# Patent

## Title
Fluorescent proteins with increased activity in cells

## Abstract

The present invention relates to fluorescent proteins, in particular green fluorescent proteins (GFPs), with increased activity in cells, and thus increased signal strength. A further aspect of the present invention relates to the use of peptides for increasing the expression and/or stability of a protein in a cell.

## Background
CROSS REFERENCE TO A RELATED APPLICATION
This application is a National Stage Application of International Application Number PCT/EP2010/004862, filed Aug. 9, 2010; which is incorporated herein by reference in its entirety.
The present invention relates to fluorescent proteins, in particular green fluorescent proteins (GFPs), with increased activity in cells, and thus increased signal strength. A further aspect of the present invention relates to the use of peptides for increasing the expression and/or stability of a protein in a cell.
Because of its easily detectable green fluorescence, green fluorescent protein (GFP) from the jellyfish Aequorea victoria has been widely used to study gene expression and protein localization. GFP fluorescence does not require a substrate or cofactor; hence, it is possible to use this reporter in a wide variety of applications and cells.
The green fluorescent protein (GFP) is a protein composed of 238 amino acids (26.9 kDa), 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 A. 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 has a typical beta barrel structure, consisting of one β-sheet with alpha helices containing the chromophore running through the centre. Inward facing sidechains of the barrel induce specific cyclization reactions in the tripeptide Ser65-Tyr66-Gly67 that lead to chromophore formation. This process of post-translational modification is referred to as maturation. The hydrogen bonding network and electron stacking interactions with these sidechains influence the colour of wildtype GFP and its numerous derivatives. The tightly packed nature of the barrel excludes solvent molecules, protecting the chromophore fluorescence from quenching by water.
Due to the potential for widespread usage and the evolving needs of researchers, many different mutants of GFP have been engineered (Shaner et al., 2005). The first major improvement was a single point mutation (S65T) reported in 1995. This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostability and a shift of the major excitation peak to 488 nm with the peak emission kept at 509 nm. This matched the spectral characteristics of commonly available FITC filter sets, increasing the practicality of use by the general researcher. A 37° C. folding efficiency (F64L) point mutant yielding enhanced GFP (EGFP) was discovered in 1995 and facilitated the use of GFPs in mammalian cells. Superfolder GFP, based on a series of mutations that allow GFP to rapidly fold and mature even when fused to poorly folding peptides, was reported in 2006. Many other mutations have been made, including colour mutants; in particular blue fluorescent protein, cyan fluorescent protein and yellow fluorescent protein derivatives. BFP derivatives contain the Y66H substitution. The critical mutation in cyan derivatives is the Y66W substitution, which causes the chromophore to form with an indole rather than phenol component. The red-shifted wavelength of the YFP derivatives is accomplished by the T203Y mutation and is due to π-electron stacking interactions between the substituted tyrosine residue and the chromophore.
Semirational mutagenesis of a number of residues led to pH-sensitive mutants known as pHluorins, and later super-ecliptic pHluorins. By exploiting the rapid change in pH upon synaptic vesicle fusion, pHluorins tagged to synaptobrevin have been used to visualize synaptic activity in neurons.
Redox sensitive versions of GFP (roGFP) were engineered by introduction of cysteines into the beta barrel structure. The redox state of the cysteines determines the fluorescent properties of roGFP.
It was an object of the present invention to provide fluorescent proteins, in particular GFPs, with increased activity in cells, and thus increased signal strength.
This object of the present invention is solved by a fluorescent protein having
an amino acid sequence which is at least 82%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, most preferably 100% identical to SEQ ID NO: 3 or

an amino acid sequence which is at least 82%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, most preferably 100% identical to a sequence wherein one to four amino acid residues of SEQ ID NO: 3 are replaced by another amino acid residue, wherein the one to four amino acid residues are selected from the group of S58, F61, Q62 and K158.

In one embodiment, the fluorescent protein is a green fluorescent protein (GFP). However, it is known to a person skilled in the art that by exchanging single amino acids, e.g. in the chromophore region, the color of a fluorescent protein can be changed, e.g. to yellow (YFP), cyan (CFP), blue (BFP) and red (RFP).
In one embodiment, the fluorescent protein has an activity which is increased as compared to the polypeptide having the amino acid sequence of SEQ ID NO: 11 (EGFP). Preferably, the increased activity results in an increased fluorescence intensity of cells containing the fluorescent protein, wherein, preferably, the fluorescence intensity is increased by at least the factor of 1.5, preferably at the least the factor of 2, more preferably at least the factor of 3, as compared to cells containing the polypeptide having the amino acid sequence of SEQ ID NO: 11.
The term “activity” as used herein is meant to refer to the activity of the fluorescent protein in a cell, which is preferably quantified by measuring the fluorescence intensity of cells containing the fluorescent protein. The activity of a fluorescent protein in a cell is dependent on its concentration in the cell and its actual fluorescence, wherein the concentration is dependent, e.g., on the expression level and/or the stability within the cell. Therefore, an “increased activity” of the fluorescent protein according to the present invention may be due to an increased expression and/or an increased stability in the cell (e.g. based on a decreased degradation rate), but also due to increased fluorescence of the protein itself Without wishing to be bound to a certain theory, the inventor believes that the increased activity, and thus increased signal strength of the fluorescent proteins according to the present invention are largely based on their increased expression in cells.
In one embodiment, S58 is replaced with T.
In one embodiment, F61 is replaced with L.
In one embodiment, Q62 is replaced with C.
In one embodiment, K158 is replaced with T.
In one embodiment, the fluorescent protein has an amino acid sequence which is identical to SEQ ID NO: 4. Preferably, the fluorescent protein has an activity which is increased as compared to the polypeptide having the amino acid sequence of SEQ ID NO: 11 (EGFP). Preferably, the increased activity results in an increased fluorescence intensity of cells containing the fluorescent protein, wherein, preferably, the fluorescence intensity is increased by at least the factor of 2, preferably at the least the factor of 3, more preferably at least the factor of 4, as compared to cells containing the polypeptide having the amino acid sequence of SEQ ID NO: 11.
In one embodiment, the fluorescent protein further comprises at its N-terminus or its C-terminus, preferably at its C-terminus, a peptide having an amino acid sequence which is at least 90%, preferably at least 95%, most preferably 100% identical to SEQ ID NO: 5 or to SEQ ID NO: 6 or to a fragment of SEQ ID NO: 6, wherein the fragment comprises at least 25 consecutive amino acids.
Preferably, the fluorescent protein which further comprises the peptide at its N-terminus or its C-terminus, preferably at its C-terminus, has an intracellular half-life of more than 10 hours, preferably of more than 15 hours, more preferably of more than 20 hours.
The term “intracellular half-life” as used herein is meant to refer to the period of time in which half of the fluorescent signal from the fluorescent protein expressed in cells disappears and half remains.
The objects of the present invention are also solved by a nucleic acid molecule coding for a fluorescent protein as defined above.
The term “nucleic acid molecule” as used herein includes DNA, such as cDNA or genomic DNA, and RNA. In a preferred embodiment, the nucleic acid molecule is DNA.
In one embodiment, the number of RNase L cleavage sites (in particular UU and/or UA dinucleotides) is reduced in the nucleic acid molecule in order to increase the expression of the encoded protein in cells, particularly in eukaryotic cells (see PCT/EP2010/000271).
The objects of the present invention are also solved by an expression construct comprising a nucleic acid molecule as defined above.
The term “expression construct” as used herein is meant to refer to an expression active PCR product or an expression vector.
The term “expression active PCR product” as used herein is meant to refer to a PCR product that is generated by PCR amplification using two primers complementary to sequences flanking the DNA sequence of interest, such as a cDNA, an open reading frame, or a gene that is contained in an expression vector, wherein the resulting PCR product contains a promoter, the DNA sequence of interest, and a termination sequence, and allows the expression of the DNA of interest, when transfected to a host cell (see also: Al-Zoghaibi et al., 2007).
Preferably, the expression vector is a plasmid, cosmid, virus, bacteriophage or another vector used conventionally e.g. in genetic engineering.
The objects of the present invention are also solved by a fusion protein comprising a fluorescent protein as defined above.
The objects of the present invention are further solved by a cell or tissue comprising a fluorescent protein as defined above, a nucleic acid molecule as defined above, an expression construct as defined above or a fusion protein as defined above.
The term “cell” as used herein refers to any prokaryotic or eukaryotic cell, wherein eukaryotic cells are preferred. Prokaryotic cells include bacteria of the species Escherichia, Streptomyces, Salmonella or Bacillus. Suitable eukaryotic cells include yeasts, such as Saccharomyces cerevisiae or Pichia pastoris, insect cells, such as Drosophila S2 or Spodoptera Sf9 cells, and mammalian cells. Mammalian cells that could be used include human HeLa, HEK293, Huh-7, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, COS 1, COS 7 and CVI, quail QCI-3 cells, mouse L cells, Bowes melanoma cells and Chinese hamster ovary (CHO) cells. Also within the scope of the present invention are primary mammalian cells or cell lines. Primary cells are cells which are directly obtained from an organism. Suitable primary cells are, for example, mouse embryonic fibroblasts (MEF), mouse primary hepatocytes, cardiomyocytes and neuronal cells as well as mouse muscle stem cells (satellite cells) and stable, immortalized cell lines derived thereof. Appropriate culture media and conditions for the above-described (host) cells are known in the art.
The term “tissue” as used herein refers to a cellular organizational level intermediate between cells and a complete organism. Hence, a tissue is an ensemble of cells, not necessarily identical, but from the same origin, that together carry out a specific function.
The objects of the present invention are also solved by a kit comprising at least one of a fluorescent protein as defined above, a nucleic acid molecule as defined above, an expression construct as defined above, a fusion protein as defined above or a cell as defined above.
The various components of the kit may be packaged in one or more containers such as one or more vials. The vials may, in addition to the components, comprise preservatives or buffers for storage.
Another aspect of the present invention relates to the use of a peptide having an amino acid sequence which is at least 90%, preferably at least 95%, most preferably 100% identical to SEQ ID NO: 5 or to SEQ ID NO: 6 or to a fragment of SEQ ID NO: 6, wherein the fragment comprises at least 25 consecutive amino acids, for increasing the expression and/or stability of a protein, in particular a recombinant protein, in a cell.
In one embodiment, the peptide is fused to the N-terminus or the C-terminus of the protein. Preferably, the peptide is fused to the C-terminus of the protein.
In one embodiment the peptide is further used as a tag, preferably for antibody recognition (“epitope tag”) and/or purification (“affinity tag”) of the protein. Just as commonly used tags (e.g. myc, HA, His), the peptide may be removed by enzymatic cleavage, if a cleavage site, e.g., a specific protease site, is inserted between the peptide and the protein.
In one embodiment, the protein is a reporter protein.
The term “reporter protein” as used herein refers to fluorescent and non-fluorescent reporter proteins including (without being limited to) green fluorescent proteins (GFP), red fluorescence proteins (RFP), yellow fluorescent proteins (YFP), blue and cyan fluorescent proteins (CFP), luciferase, secreted alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT), secreted hormone, secreted cytokine, β-galactosidase, and other fluorescent and bioluminescent proteins.
Another aspect of the present invention relates to a peptide having an amino acid sequence which is at least 90%, preferably at least 95%, most preferably 100% identical to SEQ ID NO: 5.
A further aspect of the present invention relates to a fusion protein comprising a protein to be expressed in a cell and a peptide having an amino acid sequence which is at least 90%, preferably at least 95%, most preferably 100% identical to SEQ ID NO: 5 or to SEQ ID NO: 6 or to a fragment of SEQ ID NO: 6, wherein the fragment comprises at least 25 consecutive amino acids, wherein the peptide is fused to the N-terminus or the C-terminus of the protein to be expressed in a cell. Preferably, the peptide is fused to the C-terminus of the protein to be expressed in a cell.
In one embodiment, the protein to be expressed in a cell is a reporter protein as defined above.
Preferably, the fusion protein has an intracellular half-life which is increased by at least the factor of 1.5, preferably at the least the factor of 2, more preferably at least the factor of 3, as compared to the protein expressed to be in a cell without the peptide at its N-terminus or C-terminus.
In the case of non-fluorescent proteins, the term “intracellular half-life” simply refers to the period of time in which half of the initial amount of the non-fluorescent protein expressed in cells disappears, e.g. due to degradation, and half remains.
A still further aspect of the present invention relates to a nucleic acid molecule coding for a peptide as defined above or for a fusion protein as defined above.
In a further aspect, the present invention relates to a method of increasing the expression and/or stability of a protein, in particular a recombinant protein, to be expressed in a cell, which method comprises the steps of

    
    
        providing a nucleic acid molecule coding for a fusion protein as defined above;
        inserting (i.e. cloning) the nucleic acid molecule into an expression vector; and
        transforming, transfecting or injecting the expression vector into a cell.
    
    


In a further aspect, the present invention relates to a method of increasing the expression and/or stability of a protein, in particular a recombinant protein, to be expressed in a cell, which method comprises the steps of

    
    
        providing an expression active PCR product comprising a nucleic acid molecule coding for a fusion protein as defined above; and
        transfecting or injecting the expression active PCR product into a cell.
    
    


A further aspect of the present invention relates to an expression construct comprising a multiple cloning site followed or preceded by a nucleic acid molecule coding for a peptide having an amino acid sequence which is at least 90%, preferably at least 95%, most preferably 100% identical to SEQ ID NO: 5 or to SEQ ID NO: 6 or to a fragment of SEQ ID NO: 6, wherein the fragment comprises at least consecutive 25 amino acids.
In a further aspect, the present invention relates to a method of increasing the expression and/or stability of a protein, in particular a recombinant protein, to be expressed in a cell, which method comprises the steps of

    
    
        providing a nucleic acid molecule coding for the protein to be expressed in a cell;
        inserting (i.e. cloning) the nucleic acid molecule into an expression construct as defined above; and
        transforming, transfecting or injecting the expression construct into a cell.
    
    


A further aspect of the present invention relates to an antibody against a peptide having an amino acid sequence which is at least 90%, preferably at least 95%, most preferably 100% identical to SEQ ID NO: 5 or to SEQ ID NO: 6 or to a fragment of SEQ ID NO: 6, wherein the fragment comprises at least consecutive 25 amino acids.
The term “antibody” refers to a polypeptide having affinity for a target, antigen or epitope (here: a peptide or part of a peptide as defined above) and includes both naturally-occurring and engineered antibodies. The term “antibody” encompasses polyclonal, monoclonal, human, chimeric, humanized, primatized, veneered, and single chain antibodies, as well as fragments of antibodies (e.g., Fv, Fc, Fd, Fab, Fab′, F(ab′), scFv, scFab, dAb).
A further aspect of the present invention relates to a cell comprising a peptide as defined above, a fusion protein as defined above, a nucleic acid molecule as defined above or an antibody as defined above.
A further aspect of the present invention relates to a kit comprising at least one of a peptide as defined above, a fusion protein as defined above, a nucleic acid molecule as defined above, an expression construct as defined above, an antibody as defined above or a cell as defined above.
The various components of the kit may be packaged in one or more containers such as one or more vials. The vials may, in addition to the components, comprise preservatives or buffers for storage.
As used herein, the term “percent (%) identical” refers to sequence identity between two amino acid sequences. Identity can be determined by comparing a position in both sequences, which may be aligned for the purpose of comparison. When an equivalent position in the compared sequences is occupied by the same amino acid, the molecules are considered to be identical at that position.
Preferably, non-identity (e.g. at most 18%, at most 15%, at most 10%, at most 5%, at most 4%, at most 3%, at most 2% or at most 1%) is based on amino acid exchanges which do not alter the activity or function of the fluorescent protein or peptide as compared to the fluorescent protein having the amino acid sequence of SEQ ID NO: 3 or a sequence wherein one to four amino acid residues of SEQ ID NO: 3 are replaced by another amino acid residue, wherein the one to four amino acid residues are selected from the group of S58, F61, Q62 and K158, or as compared to the peptide having the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6 or a fragment of SEQ ID NO: 6, wherein the fragment comprises at least 25 consecutive amino acids.
Generally, a person skilled in the art is aware of the fact that some amino acid exchanges in the amino acid sequence of a protein or peptide do not have any influence on the function, activity and/or (secondary or tertiary) structure of the protein or peptide at all. Amino acid sequences with such “neutral” amino acid exchanges as compared to the amino acid sequences disclosed herein fall within the scope of the present invention.
The fluorescent proteins according to the present invention show increased activity, and thus increased signal strength in cells. The fluorescent proteins according to the present invention are useful in a wide variety of applications, including the monitoring of gene expression and protein localization.
Furthermore, the inventor has surprisingly found that by fusing a peptide having an amino acid sequence which is at least 90%, preferably at least 95%, most preferably 100% identical to SEQ ID NO: 5 or to SEQ ID NO: 6 or to a fragment of SEQ ID NO: 6, wherein the fragment comprises at least 25 consecutive amino acids, to the N- or C-terminus of fluorescent proteins according to the present invention, their expression and/or stability, and thus their activity in cells is significantly increased. However, this principle is not restricted to the fluorescent proteins of the present invention, but can be used for any other reporter protein or, as a matter of fact, for any other protein to be expressed in a cell. It is especially useful for recombinant proteins, in particular for those, which are regularly hard to express in cells.



FIGURES
FIG. 1 shows the fluorescence intensity of Huh-7 cells two days after transfection with 75 ng of expression plasmids harboring different green fluorescent proteins (GFPs). Fluorescence was assessed from images captured by BD automated Bioimager and quantified using ProXcell algorithm. The values are Mean+/−SEM from four different wells.
FIG. 2 shows the fluorescence intensity of HEK293 cells one day after transfection with 75 ng of PCR products derived from expression plasmids harboring EGFP, conGFP or various conGFP mutants. Fluorescence was assessed from images captured by BD automated Bioimager and quantified using ProXcell algorithm. The values represent Mean+/−SEM from four different wells. *, ** and *** denote <0.01, <0.005 and <0.001, respectively.
FIG. 3 shows the fluorescence intensity of Huh-7 cells one day after transfection with 50 ng of expression plasmids harboring various GFP variants with or without different peptides fused to their C-termini. “PEST” refers to the destabilization domain of the murine MODC1 gene. Fluorescence was assessed from images captured by BD automated Bioimager and quantified using ProXcell algorithm. The values represent Mean+/−SEM from four different wells.
FIG. 4 shows the fluorescence intensity of Huh-7 cells two days after transfection with 50 ng of expression plasmids (grey columns) or 75 ng of PCR products (white columns) harboring various GFP variants with different peptides fused to their C-termini Fluorescence was assessed from images captured by BD automated Bioimager and quantified using ProXcell algorithm. The values represent Mean+/−SEM from four different wells.
FIG. 5 shows luciferase activity in Huh-7 cells two days after transfection with 50 ng of expression plasmids harboring firefly luciferase cDNA or DNA coding for a fusion protein consisting of firefly luciferase and peptide 2 (SEQ ID NO: 10). Luciferase activity was measured in a 96-well standard luminometer. The values represent Mean+/−SEM from four different wells.
FIG. 6 shows the fluorescence intensity of Huh-7 cells one day after transfection with plasmids harboring various GFP variants and 3, 5, and 7 hours after subsequent treatment with cycloheximide (CHX). Fluorescence was assessed from images captured by BD automated Bioimager and quantified using ProXcell algorithm. The values represent Mean+/−SEM from four different wells.



SEQUENCE LISTING
SEQ ID NO: 1 is also referred to as GFPknown and is the consensus amino acid sequence based on an alignment of six GFPs known to have very good signal strength.
SEQ ID NO: 2 is the consensus amino acid sequence based on an alignment of 26 publicly available GenBank records of various GFPs.
SEQ ID NO: 3 is also referred to as conGFP and is a combination of SEQ ID NO: 1 and SEQ ID NO: 2. More specifically, SEQ ID NO: 1 was used to fill in the gaps (Xaa) in SEQ ID NO: 2.
SEQ ID NO: 4 is also referred to as conGFP-SKQ and is the amino acid sequence of conGFP (SEQ ID NO: 3) with the mutations S58T, K158T and Q62C.
SEQ ID NO: 5 is the amino acid sequence of 23-residue artificial peptide 1.
SEQ ID NO: 6 is the amino acid sequence of 33-residue peptide 2, which is derived from Danio rerio neuronal adhesion molecule L12.
SEQ ID NO: 7 is the amino acid sequence of a fusion protein consisting of conGFP-SKQ (SEQ ID NO: 4) and peptide 1 (SEQ ID NO: 5).
SEQ ID NO: 8 is the amino acid sequence of a fusion protein consisting of conGFP-SKQ (SEQ ID NO: 4) and peptide 2 (SEQ ID NO: 6).
SEQ ID NO: 9 is the amino acid sequence of a fusion protein consisting of firefly luciferase and peptide 1 (SEQ ID NO: 5).
SEQ ID NO: 10 is the amino acid sequence of a fusion protein consisting of firefly luciferase and peptide 2 (SEQ ID NO: 6).
SEQ ID NO: 11 is the amino acid sequence of A. victoria enhanced GFP (EGFP).
EXAMPLES
1. Materials & Methods
1.1 Plasmids
The coding regions of putative GPFs and their variants were first optimized using UU/UA dinucleotide frequency reduction approach as previously described (PCT/EP2010/000271), then submitted for custom gene synthesis. The coding regions were subcloned into an expression vector under the control of CMV promoter by using SalI and BamHI restriction.
1.2 Cell Lines
HEK293 cell line was obtained from American Type Culture Collection (ATCC; Rockville, Md.) and cultured in DMEM medium (Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS and antibiotics. Huh-7 cell line was also propagated in DMEM medium with 10% FBS and antibiotics.
1.3 Reporter Transfection and Reporter Activity Assessment
Cells in 96-well clear bottom black microplates were transfected with 50-100 ng of the reporter vectors using lipofectamine 2000 reagent (Invitrogen). All transfections were performed in several replicates as indicated in the figure legends. The variance in GFP fluorescence among replicate microwells was <6%; thus, with this minimum variance, experiments do not warrant transfection normalization (Al-Zoghaibi et al., 2007). Automated laser-focus image capturing was performed using the high-throughput BD Pathway 435 imager (BD Biosciences, San Jose, Calif.). A wavelength of 482 nm was used for excitation. Image processing, segmentation, and fluorescence quantification was facilitated by ProXcell program and was previously described (al-Haj et al., 2009). Data are presented as mean values±standard error (SEM) of total fluorescence intensity in each well with replicate readings ranging from three to four as indicated in the text. Student's t-test was used when comparing two data groups while analysis of variance (ANOVA) was performed for each data set having three or more data groups.
2. Results
By alignment of the amino acid sequences of six different GFPs (from the organisms Aequorea victoria, Pontellina plumata, Pitlosarcus, Montastrea cavernosa, Renilla mullerei, Clavulariidae clavularia), which are known to have very good signal strength, a consensus amino acid sequence GFPknown (SEQ ID NO: 1) was obtained. Cells transfected with a plasmid harboring chemically synthesized DNA coding for the protein of SEQ ID NO: 1 showed no fluorescence (see FIG. 1).
An alignment of 26 publicly available GenBank records of various GFPs resulted in another consensus amino acid sequence (SEQ ID NO: 2), which, after transfection of cells with its chemically synthesized DNA, showed very poor fluorescence (data not shown).
The consensus sequence GFPknown (SEQ ID NO: 1) was used to fill in the gaps (denoted as Xaa) in SEQ ID NO: 2. The combination of SEQ ID NO:1 and SEQ ID NO: 2 resulted in conGFP (SEQ ID NO: 3). A BLAST search with the amino acid sequence of conGFP only revealed a few hits with less than 82% sequence identity, which included wildtype and artificial GFPs.
Cells transfected with conGFP-DNA showed lower fluorescence intensity than some individual wildtype GFPs (Montastrea cavernosa, Puntellina plumate), but it was significantly (i.e. at least 2-fold) higher than those of A. victoria GFPs, including EGFP (FIG. 1).
Next, DNA coding for several mutants of conGFP was synthesized and tested in HEK293 cells. The mutants included S58T, F61L, Q62C and K158T. Combinations of these mutants resulted in further increased fluorescence intensity, which was in all cases higher than that of EGFP (see FIG. 2). The triple mutant conGFP S58T K158T Q62C (also referred to as conGFP-SKQ, SEQ ID NO: 4) represented the best fluorescent protein with a fluorescence intensity which was about 5× higher than that of conGFP and about 10× higher than that of EGFP.
The performance of fluorescent proteins according to the present invention was further improved by adding peptide 1 (SEQ ID NO: 5), peptide 2 (SEQ ID NO: 6) or a fragment of peptide 2 with at least 25 consecutive amino acids to the N-terminus or the C-terminus of the proteins, more particular, a DNA sequence coding for one of these peptides was added in frame to the 5′-end or the 3′-end of the DNA coding for the fluorescent proteins.
Peptide 1 represents a consensus sequence of PEST-containing amino acid fragments of highly unstable genes. A PEST sequence is a peptide sequence which is rich in proline (P), glutamic acid (E), serine (S), and threonine (T). This sequence is associated with proteins that have a short intracellular half-life; hence, it is hypothesized that the PEST sequence acts as a signal peptide for protein degradation. Surprisingly, the addition of peptide 1 to the C-terminus of conGFP-SKQ (conGFP-SKQ-1) resulted in a dramatically increased fluorescence intensity of Huh-7 cells containing this fluorescent protein (see FIG. 3).
Similarly, the addition of peptide 2, which is derived from the Danio rerio (zebrafish) neuronal adhesion molecule L12 and comprises a PEST-like domain, to the C-terminus of conGFP-SKQ (conGFP-SKQ-2) lead to a significantly increased fluorescence intensity (FIG. 3).
The effects observed when peptide 1 or 2 are added to the N-terminus or the C-terminus of fluorescent proteins according to the present invention are not limited to these specific proteins. As shown in FIG. 4, the fusion of the peptides to commercially available GFP (“Monster GFP”) also increased the fluorescence intensity as compared to Huh-7 cells containing “wildtype” Monster GFP.
Moreover, the peptides (here: peptide 2) increased the activity of firefly luciferase in Huh-7 cells (FIG. 5).
In order to determine the intracellular stability of various GFP variants, Huh-7 cells were transfected with plasmids expressing “wildtype” EGFP, EGFP with the murine MODC1 PEST domain fused to its C-terminus (EGFP-PESTmodc), conGFP-SKQ, conGFP-SKQ-P1 and conGFP-SKQ-P2 (see FIG. 6). After 24 hours cells were treated with the protein synthesis inhibitor cycloheximide (CHX). Because of the non-specific toxicity of CHX experiments could not be extended to more than 6-8 hours. Both EGFP and EGFP-PESTmodc decayed at a significant rate. After 7 hours, there were only 51% (EGFP) and 44% (EGFP-PESTmodc) of the fluorescence activity remaining. Thus, the estimated intracellular half-life of the proteins was between three and four hours. In the case of conGFP-SQK, 87% of the fluorescence activity was remaining after 7 hours, while conGFP-SQK-P1 and -P2 (i.e. fusion proteins comprising conGFP-SQK and peptide 1 or 2 according to the present invention) were even more stable, with approximately 90% remaining after 7 hours. The half-life of conGFP-SQK appears to be more than 10 hours, more likely more than 20 hours, and the half-life of conGFP-SQK-P1 and -P2 is clearly more than 20 hours, possibly more than 24 hours. Thus, although peptides 1 and 2 of the present invention contain a PEST motif, there are no destabilization effects observed (in contrast to the murine MODC1 PEST domain; see FIG. 6).
Surprisingly, the peptides according to the present invention appear to increase the expression and/or intracellular stability of the proteins to which they are fused. An increased intracellular stability (or intracellular half-life) may be based on a decreased degradation rate of the fusion proteins.
REFERENCES


    Al-Haj, L., Al-Ahmadi, W. Al-Saif, M. Demirkaya, O., and K. S. A. Khabar. 2009. Cloning-Free Regulated Monitoring of Reporter and Gene Expression. BMC Molecular Biology 10:20.
    Al-Zoghaibi, F., T. Ashour, W. Al-Ahmadi, H. Abulleef, O. Demirkaya, and K. S. A. Khabar. 2007. Bioinformatics and experimental derivation of an efficient hybrid 3′ untranslated region and use in expression active linear DNA with minimum poly(A) regions. Gene 391: 130-139.
    Shaner, N., Steinbach, P., Tsien, R. 2005. A guide to choosing fluorescent proteins. Nat Methods 2 (12): 905-9.

## Claims

1. A fluorescent protein having:
an amino acid sequence that is at least 90%-identical to SEQ ID NO: 3 or
an amino acid sequence that is at least 90% identical to a sequence wherein one to four amino acid residues of SEQ ID NO: 3 are replaced by another amino acid residue, wherein the one to four amino acid residues are selected from the group consisting of S58, F61, Q62 and K158.

2. The fluorescent protein according to claim 1, wherein S58 is replaced with T.

3. The fluorescent protein according to claim 1, wherein F61 is replaced with L.

4. The fluorescent protein according to claim 1, wherein Q62 is replaced with C.

5. The fluorescent protein according to claim 1, wherein K158 is replaced with T.

6. The fluorescent protein according to claim 1 having an amino acid sequence which is identical to SEQ ID NO: 4.

7. The fluorescent protein according to claim 1 further comprising, at its N-terminus or its C-terminus, a peptide having an amino acid sequence that is at least 90% identical to SEQ ID NO: 5 or to SEQ ID NO: 6 or to a fragment of SEQ ID NO: 6, wherein the fragment comprises at least 25 consecutive amino acids.

8. A cDNA acid molecule encoding a fluorescent protein according to claim 1.

9. An expression construct comprising a cDNA molecule according to claim 8.

10. A fusion protein comprising a fluorescent protein according to claim 1.

11. An in vitro cell or tissue comprising a fluorescent protein according to claim 1, a cDNA molecule encoding a fluorescent protein according to claim 1, or a fusion protein comprising a fluorescent protein according to claim 1.

12. A kit comprising at least one of a fluorescent protein according to claim 1, a cDNA molecule encoding a fluorescent protein according to claim 1, or a fusion protein comprising a fluorescent protein according to claim 1.