dsFP483 from Discosoma striata) with tyrosine-derived chromophores and emission peaks between  ̃480-490 nm that had been identified in coral (Matz, 1999) were initially considered.
As used-herein, the terms “CFP” and “cyan fluorescent protein” refer to any GFP homologue with a fluorescence emission peak at less than or equal to 495 nm. As further used herein, a prefix is added to “CFP”, when referring to CFPs from specific organisms, to indicate the name of the organism from which the gene encoding the protein was originally derived. As an example, as used herein, the term “cFP484” refers to a fluorescent protein (“FP”) of the species Clavularia sp. (“c”) with a major emission maximum of 484 nm. The term “TFP” or “teal fluorescent protein” refers to a CFP of the present invention, having a tyrosine derived chromophore.
To identify amino acid positions of TFP, a numbering system is used in which the N-terminal residue is labeled 1, the second residue from the N-terminus is labeled 2, and so until the last residue of the protein is reached at the C-terminus. If residues are added or deleted from the N-terminus of the protein, the identity of each subsequent residue could change if consistently numbered from the N-terminus. Therefore, employed herein, was a numbering system in which the residues of the chromophore-forming residues in the wild-type protein are glutamine 66, tyrosine 67, and glycine 68. Preceeding and subsequent amino acids were labeled consistently with the chromophore numbering amino acids.
The naturally occurring homologues of Aequorea GFP mentioned above have emission peak shapes and fluorescent brightness comparable to Aequorea GFP and are likely tetramers and unsuitable for use as non-perturbing genetic labels (Campbell, 2002). In one example, the inventors chose cFP484 (International Application Publication No. WO0127150; Matz 1999) as the template from which to initiate the directed evolution. cFP484 is a preferred starting point as it is the least divergent from a hypothetical ‘consensus’ sequence (FIG. 3); it has the highest fluorescent brightness; and it has a single cysteine residue while dsFP483 and amFP486 each have five (Matz, 1999).
To prepare proteins of the current invention, a fully-synthetic designed gene library with a theoretical diversity of  ̃5×105 cFP484 variants (Table 1) was prepared; followed by screening. Such an approach has not been previously used for fluorescent proteins. In previous work, genes have been resynthesized with human codon usage. Libraries of gene variants have then been created by random mutation of the gene, or targeted diversification at specific positions within the gene.







TABLE 1







Rationale for design of the synthetic gene library










Residue





number
Mutated to
Codon
Rationale





His42
His, Asn,
MAS
Mutations beneficial to



Gln, Lys

tetrameric DsRed variants





(Bevis, 2002).





Not conserved in coral CFPs.


Leu44
Leu, Val,
SYC
Mutations beneficial to



Ala, Pro

tetrameric DsRed variants





(Bevis, 2002).





Not conserved in coral CFPs.


Gln66
Gln, Lys,
MWG
Mutations beneficial to


(residue of
Met, Leu

Aequorea GFP (Heim, 1995) and


chromophore)


monomeric RFPs (Shaner, 2004).





Lys or Gln present in coral





CFPs.


Arg70
Lys, Arg
ARG
Mutations beneficial to





monomeric RFPs (Campbell,





2002). Lys or Arg present





in coral CFPs.


Ala71
Ala, Val
GYC
Mutations beneficial to





monomeric RFPs (Campbell,





2002). Ala or Cys present





in coral CFPs.


Leu72
Leu, Phe,
HTC
Phe in 2 coral CFPs and Leu



Ile

in 1 coral CFP.


Phe83
Leu, Phe,
HTC
Mutations beneficial to



Ile

monomeric RFPs (Campbell,





2002; Shaner, 2004). Phe in





2 coral CFPs and Leu in 1





coral CFP.


Ile104
Ile, Thr
AYC
May disrupt A–B interface





interactions


Phe124
Leu, Phe,
HTC
Mutations beneficial to



Ile

dimeric RFPs (Campbell,





2002). Strict conservation





in coral CFPs.


Asp125
Lys, Arg
ARG
Likely to disrupt A–B





interface interactions.


Met127
Lys, Arg
ARG
Likely to disrupt A–B





interface interactions.


Met150
Met, Leu
MTG
Mutations beneficial to





monomeric RFPs (Campbell,





2002). Met in 2 coral CFPs





and Leu in 1 coral CFP.


His163
His, Gln
CAS
Mutations beneficial to





dimeric and monomeric





RFPs (Campbell, 2002; Shaner,





2004). His in 2 coral CFPs





and Ala in 1 coral CFP.


Ser179
Ser, Thr
WCC
Mutations beneficial to





dimeric RFPs (Campbell,





2002). Thr in 2 coral CFPs





and Ser in 1 coral CFP.









Preferred features of the library included: mammalian codon usage, deletion of 40 non-homologous residues from the N-terminus, addition of the 7 N-terminal and 7 C-terminal residues of Aequorea GFP, semi-degenerate codons encoding potential ‘tetramer-breaking’ mutations at 3 external positions, and semi-degenerate codons encoding potential ‘rescuing’ mutations at 11 internal positions. Sequencing of random clones revealed that 12% of the genes had mutations only at desired positions and that the true library diversity was  ̃4×106 variants. The library was used to transform E. coli and the resulting colonies were screened for fluorescence. Fluorescent colonies represented  ̃0.5% of all colonies and were approximately equally divided between colonies that fluoresced at 490 nm and colonies that fluoresced at  ̃510 nm. The most brightly fluorescent protein identified after extensive screening was a dimer with 8 mutations relative to wild type and an emission peak at 486 nm. These proteins have been described herein as ‘teal fluorescent protein’ (TFP), with a preceding ‘d’ for dimeric or ‘m’ for monomeric, and a succeeding numerical identifier, to identify variants described herein. Following this convention, the dimeric protein identified in the initial screen was designated dTFP0.1 and the 170% brighter version resulting from one round of directed evolution was designated dTFP0.2 (Table 2 and 3). Substitution of dimer interface residues Ser162 and Ser164 with lysine produced a monomeric version (FIG. 4), mTFP0.3, which retained only 15% of the brightness of its dimeric precursor. After multiple successive rounds of screening libraries (generated by random or saturation mutagenesis) for variants with improved brightness and high 480/530 nm emission ratio, mTFP0.7 was obtained that had a fluorescent brightness equivalent to Cerulean (Table 2). However, imaging an mTFP0.7-actin fusion in live HeLa cells by confocal microscopy, led to a fluorescent signal that rapidly vanished upon illumination with the 458 μm laser, due to a rapid in vitro photo-conversion to a non fluorescent state (Table 2 and 3).







TABLE 2







Fluorescent properties of CFPs and TFPs.





















Cameleon









FRET





εa * 10−3



efficiency



Absorbance
Emission
(mM−1

Brightnessc

(+/− Ca2+)
















Protein
(nm)
(nm)
cm−1)
Φb
(mM−1cm−1)
pKa
Photostabilityd
−
+



















cFP484e
456
484
35.3
0.48
17
NDf
ND
ND
ND


dTFP0.1
456
485
42
0.63
26
ND
<1
ND
ND


dTFP0.2
456
486
60
0.68
41
ND
<1
ND
ND


mTFP0.3
458
488
19
0.31
 6
ND
<1
ND
ND


mTFP0.7
453
488
60
0.50
30
4.0
<1
ND
ND


mTFP1
462
492
64
0.85
54
4.3
163g/110h
39%
49%


mECFP
433/451i
475/504i
33/30i
0.41
13/12i
4.7
 64h,j
26%
39%


mCerulean
433/451i
475/503i
43/37i
0.64
27/24i
4.7
 36g,j
30%
41%


EGFPj
488
507
56
0.60
34
6
 174h,j
ND
ND


tdTFP0.3
458
489
125
0.69
86
ND
ND
ND
ND





aExtinction coefficient.


bQuantum yield.


cProduct of Φ and ε * 10−3. Values for common FP variants have been previously tabulated (Shaner, 2005).


dTime to bleach from an initial emission rate of 1000 photons/s to 500 photons/s.


eValues from ref. (Matz, 1999).


fNot determined.


gMeasured with 10% ND filters.


hMeasured with no ND filters.


iValues for both ‘humps’ of mECFP and mCerulean are provided.


jValues from ref. (Shaner, 2005).













TABLE 3







Mutations in dimeric and monomeric TFP variants.










Library construction



Variant
strategies
Mutations





dTFP0.1
See Supplementary
Inside: H42N, L44V, L72F,



Table 1.
F124L, M150L, S179T




A–B interface: D125K,




M127K


dTFP0.2
2 generations of
Inside: D81N



random mutagenesis.
Outside: S226P


mTFP0.3
Site-directed mutagenesis
A–C interface:



at 162 and 164. Saturation
S162K, S164K



mutagenesis at 163.


mTFP0.4
Saturation mutagenesis
Inside: Q66C, C175V



at 66. Semi-saturation



mutagenesis at 175.


mTFP0.5
3 generations of random
Inside: S62T, C66G



mutagenesis.
Outside: A80P, N216S




A–B interface: K127E,




K182R


mTFP0.6
Saturation mutagenesis at
Inside: G66A, L213V



66 + 163, 66 + 197.
Outside: S2N



Semi-saturation mutagenesis
Replace 223–228 with TG



at 66 + 147, 66 + 213.


mTFP0.7
2 generations of random
Inside: V44I, Y173H



mutagenesis.
Outside: V186A




A–B interface: R123H




Mutate: N2S


mTFP0.8
Semi-saturation mutagenesis
Inside: N63T



at 62, 63, 64, 65, and 66



with screening for



photostability.


mTFP0.9
Semi-saturation mutagenesis
Inside: K142G, L150M



at 142, 144, 145, 149, 150,
(reversion to wild type),



and 161 with screening for
I161V A–C interface:



photostability.
E144D, P145A, I149R


mTFP1
2 generations of random
Outside: L141T, V158K,



mutagenesis with
Y221N, G224D



screening for photostability.









To select for photostable variants, an array of six 460 nm light emitting diodes (Lumileds, San Jose, Calif.) was constructed that provided even illumination of a Petri dish with  ̃55 mW/cm2; an irradiance that is 1400× more intense than the excitation used in previous screening and sufficient to photoconvert mTFP0.7 within seconds. The fluorescence of libraries of mTFP0.7 variants expressed in bacterial colonies was digitally imaged during exposure to intense illumination and colonies with decreased propensity to photoconvert were identified. Following several rounds of selection for variants that were photostable, bright, and retained a high 480/530 nm emission ratio, we arrived at mTFP1 that has a total of 29 mutations relative to wild type protein. In addition, mTFP1 is 2-fold brighter and more photostable than Cerulean and is insensitive to physiologically relevant changes in pH (Table 2 and FIGS. 5 and 6).
To determine the photostability of mTFP1, photobleaching experiments were done on droplets of purified protein suspended in mineral oil following the protocol of Shaner et al. (Shaner, 2005). This method is designed to approximate the conditions of a typical wide-field microscopy experiment while rigorously accounting for differences in the spectral properties of the fluorescent proteins as well as the optical properties of the microscope. Using this method, the time for bleaching from an initial emission rate of 1,000 photons/sec/molecule down to 500 photons/sec/molecule (t1/2) was determined to be 163 s when 10% neutral density filters were used. With no neutral density filters the t1/2 was 110 s for mTFP1. For the sake of comparison, the t1/2 for Cerulean with 10% neutral density filters is 36 s and the t1/2 for EGFP with no neutral density filters is 174 s (Shaner, 2005). As previously herein (Shaner, 2005), Cerulean displays an illumination intensity-dependent fast bleaching component that can decrease the intensity to 60% of its initial value within the first few seconds of imaging under typical conditions (Shaner, 2005 and P. Steinbach, personal communication). No fast bleaching component was observed for mTFP1.
Expression of mTFP1-actin fusion in mammalian cells was performed to demonstrate its suitability for use as non-perturbing fusion partner (FIG. 7). The fusion protein localized correctly and there was no significant decrease in intensity upon illumination with the 458 nm laser.
Typical fluorescence filter sets used to image Cerulean are adequate but suboptimal for mTFP1. In one embodiment, to take advantage of mTFP1's improved brightness, a 445/30 nm excitation filter, a 470 nm beamsplitter, and a 495/30 nm emission filter may be used (FIG. 7B and Table 4). This combination gives a 2.6-fold increase in fluorescent signal relative to mCerulean imaged using a standard CFP set (e.g. a 436/20 nm excitation filter, a 455 nm beamsplitter, and a 480/40 nm excitation filter). This preferred set has been used in combination with a new YFP emission filter (545/30 nm), to demonstrate that mTFP1-YC3.3 can be practically employed in live cell FRET imaging. The gene encoding mTFP1-YC3.3 was cloned into a mammalian expression vector with an N-terminal signal sequence and a C-terminal endoplasmic reticulum (ER) retention peptide (Miyawaki, 1997). FRET imaging of transiently transfected HeLa cells on an Zeiss Axiovert 200M equipped with a digital CCD camera revealed a typical pattern of endoplasmic reticulum localization and robust ratiometric responses to induced changes in the free Ca2+ concentration. This result demonstrates that the spectral distinction between mTFP1 and mCitrine are sufficient for these proteins to be used as an advantageous new FRET pair that can be imaged on a standard epifluorescence microscope equipped with appropriate bandpass filter sets.







TABLE 4





Experimentally determined relative fluorescence intensities


for identical concentrations of mTFP1, mCerulean,


and mCitrine imaged on an epi-fluorescence microscope


with xenon arc lamp illumination.



















Intensity relative




to mCerulean imaged




with a HQ436/20




excitation filter and



Excitation
D480/40 emission filtera











Protein
filtera
Beamsplittera
D480/40
HQ495/30





mTFP1
D436/20
455DCLP
1.3
1.5



HQ445/30
470DCXR
NDb
2.6


mCerulean
D436/20
455DCLP
1.0
0.8



HQ445/30
470DCXR
NDb
1.3


















Relative intensity





TFP or CFP
passed by YFP



Excitation
Beam-
emission
emission filtera,c












Protein
filtera
splittera
filtera
HQ535/30
HQ545/30





mTFP1
D436/20
455DCLP
HQ495/30
0.39
0.30



HQ445/30
470DCXR
HQ495/30
0.39
0.30


mCerulean
D436/20
455DCLP
D480/40
0.35
0.30



HQ445/30
470DCXR
HQ495/30
0.41
0.34


mCitrine
HQ500/20
Q515LP
NDb
1
0.91



D436/20
455DCLP
NDb
0.12
0.07



HQ445/30
470DCXR
NDb
0.15
0.09





aAll filters and beamsplitters were purchased from Chroma Technology Corp. Filters are designated with Chroma part numbers.


bNot determined.


cFor mTFP1 and mCerulean, intensities are relative to the intensity in the indicated TFP or CFP emission channel. For mCitrine, all intensities are relative to the intensity obtained with a HQ500/20 excitation filter and HQ535/30 emission filter.






mTFP1's suitability as a FRET donor to Citrine was also demonstrated. Analogous versions of yellow Chameleon 3.3 (YC3.3) containing either mECFP, mCerulean, or mTFP1 were constructed and their FRET efficiency in the absence and presence of Ca2+ was determined (Table 2). FRET efficiencies were accurately determined by trypsinolysis under conditions where the fluorescent proteins themselves remain intact but the calmodulin-M13 domain is cleaved. FRET efficiencies (E) for mECFP and mCerulean in Table 2 were calculated using the formula E=1-(fluorescence at 475 nm before trypsinization/fluorescence at 475 nm after trypsinization). For mTFP1, the fluorescence before and after trypsinization was measured at 490 nm. All three reporters increased FRET efficiency upon Ca2+-binding but the mTFP1 version was notable for its significantly higher efficiencies in both the Ca2+-bound and Ca2+-free states. The trend in experimental FRET efficiencies, mECFP<mCerulean<mTFP1, is consistent with our calculated Ro values of 5.0 nm for mECFP, 5.3 nm for mCerulean, and 5.7 nm for mTFP1. It is important to note that the original YC3.3 contained ECFP and had been optimized for maximum FRET difference between the Ca2+-bound and Ca2+-free states. It is therefore unsurprising that switching to an mTFP1 donor (with a different dipole orientation and increased Ro value) results in a slightly decreased dynamic range. This is almost certainly because the protein was empirically optimized (Miyawaki, 1997 and 2000) for interfluorophore distance changes centered on the Ro of ECFP (where there is the strongest dependence of FRET efficiency on distance). It is believed that if mTFP1-YC3.3 was to be subject to similar systematic optimization, the dynamic range will be significantly improved.
Finally, the utility of mTFP1 for use in fluorescence lifetime imaging (FLIM) was demonstrated by measuring the fluorescence lifetimes of mECFP, mCerulean, and mTFP1 under identical in vitro conditions (FIG. 8). In contrast to mCerulean, which is best described as a poor fit of a single lifetime (Rizzo, 2004), mTFP1 is unambiguously best fit as single lifetime (τ=3.3 ns, χ2=1.1). Attempts to fit the mTFP1 lifetime decay data with a double exponential converged to solution in which τ1=τ2=3.2 ns; strong evidence for the single fluorescent lifetime. Repeating the experiment under ‘magic angle’ conditions had no effect on the observed lifetimes or statistics. Analogous experiments with mECFP and mCerulean resulted in data that was best fit as double exponentials (for mECFP τ1=3.7 ns (80%), τ2=1.7 ns (20%), χ2=1.1; for mCerulean c=3.7 ns (80%), τ2=1.9 ns (20%), χ2=0.99). As previously reported (Rizzo, 2004), mCerulean could also be adequately fit with a single exponential (τ=3.5 ns, χ2=1.1), though the residuals were consistently unsatisfactory.
The approach described herein is advantageous in that it permits the discovery of mutants with combinations of mutations that are only beneficial when they occur together. Such variants could not be discovered by a stepwise approach, since it is possible that the individual mutations are not beneficial (or might even be detrimental) by themselves. For example, dTFP0.1 contains six mutations (H42N, L44V, L72F, F124L, M150L, S179T) in the interior of the protein. Through the use of random mutagenesis, such a combination of mutations would be so exceptionally rare that it would never be discovered by any sort of conventional library screening procedure. In addition, even six rounds of random mutagenesis (with an average of one mutation per gene in each round) would probably not lead to this same variant, unless each mutation in isolation resulted in an improvement.
The engineered proteins described herein differ from the wild-type protein in a number of respects. For example, the proteins of the present invention are monomers, and are brighter than the wild-type protein. The proteins also lack the first forty amino acids of cFP484, and the long N-terminal sequence of cFP484. Furthermore, the monomeric versions of TFP described herein lack the last six amino acids of cFP484. The corresponding sequences of Aequorea GFP have been appended to the N- and C-termini of the TFP variants.
The chromophore of cFP484, from which the monomers of the present invention were ultimately derived, was obtained from the amino acid sequence glutamine-tyrosine-glycine (QYG). The tyrosine and glycine are absolutely essential, and are present in all naturally-occurring fluorescent proteins of all colors. The preceding residue, glutamine, is also very important, but tends to vary among different colors. Nevertheless, different proteins of the same color can have different residues at this position. For example, the other two naturally-occurring cyan fluorescent proteins have a glutamine at this position. In certain of the monomers of the present invention, mutations have been introduced at this position, and particular residues have been found to be preferable to the wild-type glutamine residue. By way of example, mTFP0.4 has a cysteine, mTFP0.5 has a glycine, and mTFP0.1 has an alanine.
Tryptophan residues located in the vicinity of the chromophore sequence do not have a particular significance with respect to the chromophore structure. The engineered cyan fluorescent proteins, avCFP and Cerulean, have a chromophore structure that is derived from the sequence threonine-tryptophan-glycine.
The proteins of the present invention are advantageous, relative to cFP484, in that: (1) they are monomers; (2) they have a higher extinction coefficient; (3) they have a higher quantum yield; (4) they have mammalian codon usage; (5) high brightness; (6) improved photostability; (7) efficient intra- and inter-molecular FRET with a yellow or orange acceptor fluorescence protein; (8) dual color imaging in combination with an orange or red fluorescence protein and (9) a single fluorescence lifetime. For dual color imaging in combination with a YFP, mTFP1 could provide a brighter fluorescent signal than existing Aequorea-derived CFPs but the bleedthrough into the acceptor emission channel may be only slightly better (or possibly worse) depending on the specific filter combination in place. In addition, these proteins have N- and C-terminal sequences corresponding to the N- and C-terminal sequences of Aequorea GFP. This facilitates subcloning, since any DNA primer that can be used to perform PCR on the widely-available Aequorea GFP will also work with the TFPs of the present invention. These proteins are also optimized for expression at 37° C., and may have lower toxicity to live cells. Furthermore, these proteins have a narrow emission peak; this is advantageous, relative to Aequorea-derived CFP. It is believed that these proteins will be useful in research, particularly in the areas of cell biology, physiology, oncology, and biochemistry.
In view of the foregoing, the present invention provides an isolated nucleic acid composition comprising a nucleic acid sequence encoding a non-oligomerizing Clavularia teal fluorescent protein (TFP) variant having a tyrosine-derived chromophore. Also provided is any fragment or derivative of the isolated nucleic acid composition of the invention. The complete DNA sequence of Clavularia sp. fluorescent protein FP484 may be obtained from Accession No. AF168424.
The recently reported crystal structure of amFP486 revealed the presence of a positively charged histidine near the chromophore that may limit charge transfer in the excited state and thereby give rise to the blue-shift [44]. Arg70, Glu148, His197, and Glu215. Without being bound by a theory, it is likely that the Arg/Lys degeneracy at position 70 was responsible for the 50/50 split between green and cyan FPs in the initial library.
As used herein, a “nucleic acid” or “polynucleotide” includes a nucleic acid, an oligonucleotide, a nucleotide, a polynucleotide, and any fragment, variant, or derivative thereof. The nucleic acid or polynucleotide may be double-stranded, single-stranded, or triple-stranded DNA or RNA (including cDNA), or a DNA-RNA hybrid of genetic or synthetic origin, wherein the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides and any combination of bases, including, but not limited to, adenine, thymine, cytosine, guanine, uracil, inosine, and xanthine hypoxanthine. The nucleic acid or polynucleotide may be combined with a carbohydrate, a lipid, a protein, or other materials. A nucleic acid sequence of interest may be chemically synthesized using one of a variety of techniques known to those skilled in the art, including, without limitation, automated synthesis of oligonucleotides having sequences which correspond to a partial sequence of the nucleotide sequence of interest, or a variation sequence thereof, using commercially-available oligonucleotide synthesizers, such as the Applied Biosystems Model 392 DNA/RNA synthesizer.
One nucleic acid composition of the present invention is the nucleotide sequence encoding mTFP0.86 (SEQ ID NO: 2; FIG. 10A). Another nucleic acid composition comprises the sequence encoding mTFP1 (SEQ ID NO: 6, FIG. 10B). Accordingly, in one embodiment of the present invention, the isolated nucleic acid composition comprises a nucleic acid sequence having at least about 60% homology with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 6. In another embodiment of the present invention, the isolated nucleic acid composition comprises a nucleic acid sequence having at least about 75% homology with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 6. In still another embodiment of the present invention, the isolated nucleic acid composition comprises a nucleic acid sequence that is substantially the same as, or identical to, the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 6. In one embodiment, the nucleic acid composition comprises any nucleic acid sequence which encodes any protein of the present invention, including proteins having the ammo acid sequence SEQ ID NO: 3 or SEQ ID NO: 7. In certain embodiments, the nucleic acid sequence may be re-synthesized such that it is compatible with mammalian (e.g., human) codon usage, or the sequence may be resynthesized to be optimised for non-mammalian codon preferences.
The present invention further provides an isolated nucleic acid, including any mimetic or complement thereof, that hybridizes under stringent conditions to the nucleic acid composition described herein. The “complement” of a nucleic acid sequence refers, herein, to a nucleic acid molecule which is completely complementary to another nucleic acid, or which will hybridize to the other nucleic acid under conditions of stringency. Stringent (e.g., high-stringency) conditions are known in the art (see, e.g., Maniatis, 1989; Ausubel, 2001). Stringent conditions are sequence-dependent, and may vary depending upon the circumstances.
The present invention further provides a method of engineering a nucleic acid sequence encoding a non-oligomerizing Clavularia teal fluorescent protein (TFP) variant having a tyrosine-derived chromophore, by screening a fully-synthetic gene library. In one embodiment, the gene library comprises the nucleotide sequence of SEQ ID NO: 1 (FIG. 11).
The present invention also provides a vector comprising a nucleic acid sequence encoding a non-oligomerizing Clavularia teal fluorescent protein (TFP) variant having a tyrosine-derived chromophore. Also provided is a host cell (e.g., a mammalian cell) comprising the vector.
In one embodiment of the present invention, the vector is a plasmid, although it is to be understood that other types of vectors, such as cosmids and phagemids, may also be used for the purposes of the present invention. The term “plasmid”, as used herein, refers generally to circular double-stranded DNA, which is not bound to a chromosome. The DNA may be a chromosomal or episomal-derived plasmid. The plasmid of the present invention may optionally contain a terminator of transcription; a promoter; and/or a discrete series of restriction-endonuclease recognition sites, located between the promoter and the terminator. In the plasmid, a polynucleotide insert of interest (e.g., one encoding a non-oligomerizing Clavularia teal fluorescent protein (TFP) variant having a tyrosine-derived chromophore) should be operatively linked to an appropriate promoter, such as its native promoter or a host-derived promoter, such as the E. coli lacZ promoters, the trp and tac promoters, the τ3 and τ7 promoters, or the CMV promoters. Other suitable promoters will be known to the skilled artisan.
The vector of the present invention may comprise cDNA encoding a non-oligomerizing Clavularia teal fluorescent protein (TFP) variant having a tyrosine-derived chromophore. As used herein, the term “cDNA” refers to an isolated DNA polynucleotide or nucleic-acid molecule, or any fragment, derivative, or complement thereof. The cDNA may be double-stranded, single-stranded, or triple-stranded, it may have originated recombinantly or synthetically, and it may represent coding and/or noncoding 5′ and/or 3′ sequences.
The vector of the present invention may be useful in a method for expressing the nucleic acid sequence in mammalian cells or non-mammalian cells. In one embodiment, the nucleic acid of the vector is expressed as a tandem genetic fusion to another protein.
The present invention further provides a non-oligomerizing Clavularia teal fluorescent protein (TFP) variant comprising a tyrosine-derived chromophore, as well as any derivative, fragment, or homologue thereof. The amino acid sequence of Clavularia sp. fluorescent protein FP484 may be obtained from Accession No. AF168424. The TFP variant of the present invention may be a monomer or a dimer.
In one embodiment of the present invention, the TFP variant includes a chromophore comprising the amino acid sequence tyrosine-glycine (YG). By way of example, the chromophore may comprise the amino acid sequence glutamine-tyrosine-glycine (QYG); the chromophore may also comprise the amino acid sequence cysteine-tyrosine-glycine (CYG), glycine-tyrosine-glycine (GYG), or alanine-tyrosine-glycine (AYG), as in variants mTFP0.7, mTFP0.75, and mTFP0.86, respectively.
Furthermore, the TFP variant of the present invention may comprise an amino acid sequence having at least about 60% homology with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 7 (FIGS. 12A and 12B). In one embodiment, the TFP variant comprises an amino acid sequence having at least about 75% homology with the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 7. In another embodiment, the TFP variant comprises an amino acid sequence, which is substantially the same as, or identical to, the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 7. The TFP variant of the present invention may also comprise at least one of the following mutations: H42N, L44V, L72F, F124L, M150L, and S179T.
As discussed above, the inventors' variant proteins are advantageous in that they exhibit enhanced brightness. The TFP variant of the present invention may have a fluorescence emission that is blue-shifted relative to the wild-type cFP484 protein. In one embodiment, the TFP variant of the present invention has a wavelength of maximum fluorescence emission that is less than about 504 nm. In another embodiment, the TFP variant of the present invention has an excitation spectrum ranging from about 350 to 500 nm and an emission spectrum ranging from about 450 to 600 nm. In still another embodiment, the TFP variant has an excitation maximum ranging from about 450 to 460 nm and an emission maximum ranging from about 485 to 495 nm.
The present invention further provides a tandem dimer comprising two TFP dimers, operatively linked by a peptide linker. Thus, the dimers may be fused into a single non-oligomerizing gene product.
An exemplary tandem dimer of the present invention is tdTFP0.3. Accordingly, in one embodiment of the present invention, the tandem dimer is encoded by a DNA sequence comprising the nucleotide sequence of SEQ ID NO: 4 (FIG. 13). In another embodiment of the present invention, the tandem dimer comprises the protein sequence of SEQ ID NO: 5 (FIG. 14).
Additionally, the present invention provides an antibody that specifically binds to a TFP variant, as described herein. The antibody of the present invention may be polyclonal or monoclonal, and may be produced by techniques well known to those skilled in the art. Polyclonal antibody, for example, may be produced by immunizing a mouse, rabbit, or rat with purified protein. Monoclonal antibody then may be produced by removing the spleen from the immunized mouse, rabbit, or rat, and fusing the spleen cells with myeloma cells to form a hybridoma which, when grown in culture, will produce a monoclonal antibody.
The antibodies used herein may be labelled with a detectable marker or label. Labelling of an antibody may be accomplished using one of a variety of labelling techniques, including peroxidase, chemiluminescent labels known in the art, and radioactive labels known in the art. The detectable marker or label of the present invention may be, for example, a non-radioactive or fluorescent marker, such as biotin, fluorescein (FITC), acridine, cholesterol, or carboxy-X-rhodamine, which can be detected using fluorescence and other imaging techniques readily known in the art. Alternatively, the detectable marker or label may be a radioactive marker, including, for example, a radioisotope. The radioisotope may be any isotope that emits detectable radiation. Radioactivity emitted by the radioisotope can be detected by techniques well known in the art. For example, gamma emission from the radioisotope may be detected using gamma imaging techniques, particularly scintigraphic imaging.
The present invention is described in the following Examples, which are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.
EXAMPLES
Example 1
General Methods and Materials
The synthetic gene library of Clavularia cFP486 variants was commissioned from the DNA Technologies Unit at the NRC Plant Biotechnology Group (Saskatoon, SK). All synthetic DNA oligonucleotides for cloning and construction of subsequent libraries were purchased from Sigma-Genosys Canada (Oakville, ON). PCR products and products of restriction digest were routinely purified using the QIAquick PCR-purification kit according to the manufacturers protocols (Qiagen). Restriction enzymes were purchased from either Invitrogen or New England Biolabs. The cDNA sequences for all TFP variants and fusion constructs was confirmed by dye terminator cycle sequencing using the DYEnamic ET kit (Amersham Biosciences). Sequencing reactions were analyzed at the University of Alberta Molecular Biology Service Unit. The proteins mECFP and mCerulean have the A206K mutation in addition to their characteristic substitutions (Zacharias, 2002; Rizzo, 2004 and 2005).
Example 2
Selection of the Starting Template
Prior to the present invention, there were three reported naturally-occurring fluorescent proteins with tyrosine-derived chromophores and wavelengths of maximum fluorescence emission at less than 490 nm (Matz, 1999): ClavCFP (a.k.a. ClavFP484) from Clavularia sp., dsCFP (a.k.a. dsFP483) from Discosoma striata, and amCFP (a.k.a. amFP486) from Anemonia majano. At least one other CFP, mcCFP (a.k.a. mcCFP477) from Montastrea cavemosa, has been recently reported (Sun et al., FEBS Lett., 570:175-83, 2004). One other CFP, asCFP (a.k.a. MiCy) from Acropara sp., has a wavelength of maximum fluorescence of 495 nm, and, therefore, is essentially green (as opposed to cyan) in its fluorescence (Karasawa et al., Biochem. J., 381:307-12, 2004). The naturally-occurring CFPs have quantum yields (QY) of 0.24-0.48 and extinction coefficients (EC) of 24,000-40,000M-1 cm-1. All of the naturally-occurring CFPs are oligomers, with some reported to be trimers and some reported to be tetramers. Protein-sequence alignments of ClavCFP, dsFP483, and amFP483 revealed that, of 227 structurally-aligned residues, there are 78 residues that are conserved in all 3 CFPs (FIG. 3). There are an additional 91 residues that are conserved in two of the three CFPs.
Considering only these positions, the inventors determined that ClavCFP is the variant at 19%, dsFP483 at 34%, and amFP486 at 47%, of the 91 positions. Thus, ClavCFP is the closest to a “consensus” sequence. ClavCFP has one cysteine (position 175 with DsRed numbering), while each of dsFP483 and amFP486 has five cysteines. Multiple cysteine residues are undesirable, due to the unpredictable effects of thiol oxidation in applications where the CFP is targeted to an oxidizing subcellular environment—such as the secretary pathway or the bacterial periplasm. Accordingly, the inventors concluded that ClavCFP is a preferred template upon which to base this directed evolution.
Example 3
Library Construction and Mutagenesis
The inventors commissioned the synthesis of the gene library encoding the designed protein library from the DNA Technologies Unit at the NRC Plant Biotechnology Group. The important features of the designed gene included: mammalian optimized codon usage, deletion of forty residues from the N-terminus, addition of the seven N-terminal and seven C-terminal residues of avGFP, semi-degenerate codons encoding potential “tetramer-breaking” mutations at three external positions, and semi-degenerate codons encoding potential “rescuing” mutations at eleven internal positions (FIGS. 11, 15, and 16). Library quality was verified by sequencing 77 independent clones, of which nine (12%) were confirmed to contain mutations at only the desired positions.
The initial synthetic gene library was digested with Xho1 and EcoR1 and ligated into similarly digested pBAD/HisB vector (Invitrogen). Subsequent libraries with saturation mutagenesis at a particular residue were constructed by either by an overlap-extension PCR method (Campbell, 2002) or the Quikchange protocol (Stratagene). Randomly mutated libraries were constructed by error-prone PCR as previously described (Campbell, 2002) under conditions optimal for 3 mutations per 1,000 bp (Fromant, 1995). Full-length gene libraries resulting from overlap-extension PCR or error-prone PCR were ligated into the XhoI and EcoR1 restriction sites of pBAD/HisB. Regardless of library assembly method, electrocompetent Escherichia coli strain DH10B (Invitrogen) was transformed and plated on Luria-Bertani (LB)/agar plates supplemented with ampicillin (0.1 mg/ml) and L-arabinose (0.02%). Plates were incubated for 14 h at 37° C. prior to screening.
Example 4
Screening
The system for imaging the fluorescence of bacterial colonies grown on 10 cm Petri dish is a custom built device similar to one previously described6. Briefly, the light from a 175W xenon-arc lamp (Sutter) is passed through a 426 nm to 446 nm bandpass filter (Chroma) and into a bifurcated fiber optic bundle (Newport). Light exiting the fiber optic bundle illuminates (0.04 mW/cm2) a 10 cm dish placed in a recessed holder on the bench top. For all screening up to the identification of mTFP0.6, colony fluorescence was screened by viewing illuminated plates through a pair of custom goggles fitted with image quality 3 mm thick GG455 glass (Chroma). When viewed through these goggles, colonies fluorescing at 480-490 nm have a distinctly bluish hue and are easily distinguishable from ‘greenish’ colonies emitting at 500-510 nm. For the identification of mTFP0.7, colony fluorescence was digitally imaged with a Retiga 1300i 12-bit CCD camera (QImaging) fitted with filter wheel (Sutter) that contains both a 480/40 nm and a 530/30 nm bandpass filter. Through the use of custom macros for Inage Pro Plus (Media Cybernetics), images in both emission channels were acquired and the fluorescence intensities of all colonies were individually integrated. Colonies with high 480/530 nm intensity ratios and high brightness at 480 nm were selected for further characterization.
For the identification of mTFP0.8, mTFP0.9, and mTFP1, the screening protocol was modified in order to select for photostability. We equipped six Royal Blue (peak emission at 455 nm) LuxeonTM V light emitting diodes (LEDs) (Lumileds Lighting) with narrow beam lenses (Fraen) and positioned them to evenly illuminate (55 mW/cm2) the 10 cm dish in the imaging system described above. Through the use of a custom serial port connection, the LEDs could be switched on and off at computer controlled intervals. Through the use of a custom serial port connection, the LEDs could be switched on and off at computer controlled intervals. An Image Pro Plus macro (Media Cybernetics) was used to automate acquisition and processing. For each plate, fluorescence images were acquired following a series of programmed intervals of intense illumination. Using this system, we could identify colonies with decreased propensity to photo-convert or otherwise photo-bleach.
For all screening protocols, colonies with more intense fluorescence or decreased propensity to photo-convert were picked and cultured overnight in 4 ml LB media containing ampicillin and arabinose. The following day 0.1 ml of each culture was dispensed into a 96-well plate (Nunc) and the full emission spectra of each variant measured with a Safire2 plate reader (Tecan). Variants with the most blue-shifted and intense emission peak were used as templates in the subsequent round of library construction.
Example 5
Protein Purification and Characterization
To prepare proteins in sufficient quantity for characterization, E. coli strains DH10B or LMG194 were transformed with the pBAD/His B expression vector containing the gene of interest. A single colony was used to inoculate a 4 ml culture that was allowed to grow overnight (37° C., 225 rpm) before being diluted into 1 L of LB media containing ampicillin (0.1 mg/ml) and arabinose (0.2%). The culture was allowed to grow for 12 h before cells were harvested by centrifugation and lysed by French Press. Proteins were purified by Ni-NTA chromatography (Amersham). Cameleon constructs were further purified by gel filtration chromatography using a HiLoad 16/60 Superdex 75 μg column (GE Healthcare). Proteins were dialyzed into 50 mM Tris, pH 7.5.
The oligomeric structure of mTFP variants was determined by gel filtration chromatography on a HiLoad 16/60 Superdex 75 μg gel filtration column. Samples of the dimeric dTomato and the monomeric mCherry proteins (Shaner, 2004) were expressed and purified as described above and used as size standards. The AKTA basic liquid chromatography system (GE Healthcare) can monitor multiple wavelengths simultaneously. Purified TFP variants were mixed with either dTomato or mCherry and their respective elution profiles monitored separately at 450 nm and 550 nm, respectively.
Example 6
Spectoscopy
Absorption spectra were recorded on a DU-800 UV-visible spectrophotometer (Beckman). Quantum yields for TFP variants were measured using fluorescein in 10 mM NaOH as the reference standard (Brannon, 1978). Extinction coefficients were measured by the alkali denaturation method (Shaner, 2004; Ward, 1998). A QuantaMaster spectrofluorometer (Photon Technology International) was used to acquire the emission spectra (excitation at 435 nm) shown in FIG. 8 and FIG. 17. All emission spectra have been corrected for the instrument response. Fluorescence lifetimes were determined on a TimeMaster time-resolved spectrofluorometer (Photon Technology International), which uses the stroboscopic optical boxcar technique (James, 1992). Lifetimes were determined in 10 mM Tris pH 8.0 with 1 mM EDTA and 50 mM NaCl at a protein concentration of 0.1 μM. A nitrogen dye laser at 440 nm was used for excitation, and the emission monochromator was set to either 480 nm (for mECFP and mCerulean) or 490 nm for mTFP1. A 480/40 nm bandpass filter was used on the emission channel to further minimize scattered light. Slits were adjusted as necessary to obtain adequate fluorescent intensity when using polarizing filters at ‘magic angle’ conditions or lower concentrations of fluorescent protein. The photostability of mTFP1 under arc lamp illumination was determined using the method of Shaner et al. (Shaner, 2005).
To determine the pH-dependence of the fluorescence emission of mECFP, mCerulean, and mTFP1, each protein (stock solution of 1 mg/ml in 5 mM Tris pH 7) was diluted 1:100 in a 96-well plate black clear bottom plate (Corning) containing 0.1 ml buffer (100 mM) at pH values ranging from 2 to 9. Full emission spectra at each pH were acquired with a Safire2 plate reader (Tecan). The relative fluorescence at each pH was measured at the peak wavelength.
Example 7
Cameleons and Actin-Fusion for Imaging in Mammalian Cells
To construct expression vectors for cameleon variants, the gene encoding yellow cameleon3.3 (YC3.3) was first inserted into the Xho1 and EcoR1 sites of the pBAD/His B bacterial expression vector (Griesbeck, 2001). The cDNAs encoding mECFP, mCerulean, and mTFP1 were each PCR amplified with primers that added a 5′ Xho1 and a 3′ Sph1 site. To maintain identical linker lengths, the 3′ Sph1 site was appended immediately after the codon encoding Ala227 of mECFP and mCerulean or after the codon encoding the structurally aligned Arg220 of mTFP1. The purified PCR products were digested and ligated into the similarly digested YC3.3 gene. Cameleons expressed in E. Coli were expressed and purified as described herein. To create the mTFP1-YC3.3 mammalian cell expression vector, the full-length gene in pBAD/His B was PCR amplified with a 5′ primer that appended a HindIII restriction site, a Kozak sequence (gccaccgccATGc, where ATG is the start codon (SEQ ID NO: 17), and the endoplasmic reticulum (ER) targeting sequence of calreticulin (MLLSVPLLLGLLGLAAAD) (SEQ ID NO: 18). The 3′ primer appended the ER retention signal (KDEL) followed by an EcoR1 restriction site. The PCR product was digested with HindIII/EcoR1 and ligated with appropriately digested pcDNA3 (Invitrogen).
To create the mTFP1-actin mammalian expression plasmid, the gene encoding mTFP1 was PCR amplified with a 5′ primer encoding an Nhe1 site and a 3′ primer encoding an Xho1 site. The purified and digested PCR product was ligated into the pEGFP-actin vector (Clontech) that had been previously digested with the same restriction enzymes to excise the EGFP coding sequence. DNA was purified by Plasmid Midi kit (Qiagen). HeLa cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS (Sigma) at 37° C. Cells in 35 mm imaging dishes were transfected with 4 μg plasmid DNA mixed with 10 μg PEI in 0.5 ml OptiMEMTM (Invitrogen) and serum was added after 3 h. Approximately 14 h later the medium was exchanged for Hanks' Balanced Salt Solution (HBSS) containing no calcium chloride, magnesium chloride, magnesium sulfate, or phenol red (Invitrogen) or PBS and the cells were imaged. HeLa cells expressing mTFP1-β-actin or mTFP1-α-tubulin were imaged using a LSM510 confocal microscope (Zeiss) equipped with a 5 mW 458 nm excitation laser. HeLa cells expressing mTFP1-YC3.3 were imaged with a Zeiss Axiovert 200M epi-fluorescence inverted microscope equipped with a xenon arc lamp and a monochrome Retiga 2000R 12-bit cooled CCD camera (QImaging). The external excitation filter wheel, excitation shutter, and emission filter wheel are controlled through a Lambda 10-3 controller (Sutter). Only dichroic mirrors are housed in the motorized reflector turret. The QED In Vivo software package (Media Cybernetics) is used for automated computer control of all microscope hardware and for quantitative image analysis. To create the mTFP1-α-tubulin expression vector, an identical procedure was used to replace the EGFP gene in pEGFP-tub (Clontech) with the gene encoding mTFP1.
Example 8
Development of the Variants
The sequence of mutations and changes that led to the variants described herein can be summarized as follows:

    
    
        1. cCFP484: initial wild-type sequence;
        2. dTFP0.10: wild-type cCFP484 (a.k.a. TFP) with additional mutations H42N, L44V, L72F, F124L, D125K, M127K, M150L, S179T;
        
            deleted 1st 40 residues, and appended MVSKGEE (SEQ ID NO: 19) to the start (N-terminus) of the protein;
            appended GMDELYK (SEQ ID NO: 20) to the end (C-terminus) of the protein;
        
        
        3. dTFP0.20: dTFP0.10 with additional mutations D81N, S226P;
        4. dTFP0.30: dTFP0.20 with additional mutations Q66G, K182R;
        5. tdTFP0.30: genetically fused 2 copies of dTFP0.3, in which GMDELYK (SEQ ID NO: 20) was deleted from the C-terminus of the first copy, and MVSKGEE (SEQ ID NO: 19) was deleted from the N-terminus of the second copy;
        
            a linker sequence of TGSTLVSGSGTA (SEQ ID NO: 21) was used;
            the S226P mutation was reverted to P in the first copy;
        
        
        6. mTFP0.3: dTFP0.2 with additional mutations S162K, S164K;
        7. mTFP0.4: mTFP0.3 with additional mutations Q66C, C175V;
        8. mTFP0.5: mTFP0.4 with additional mutation S62T, C66G, A80P, K127E, K182R, N216S
        9. mTFP0.6: mTFP0.4 with additional mutation S2N, G66A, L213V
        
            replaced residues L223 to A228 (sequence LLPSQA (SEQ ID NO: 22)) with sequence TG;
        
        
        10. mTFP0.7: mTFP0.6 with additional mutations N2S (reversion), V44I, R123H, Y173H, V186A;
        11. mTFP0.8: mTFP0.7 with additional mutation N63T
        12. mTFP0.9: mTFP0.8 with additional mutation K142G, E144D, P145A, I149R, L150M (reversion to wild-type sequence), I161V;
        13. mTFP1.0: mTFP0.9 with additional mutation L141T, V158K, Y221N, G224D.
    
    


Discussed below are results obtained by the inventors in connection with the experiments of Examples 2, 3 and 8:
Results of Directed Evolution
Extensive screening of the synthetic gene library resulted in identification of a brightly-fluorescent dimer (quantum yield (QY)=0.73, extinction coefficient (EC)=42,000 M-1 cm-1) with eight mutations relative to the wild-type sequence (FIGS. 18 and 19). Directed evolution was used to evolve the dimer to a substantially-brighter variant (QY=0.89, EC=60,000 M-1 cm-1). Site-directed mutagenesis of the residues in the predicted dimer interface produced a monomeric (QY=0.41, EC=19,000 M-1 cm-1) version that required many rounds of directed evolution in order to rescue and improve the fluorescent brightness. Currently, mTFP0.86 has a QY=0.65 and an EC=59,000 M-1 cm-1; mTFP0.86 is 3.6-fold brighter than avCFP, and 1.4-fold brighter than Cerulean (Rizzo, 2004).
Evaluation of mTFP as a Fret Donor
Analogous versions of yellow cameleon 3.3 (YC3.3), containing either ECFP, Cerulean, or an mTFP-0.6, were constructed, and their response to Ca2+ was determined (Miyawaki et al., Nature, 388:882-87,1997). Cameleon is a Ca2+ reporter that has been optimized to maximize the change in ratio between an ECFP donor and a YFP acceptor. Therefore, it is not surprising that substituting mTFP0.6 for ECFP resulted in a decreased % ratio change. However, the high FRET efficiencies observed with mTFP0.6, in both the presence and absence of Ca2+, showed that it is a very good FRET donor to citrine (FIG. 8 and Table 5).







TABLE 5







Summary of ratio changes and FRET efficiency changes


for “cameleon-type” calcium sensors, based


on either avCFP, Cerulean, or mTFP0.6.











ECFP
Cerulean
mTFP0.6














yellow/
FRET
yellow/
FRET
yellow/
FRET



cyan
effi-
cyan
effi-
cyan
effi-



ratio
ciency
ratio
ciency
ratio
ciency

















−Ca2+
1.48
14%
1.28
13%
0.94
25%


+Ca2+
3.59
37%
2.93
35%
1.79
38%










% ratio
242%
230%
190%


change


Δ FRET
 23%
 22%
 13%


efficiency









Outlook
Both mTFP0.6 (labelled mTFP0.6) and Cerulean can be fitted to a single exponential fluorescence lifetime decay (FIG. 21). The inventors have inserted mTFP0.6 (labelled mTFP0.6) into cameleon-type constructs, and demonstrated that it is an excellent FRET donor to Citrine. It is anticipated that mTFP can be optimized, and that a detailed comparison can be made to ECFP and Cerulean for multicolor imaging, FRET imaging, and FLIM experiments in live cells. mTFP will complement the existing preferred avGFP and YFP variants better than avCFP or Cerulean currently do (FIG. 22).
While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art, from a reading of the disclosure, that various changes in form and detail can be made without departing from the true scope of the invention in the appended claims.
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## Claims

1. An isolated nucleic acid encoding the amino acid sequence of SEQ ID NO: 7.

2. The isolated nucleic acid of claim 1, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 6.

3. The isolated nucleic acid of claim 1 which is compatible with mammalian codon usage.

4. The isolated nucleic acid of claim 3, which is compatible with human codon usage.

5. The isolated nucleic acid of claim 1, wherein the encoded polypeptide has a chromophore capable of fluorescence with an excitation maximum ranging from 450 to 460 nm and an emission maximum ranging from 485 to 495 nm.

6. The isolated nucleic acid of claim 1, wherein the encoded polypeptide is monomeric.

7. A vector comprising the nucleic acid of any one of claims 1, 2, 3, 4, 5 and 6.

8. The vector of claim 7, which is a plasmid.

9. The vector of claim 7, wherein the nucleic acid is cDNA.

10. An isolated host cell comprising the vector of claim 7.