NL1012782C2 - New variant of the green fluorescent protein gene, useful for expressing labels, particularly for genetic or expression labeling, has e.g. better solubility or shifted excitation spectrum - Google Patents

Abstract

Variant (I) of the green fluorescent protein (gfp) gene, and nucleic acid constructs containing it, also their equivalents or analogs, and their fragments, having at least one mutation that encodes a protein with better solubility, a red or blue shift in the excitation spectrum, better dual-excitation properties or stronger fluorescence, relative to wild-type green fluorescent protein (GFP). Variant (I) of the gfp gene, and nucleic acid constructs containing it, also their equivalents or analogs, and their fragments, having at least one mutation that encodes a protein with: (i) better solubility; (ii) a red or blue shift in the excitation spectrum; (iii) better dual-excitation properties; (iv) stronger fluorescence relative to wild-type green fluorescent protein (GFP). The mutations are in codons encoding: (i) amino acids (aa) 99, 153 and/or 163 (improved solubility); (ii) aa 65, 66 and/or 67 (red/blue shift); (iii) aa 163, 167 and/or 175 (dual excitation) and (iv) aa 64 (stronger fluorescence. Independent claims are also included for the following: (1) variants (II) of GFP encoded by, or produced by expression of, (I); and (2) organism (plant, animal, human, alga, yeast, bacterium or other microorganism), or derived cells or cell lines, that contain (I) and/or express (II).

Description

Improved gfp variant, usable as a marker, and g / p construct.

The present application relates to a variant of the gfp gene, which can be used as a label or marker in (micro) biological, biochemical and / or molecular biological techniques, in particular as a genetic marker and / or expression marker in vivo.

The application further relates to a nucleic acid sequence or genetic construct encoding the improved g / p variant of the application.

The application further relates to the fluorescent protein encoded by this variant of the gfp gene, as well as the use of the gfp variant according to the invention and / or the protein encoded thereby as a label or marker.

The wild-type gfp gene, the protein encoded thereby, as well as its use as a label or marker, are known, inter alia, from the prior art mentioned below. The wild-type gfp gene, which comes from the jellyfish Aequorea victoria, is about 720 bp in size and encodes a fluorescent protein ("Green Fluorescent Protein" or "GFP" of about 238 amino acids.

A number of variants of the wild-type gfp gene and GFP protein and their use as a marker or label have also been described in the prior art, for which reference is made to the prior art listed in Table 1, as well as, for example, WO 95 / 07463, WO 95/21191, WO 96/23810, WO 96/23898, WO 96/27675, WO

97/11094, WO 97/20078, WO 97/41228, WO 97/42320, WO 98/06737, WO 98/21355, WO 98/36081, US-A-5,491,084, US-A-5,625,048, US-A- 5,741,668, US-A-5,777,079, US-A-5,804,387 and US-A-5,811,238.

The object of the invention is to provide an improved GFP variant. More specifically, the object of the invention is to provide an improved gfp variant, which - compared to both the wild-type gfp and the known variants thereof - exhibits a combination of the following properties: improved solubility of the encoded protein, with especially in biological fluids or media such as, for example, present in the (living) cells in which the mutant gfp is expressed; enhanced fluorescence of the encoded protein; 1012782 2 shifting the (main) excitation peak from / for the encoded protein to the red or the blue end of the electromagnetic spectrum; (amplified) double excitation peak of the encoded protein; and / or 5 - improved compatibility of the gene or protein with (heterologous) biological systems in vivo, such as improved expression.

The invention therefore provides in a first aspect a variant of the gfp gene, or a nucleic acid sequence encoding it, which variant comprises, relative to the wild-type sequence: 10 - at least one mutation in at least one of the codons encoding amino acid positions 99,153 and / or 163 such that the protein encoded by the variant gene exhibits improved solubility compared to the wild type GFP; at least one mutation in at least one of the codons encoding 15 amino acid positions 65, 66 and / or 67 such that the excitation spectrum of the protein encoded by the variant gene is shifted towards the red or blue end of the spectrum, compared to the wild type GFP; at least one mutation in at least one of the codons encoding amino acid positions 163,167 and / or 175 such that the protein encoded by the variant gene 20 exhibits improved dual excitation characteristics compared to the wild type GFP; and preferably also at least one mutation in the codon encoding amino acid position 64 such that the protein encoded by the variant gene exhibits enhanced fluorescence compared to the wild type GFP.

Preferably, the mutations at positions 65, 66 and / or 67 are further such that the encoded protein also exhibits enhanced fluorescence (i.e., emission) compared to the wild type GFP, especially at wavelengths between 450 and 550 nm.

In a further aspect, the invention provides a nucleic acid sequence or genetic construct encoding the improved gfp variant of the invention.

This sequence / construct can be in the form of a DNA or RNA sequence and is preferably in the form of a double-stranded DNA. For example, the sequence / 1012782 3 construct may be in the form of a synthetic DNA - obtained, for example, as described below - which may or may not be integrated into the (genomic) DNA of a host organism; in a form that can independently maintain and / or replicate in a host organism; and / or in a form that can be stably transferred from one host organism to another (eg, by conjugation or at a cross to a next generation), such as a vector or a plasmid. The sequence / construct may further be in a form suitable for transformation of an intended host organism, such as a plasmid or other transformation vector.

The nucleic acid sequence / construct of the invention may further comprise other known nucleic acid sequence or genetic construct elements, such as promoters or other regulatory elements, terminators, transcriptional and / or translational enhancers, integration factors, signal sequences and the like, as well as sequences encoding other proteins or genetically determined properties, optionally as a fusion with the gfp variant according to the invention, as further described below.

The invention further relates to the use of the variant according to the invention and / or the fluorescent protein encoded by this variant as a label or marker, in particular as a genetic marker or expression marker in vivo. Typically, this use includes at least a step of transforming a host organism or a cell, part or tissue thereof with a nucleic acid sequence or genetic construct encoding a gfp variant of the invention; and / or at least one step in which a gfp variant of the invention is expressed in a host organism, or a cell, part or tissue thereof.

Further aspects and preferred embodiments of the invention will become apparent from the description below.

The gfp variants of the invention preferably comprise a combination of the following mutations, relative to the wild-type sequence: at least one mutation in each of the codons encoding amino acid positions 99,153 and / or 163, such that encoded protein exhibits improved solubility compared to the wild type GFP; at least one mutation in at least one of the codons encoding amino acid positions 65,66 and / or 67, and preferably in the codon encoding amino acid position 65, such that the excitation spectrum of the encoded protein 1012782 4 is shifted to the red end of the spectrum, compared to the wild type GFP; at least one mutation in each of the codons encoding amino acid positions 163,167 and / or 175 such that the encoded protein exhibits improved dual excitation characteristics compared to the wild type GFP; and at least one mutation in the codon encoding amino acid position 64 such that the encoded protein exhibits enhanced fluorescence compared to the wild type GFP.

The mutations at positions 65, 66 and / or 67 are again preferably such that the encoded protein also exhibits enhanced fluorescence (ie emission) compared to the wild type GFP, especially at wavelengths between 450 and 550 nm.

The above mutations may comprise one or more insertions, deletions and / or substitutions of one or more nucleotides in / of the codons encoding the above amino acid positions such that the mutant sequence thus obtained encodes a GFP variant with an altered or inserted amino acid at said positions, and / or wherein one or more amino acids have been removed at these position (s), always relative to the amino acid sequence of the wild type GFP; or? 0 'and combination of one or more such mutations. The mutations include at. choose one or more substitions such that the mutant codon encodes another amino acid present at the appropriate position in the wild-type GFP.

Preferred amino acid substitutions are shown in Table 2 101 2782 below. TABLE 2

Amino acid position Wild-type Variant according to the invention

64 F L or M

65 S C, T, G or A

66 67

99 F S

153 M Great A

163 V A.

167 I T

175 S G

Particularly preferred is 65 S to T, more particularly in combination with 64 F to L, which can give considerably enhanced fluorescence. Furthermore, 153 M to A is also preferred. A preferred example of this preferred embodiment is listed in Table 1.

Those skilled in the art will be able to choose - based on the genetic code and its known degeneration, as well as the present description - codons encoding these and other suitable amino acids / amino acid substitutions useful in the invention. Whether these substitutions lead to the desired improved properties can be determined, for example, by expressing the encoded protein and comparing its properties to the wild-type GFP and the known GFP variants, according to methods known per se.

The mutations at positions 99, 153 and / or 163 according to the invention result in improved solubility of the mutant GFP in biological fluids or media, in particular as occurring in the (living) cells in which the mutant GFP is expressed, such as the cytosol fraction. For this, the solubility in water, in a physiological buffer or solution, or an aqueous cell extract can be taken as a measure 101 2782 6, for example in a (comparative) in vitro test. The invention is not limited to an explanation of this improved solubility; for example, this can be caused by the presence of amino acids with hydrophilic (er) side chains, or by a change in the tertiary structure of the protein, such as an altered folding / three-dimensional structure.

The mutations at positions 163, 167 and 175 of the invention result in improved "dual excitation" characteristics of the protein. What is meant by this is shown in Figure 1, which shows the excitation and emission spectra of the wild type GFP, of some GFP variants of the prior art and of a GFP variant according to a preferred embodiment of the invention.

As shown in Figure 1, the wild-type GFP exit spectrum includes a major peak, indicated by (I), and a secondary peak, indicated by (II). Improved "double excitation" according to the invention means that - as also shown in Figure 1 peak (II) in the GFP variants according to the invention is enhanced relative to peak (I), i.e. in absolute sense or relatively

This allows the GFP variant according to the invention to be irradiated at two wavelength ranges, i.e. at a wavelength corresponding to peak (I) and / or a wavelength corresponding to peak (II), where the wild-type GFP mainly at peak (I) must be irradiated (as shown in Figure 1, peak II 20 in wild-type GFP is negligible, so that irradiation at a corresponding wavelength will generally make little sense).

The possibility of (also) irradiating peak (II) in the GFP variant may be useful, as it allows the occurrence of autofluorescence (ie fluorescence of components of the biological sample to be measured other than the GFP, which may occur in particular at wavelengths corresponding to peak (I)) can be avoided. Such autofluorescence may be particularly problematic when wild-type GFP or known GFP variants are used as an in vivo marker in eukaryotic tissue or eukaryotic cells, such as plant cells; the GFP variants according to the invention are therefore particularly suitable for this application.

Moreover, as shown in Figure 1, in the preferred GFP variants according to the invention, peak (II) will have a greater (relative) intensity 101 2782 7 than peak (I), which is a further reason why irradiation of these GFP variants at peak (II) rather than at peak (I) may be preferable.

According to a preferred embodiment of the invention, the GFP variant according to the invention has an excitation / emission spectrum as shown in Figure 1, 5 ie with a first excitation peak (peak (I)) between about 355 and 435 nm (with a maximum of about 395 nm) , a second excitation peak (peak (II)) between 450 nm and 520 nm (with a maximum of about 490 nm), and an emission peak between about 470 nm and 550 nm (with a maximum of about 510 nm). Here, the 2nd excitation peak is enhanced relative to the wild type GFP (i.e., plotted as relative intensity 10 as in Figure 1), while the emission is also boosted relative to the wild type (plotted again as relative intensity). In addition, the GFP variants according to the invention additionally (compared to, for example, the known "eGFP") also exhibit an excitation peak between about 355 and 435 nm (i.e. "double exitation").

More preferably, in the GFP variants of the invention, "peak (II)" is at least the same size as "peak (I)", and preferably at least 1.5 times greater, and more preferably at least 2 times so large, where the "magnitude" of the peaks indicates the (relative) intensity of the maxima in the exit spectrum (ie at the wavelengths of the respective maxima, which may be different for the different GFP variants).

The g #? Variant according to the invention preferably comprises one or more substitutions in amino acids 65,66 and / or 67 - which encode the chromophore - such that the exit spectrum of the expressed GFP variant has shifted towards the red part of the spectrum (so-called "red-shift"), compared to the wild-type GFP. Preferably, a single substitution at amino acid position 65 is used for this.

Furthermore, according to the invention, an amino acid substitution at position 66 - whether or not in combination with substitutions at position 65 and / or 67 - can optionally also be used, which may lead to shifting of the exit spectrum towards the blue part of the spectrum ("blue shift"). "), whereby a so-called" BFP "or" Blue Fluorescent Protein "can be obtained. For this, mutations 66 Y to H and 68 V to I can be mentioned, for example in combination with 64 F to M.

Another mutation that can be named for amino acid 66 is Y to W.

1012782 8

By "shifting the exit spectrum to the red or blue end of the spectrum" is meant here that the main wavelength (s) at which the mutant GFP according to the invention can be irradiated lies at a longer wavelength (in nm) (shift towards the red end) or at a shorter wavelength (shift towards the blue end), relative to the spectrum of the wild type. In this case, a shift of 10 nm, preferably 15-25 nm, can give a significant improvement in the (usage) properties of the variant, for example because the wavelength (s) to be used for the mutation excitation are better aligned with the commonly used (measurement equipment. For example, the wild-type GFP exit spectrum has an optimum at about 395 nm, while this is the optimum for a mutant (red shift mutant) according to the invention at the red end of the spectrum at about 490 nm. nm, which corresponds better to the wavelength of 488 nm which is widely used in measuring equipment Such a small shift in the excitation spectrum, in particular towards longer wavelength (s), can also lead to the biological sample to be measured less autofluorescence and / or light scattering.

The aforementioned shift can be obtained both by the fact that the entire exit spectrum is "shifted" in wavelength compared to the wild type, or only one peak of this, optionally including a simultaneous enhancement of this peak (notably through the improved "double exitation") can make a contribution. For example, this is shown for a "red shift" mutant according to the invention in Figure 1, where peak (I) has the same maximum as the wild type GFP, but peak (II) has both shifted to a longer wavelength (by about 15-20 nm), if also enhanced, relative to the wild type.

Wavelengths that can be used for the excitation of the GFP mutants according to the invention are shown in Table 3. The maximums are always stated, with a range of + 40 nm, preferably + 20 nm, more preferably + 10 nm. is maintained in relation to the stated maximum. A wavelength in brackets indicates a minor peak, while a mutant with "double excitation" properties lists the maximums for both peaks. The wild type is also listed for comparison.

1012782 9 TABLE 3 GFP maximum wild type 395 (470) ls .: "red shift" 395 and 490 ls: "blue shift" 380

Furthermore, the fluorescence can be enhanced by the much more preferred further substitution at position 64, for example F to M or preferably L.

In addition to the above mutations that lead to shifts in the excitation spectrum, the g (p variant according to the invention may comprise one or more mutations that lead to a change in the emission spectrum, ie in the "color" of the protein. a so-called "YFP" or "yellow fluorescent 10 protein" is obtained, ie a GFP variant radiating with a "green-yellow" color, instead of the "green" color of the wild type GFP. For this, mutations can be mentioned such as (expressed as the encoded amino acid) 65 S to G, 68 V to L, 72 S to A, and / or 203 T to Y or I, or an appropriate combination thereof.

In some GFP variants, a change in the emission spectrum can also be accompanied by a change in the excitation spectrum. For example, the combination of 65 S to G, 68 V to L and 72 S to A can lead to a variant with a redshift in both the excitation and emission spectrum, while the combination of 64 F to M, 66 Y to H or W and 68 V to I can lead to a variant with a blue shift in both spectra. YFPs can also display - in addition to the change of the emitted “color” - a shift in (one of) the excitation peak (s), such as a redshift.

The other codons in the sequence of the g / p variant according to the invention are preferably such that they encode the corresponding amino acids of the wild-type GFP, or the known variants thereof. For this purpose, these codons may be the same as in the wild-type g / p sequence or known variants, or codons equivalent to it by the known degeneracy of the genetic code. For example, the g / p variant of the invention may be encoded by a sequence or construct (herein referred to as "equivalent sequences") which in the entirety or in one or more parts thereof consists of codons / nucleotides that differ of the wild-type sequence, but which encode the same amino acids, such that the sequence as a whole encodes the amino acid sequence of the wild-type GFP with the mutations indicated above that define the gfp variant of the invention.

For example, a fully or partially synthetic sequence can be used, which has an increased or decreased, and preferably decreased, A-T content compared to the wild type g / p sequence. The use of such a synthetic, semi-synthetic or partially synthetic sequence may lead to improved expression in, and / or improved compatibility with, the intended host organism, as discussed further below.

In addition, the g / p variant of the invention may also include one or more mutations in the codons corresponding to amino acid positions other than 64.65, 99, 153, 163, 167 and 175, ie in those amino acids that are in the GFP variants according to the invention may be the same as in the wild type. These mutations may include one or more insertions (such as an insertion of valine or alanine between amino acid position 1 and amino acid position 2 in the wild type sequence), deletions and / or substitutions of one or more nucleotides, or a combination thereof, such that the resulting mutant sequence (referred to herein as "analogous sequence") encodes a GFP variant with one or more altered, one or more inserted, and / or one or more deleted amino acids, always relative to the amino acid sequence of the wild-type GFP; or a combination of one or more such mutations.

Here, these mutations are preferably such that the sequence thus obtained still encodes a fluorescent GFP-like protein. More preferably, the 25 g / p variant according to the invention is such that at least 50%, preferably at least 70%, more preferably at least 90% of the amino acids present correspond to the amino acids corresponding to the wild-type GFP (disregarding amino acid positions 64, 65.99, 153, 163, 167 and 175, and a deletion and / or insertion of one amino acid is considered a single mutation).

A number of amino acid substitutions known per se - compared to the wild type GFP - that can be used - alone or in combination - in the invention are shown in Table 4.

101 2782 11 TABLE 4

Amino Acid Wild-type Mutation

2 S G

80 Q R

123 I V

145 Y F or H

146 N I

148 H R

202 S F

203 T I or Y

212 N K.

231 H L

238 K E, N or Q

Other possible known per se, but less preferred substitutions are described, for example, in WO 95/21191, WO 96/23810, WO 96/27675, WO 97/11094, WO 98/06737, WO 98/21355, WO 98 / 36081, US-A-5,625,048, US-A-5,741,668, US-A-5,777,079.

The invention further comprises fragments of the variant sequences according to the invention, ie nucleic acid sequences and / or constructs encoding one or more contiguous or non-contiguous parts of the GFP variants according to the invention. However, these fragments are preferably sized to still encode at least preferably at least 80%, more preferably at least 90% of the amino acids of the GFP variant of the invention, and still at least mutations of the invention described above contain, ie at positions 64-67.99,153,163,167,175 of the corresponding "complete" GFP variant of the invention.

In particular, such fragments include variants having one or more contiguous codons removed from the 3 'and / or 5' end such that in the encoded protein one or more of amino acids 1-20 and / or 218-238 101 2782 12 have been removed, compared to the widl type sequence. Preferably, only one or more of amino acids 1-7 and / or 229-238, more preferably only one or more of amino acids 1-2 and / or 232-238, are removed.

A preferred nucleic acid sequence and amino acid sequence encoding the variant of the invention are shown in SEQ ID NO.1 and SEQ ID NO, respectively. 2. As mentioned, the invention also includes equivalent nucleic acid sequences encoding the same amino acid sequence, and nucleic acid sequences with and or more mutations outside amino acid positions 64-67, 99, 153, 163, 167, 175 in SEQ ID 1 and / or 2, as well as fragments of all these sequences.

An example of a vector preferably used according to the invention is shown in Figure 2.

The (nucleic acid sequences and constructs encoding the) variants of the invention can be obtained in a manner known per se, such as by synthesis with an automated nucleic acid synthesizer; By applying (point) mutations in a nucleic acid encoding a wild-type gfp and / or one of the known g / p variants; and / or by suitably combining parts of the nucleic acid sequences encoding the wild-type gfp gene and the known gfp variants, that is, using one or more restriction enzymes and ligases. These and other suitable techniques are described in Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory (1989) or F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987), as well as, for example, WO 95/21191, WO 96/23810, WO 96/27675, WO 97/11094, WO 98/06737, WO 98/21355, WO 98/36081, US-A-5,625,048, US-A-5,741,668 and US-A-5,777,079.

However, the gfp variants of the invention are preferably prepared by one or more PCR reactions, the desired mutations using modified primers (ie, with an appropriate "mismatch" to the template ("template") used). starting sequence (s)) are incorporated into the nucleic acid sequences obtained by amplification at the above-mentioned positions. Here, as explained in more detail in the Experimental Section, one or more separate PCR reactions are generally carried out, each of which leads to a part of the total sequence of the g / p variant according to the invention, or leads to the entire sequence. , 1012782 13 wherein the primers with the desired mutation (s) will form the ends of the amplified fragments obtained. These fragments are then combined, optionally with one or more parts of the wild-type g-1 sequence or of the sequence (s) of known gjp variants, whereby the complete g / p variant according to the invention is obtained.

The PCR reactions and the further operations, such as combining the different fragments, can be performed in a manner known per se, for example as described below or with the techniques of US-A-4,683,202; Saiki et al., Science 239 (1988), 487-491 or PCR Protocols, 1990, Academic Press, San 10 Diego, California, USA. For the starting sequence (s) used as template in the PCR reaction, one or more suitable fragments of the wild-type gfp sequence can be used, and / or one or more fragments of known gfp variants, which optionally already contain one or more of the desired mutations.

The nucleic acid sequence of the g #? Variant of the invention is preferably such that it is compatible with the cells, tissue or organism in which it is to be expressed. In particular, this means that - after expression of the gene - a fluorescent protein is formed / obtained, preferably without toxic or immune reactions.

For example, it is known that expression of the wild-type g #? Sequence in plants such as Arabidopsis can lead to a partially or even completely disrupted formation of the fluorescent protein. This is believed to be caused by Arabidopsis "recognizing" parts of the wild-type sequence after transcription as intron sequences, which are then broken down. This can be overcome by using a variant sequence with one or more altered codons which are not recognized by the host organism as (part of an) intron sequence relative to the wild-type gene sequence. Examples of suitable modified codoris are given in the prior art listed in Table 1, and include, for example, the use of modified codons at base positions 237-447,237-540. An entirely synthetic sequence with completely modified codons relative to the wild-type sequence can also be used. For example, G #? Variants of the invention with such modified codons can be obtained by using one or more corresponding sequences as starting sequence (s) during the PCR reaction, as described above.

101 2782 14

The fluorescent proteins encoded by the g / p variants can then be obtained by expressing the sequence thus obtained in a suitable host organism, preferably in a microorganism such as a bacterium, and the GFP variant on a known per se manner, for example, for which reference is made to WO 95/07463 and WO 96/23810.

The g / p variant according to the invention and the fluorescent protein encoded thereby can be used in the same manner and for the same applications as the wild-type gfp gene and protein and the already known variants thereof, i.e. as a label or marking in (micro) biological, biochemical and / or molecular biological techniques. For example, the protein encoded by the g / p variants of the invention can be used as a fluorescent marker or label in in vitro essays and assays, including hybridization essays and immunological assays (such as ELISAs) and the like.

However, the g / p variant and the protein encoded by it are particularly suitable as a marker and / or label for use in vivo, ie in eukaryotic or prokaryotic organisms such as plants, animals, humans, bacteria, fungi, algae, yeast and other micro-organisms, as well as in tissues or cells of these organisms or cell lines derived from them.

For example, the g / p variant according to the invention can be used as a genetic marker, ie to indicate that one or more nucleic acid sequences, genes and / or genetically determined properties have been transferred in or to an intended organism. Examples are the transformation of prokaryotic or eukaryotic cells or organisms (in which the transformation vector used or not will also comprise the g / p variant according to the invention in addition to the desired sequence (s)), or the transmission of one or more properties in a cross from the parent generation to the progeny (where the sequences for gfp variant and the desired trait will be related such that the presence of the g1 variant in the progeny is indicative of obtaining the desired trait).

The g #? Variant of the invention may further be used as a "reporter" to indicate that a desired sequence, gene or property is expressed in an organism, tissue, or cell, wherein the expression of the g / p variant and the desired sequence / gene / trait will be under control of the same control elements, or under control of control elements induced by the same factor (s).

Another possible application is expression analysis, such as research into the action of control elements such as promoters and enhancers, for example for monitoring / determining expression over time or under the influence of inducing factors (including harmful factors such as diseases, stress, heavy metals or drought), and / or the expression in different tissues or organs or in different stages of development of an organism.

Furthermore, the gfp variant can, for example, be used for location analysis, in particular to determine which parts or tissues of the plant, which cells or which parts / organelles of the cell a specific gene product or expression product is targeted (eg at due to the presence of a certain leader sequence). As a rule, this product will be expressed as a fusion with the g / - variant according to the invention. Another example of such location analysis includes, for example, tracking over time the spread of a virus or similar pathogen through a plant.

For further possible applications of the mutant gfp according to the invention reference is made to the above literature, in particular WO 95/07463, WO 97/11094, WO 97/42320, WO 98/06737 and in particular the applications in plants described in WO 97/41228.

The variant according to the invention can be introduced into these organisms in the form of a suitable construct or a suitable vector with which this organism can be transformed, including known transformation vectors such as plasmids.

Examples of suitable techniques for transforming plants are, for example, transformation with Agrobacterium, or transformation with "naked" DNA, for example by particle bombardment or by protoplast transformation by, for example, electroporation or treatment with PEG. Examples of suitable vector systems for use with Agrobacterium are, for example, binary vectors such as pBI 121 and derivatives thereof; co-integrate vectors such as pGV 1500 and derivatives of pBR322. Suitable vector systems for naked DNA transformation are, for example, high copy number E. coli vectors such as pUC vectors and pBluescript II (SK +) vectors. Any other per se known systems for the transformation 101 2782 16 of plants can also be used, for example as stated in WO 97/41228. Suitable transformation systems for human and human cells, animals and animal cells, as well as bacteria, yeast (cells), algae and viruses are described, for example, in WO 95/07463, WO 96/23810, WO 95/07463, WO 95/21191, WO 5 97/11094, WO 97/42320, WO 98/06737 and WO 98/21355.

The constructs of the invention may, in addition to the nucleic acid sequence encoding the g / p variant of the invention, also comprise a promoter operably linked to the nucleic acid sequence encoding the gfp variant of the invention, which meant that the promoter is able to induce / control expression of the gfp ariant in the desired organism. This requires at least the g #? Variant to be in the correct reading frame and orientation with respect to the promoter.

The promoter can be any suitable promoter active in the desired organism, including constitutive and inducible promoters. Examples of suitable promoters for use in plants and plant cells are, for example, constitutive promoters such as the nos promoter, the CaMV 35S promoter, the actin promoter and the mas (mannopine synthase) promoter or inducible promoters such as the WIN ("wound inducible") promoter and the HMGR (HydroxymethylglutarylCoA reductase) promoter. Other suitable promoters for use in plants are disclosed, for example, in WO 97/41228.

Suitable promoters for use in human and human cells, animals and animal cells, as well as bacteria, yeast (cells), algae and viruses are described, for example, in WO 95/07463, WO 96/23810, WO 95/07463, WO 95/21191, WO 97/11094, WO 97/42320, WO 98/06737 and WO 98/21355.

Furthermore, promoters can be used which are induced only in a specific part or tissue of an organism, such as only in the leaves or roots of a plant; or promoters that are induced only during a certain developmental stage of the organism; or promoters that are induced only under specific circumstances, such as the appearance of certain disease pictures or immune responses. Examples of suitable tissue specific promoters are the patatin promoter, GBSS promoter and plastocyanin promoter, as well as the tissue specific promoters for use in plants disclosed in WO 97/41228; an example of a promoter that is activated during a certain development phase of the plant is the S AG12 promoter (from the Arabidopsis "senescence associated gene").

In the construct of the invention, the g #? Variant may be preceded by one or more sequences encoding signal proteins, such as pre, pro or prepro sequences, such that the variant GFP is expressed as a fusion with these signal proteins. is being brought. This may be of particular importance when the construct of the invention is used in eukaryotic organisms such as plants and animals, and / or to obtain the post-translational modifications, such as those leading to the formation of the chromophore.

The signal sequence may also be a sequence that causes the expression product to specifically target certain parts and / or organelles within the cell. Example is the N-terminal signal sequence MKTNLFLFLIFSLLLSLSSAEF in combination with the C-terminal sequence HDEL, which causes the expressed protein to target the endoplasmic reticulum. This could prevent or reduce potential toxic or defense responses in the cells, tissues and / or organisms in which the gfp gene is expressed. Other examples of signal sequences for use in plants are given, for example, in WO 97/41228.

For the further control elements which may be present in the constructs according to the invention, such as terminators, transcriptional and / or translational enhancers and integration factors, are mentioned for example in WO 95/07463, WO 96/23810, WO 95/07463, WO 95 / 21191, WO 97/11094, WO 97/42320, WO 98/06737 and WO 98/21355.

According to a particular embodiment, the construct according to the invention encodes a fusion of the g / p variant with one or more further desired genes, wherein the variant GFP can suitably act as a "reporter" for the expression of - or more generally for the presence of these genes.

For example, these genes can encode proteins or peptides that provide the transformed organism with one or more improved properties, such as resistance to diseases, stress, heavy metals and the like. The mergers can also be used for expression analysis or biological function studies.

Examples of genes / proteins that can be expressed in plants or plant cells as a fusion with the GFP variant of the invention are 1012782 18 (defined as the final fusion) β-glucuronidase / GFP; plant plasma membrane protein AIR1 / GFP6, or phragmoplastin / GFP.

In the construct of the invention, such a fusion can also be preceded by one or more sequences encoding signal proteins, such as the signal sequences mentioned above, such that the GFP / protein fusion is expressed as a further fusion with these signal proteins. This may be of particular importance when the construct of the invention is used in eukaryotic organisms such as plants and animals, and / or to obtain the post-translational modifications to both the GFP variant and the fused protein, including of cleavage of the GFP and the protein.

The presence of the gfp variant in a cell, tissue or host organism can be demonstrated in a manner known per se, in particular by expressing and determining the presence of the encoded GFP, preferably in a per se manner such as by fluorescent techniques. As a rule, the GFP variant according to the invention is irradiated with light / radiation of a suitable wavelength, after which the fluorescence obtained is determined, i.e. optically, with a microscope, or with suitable equipment. The degree of fluorescence can also be a measure of the degree of expression.

The invention will now be elucidated with reference to the following

Experimental Section.

Experimental Section

In the early 1990s, the wild-type (wt) gfp gene from the jellyfish (Aequorea 25 victoria) was discovered and characterized. Since then, the use of this gene has made important contributions in both scientific and applied biotechnology research.

The gfp gene encodes Green Fluorescent Protein (GFP), a protein with a number of unique properties. Wt-GFP is a 27 kDa monomer and is made up of 30 238 amino acids. The protein emits green light (maximum emission at about 510 nm) when it is irradiated with UV (maximum absorption at about 395 nm) or blue (maximum absorption at about 470 nm) light. The light absorbing portion of wt-GFP, the chromophore, consists of a circularized tripeptide and can fluoresce 1012782 19 only when it is covalently bonded to and completely entrapped by the rest of the protein. The chromophore is derived from S65-Y66-G67 of the primary protein sequence and is formed by modifications after translation of the gfp gene.

The functionality of the gfp gene, and variants derived from it, has already been demonstrated in many other organisms, such as bacteria, fungi, animals, humans and plants. The demonstrated fluorescence in these organisms does not appear to depend on certain (added) co-factors or substrates (other than oxygen). In addition, GFP appears to be very stable. Even at high heat (up to 65 ° C), extreme pH 10 (between 5.5 and 12), many organic solutions and some denaturing agents, GFP continues to fluoresce.

All of these properties together make GFP an ideal in vivo reporter for use in plants. Other commonly used plant reporters, such as beta-glucuronidase (GUS) and luciferase (LUC), require the addition of substrate to detect reporter activity. In the case of GUS, it is even destructive to the material to be analyzed due to cytotoxic effects.

Many other applications with GFP are conceivable. For example, GFP can be used instead of GUS or LUC as a reporter for selecting transformed plants. Transformants are easier to identify in a non-destructive way, and large numbers of plants can be analyzed more quickly, and the cost savings that could be achieved with this could also make it possible to transform less economically interesting crops.

GFP can also be used as a so-called fluorescent marker ("fluorescent tag") for other proteins. This takes advantage of the property that GFP can remain functional when the gfp gene is fused in frame at the amino or carboxy terminal end of another protein. Subcellular activities of such protein labeled in this way, such as gene expression, protein-protein interactions, protein trafficking and protein localization, can thus be studied.

Another conceivable application with GFP is the monitoring of pathogens in the plant on both a macro and a microscopic level. For example, viral infections could be studied by replacing a virus's coat protein genes with a g / p gene.

101 2782 20

The utility of the original (wt) gfp gene in Arabidopsis, and probably other plants, is limited by the presence of a cryptic intron, resulting in a non-functional GFP through aberrant splicing. This problem can be solved for plants by adjusting the codon usage. Another reason to adjust the codon usage may be that, by lowering the relatively high A / T content in the wt-gfp gene, better expression in plants is obtained. Furthermore, it is possible to provide plant gfp variants with a signal peptide, whereby GFP is sent to the lumen of the ER, for example, to prevent an assumed toxic effect in the cytoplasm or nucleoplasm, and / or to regenerate the transformed plants to run better. A possible disadvantage of localization or retention signals, however, is that such a GFP may no longer be used for localization studies (microscopic) within the cell.

Another goal in designing gfp variants is to minimize the toxic by inhibiting aggregation in the cell. So-called "soluble modified" (sm) gfp variants can be obtained, which can also increase fluorescence, because soluble GFP is and aggregated GFP is not functional.

G / p variants can essentially be divided into four classes, based on different excitation / emission spectra: 1) GFP; 2) red shift (RS) GFP; 3) YFP 20 (Yellow Fluorescent Protein) and 4) BFP (Blue Fluorescent Protein). For example, red shift and Blue Fluorescent Protein variants can be obtained by mutations in which the wt amino acids S65 and Y66 (but not usually G67) of the chromophore are substituted. Emission of the color blue, rather than green, by Blue Fluorescent Protein variants is mainly due to substitution of wt-25 Y66, in particular H66 or W66 (i.e. mutation Y66H and Y66W).

The chromophore flanking wt-amino acid F64 can optionally also be substituted for all the classes mentioned.

GFP variants of the invention of the Yellow Fluorescent Protein type can be obtained, for example, by mutations in which (in relation to the wild-type sequence) the amino acids S65, V68, S72, and / or T203 are substituted.

As already mentioned, these "YFPs" will emit with a yellow / green color, rather than with the green color of the wild type.

1012782 21

The choice for a certain GFP variant will depend on the application for which a certain variant is used. In the case of plants, autofluorescence of plant parts must be taken into account. Some GFPs and BFPs known from the prior art appear to be difficult to detect in root. smRS-5 GFP and smGFP, on the other hand, are detectable in root. In addition, redshift g #? Variants may exhibit more intense fluorescence.

For example, the utility of a variant of the invention can be judged based on the following criteria: 1- Adapted codon usage (no cryptic intron and reduced A / T content for higher expression in eukaryotes).

2- Red-shift (excitation peak more in accordance with bandwidth used filters, higher excitation coefficient, faster chromophore formation, less burden of autofluorescence in plant parts) 3- Preferably no signal peptides (useless when a fused protein needs to be localized) 4- Mutations that achieve a higher fluorescence (whether or not by increasing the "solubility" of GFP in the cell (aggregated GFP is usually little or inactive).

None of the prior art variants listed in Table 1 satisfactorily meet all of these criteria.

Example 1: Construction g / b variant according to the invention (smeg / pk

The known sequence of the egfp gene (from pEGFP, Clontech) was started from. In this sequence, the so-called "soluble modification" mutations were F99S, M153T, V163A and S175G and the "dual excitation peak" mutation I167T

introduced. The mutation H231L (meaning unknown) present in the starting sequence was restored to its original state (L231H). A 6th mutation, K238Q, arose from the chosen cloning strategy introducing a Λ / wNI restriction site into the Qgfp gene. However, this mutation does not fall within the preferred sustained minimum range of gfp (amino acid 7 to 229) for fluorescence, so that this mutation does not (negatively) affect the level of fluorescence.

To introduce the mutations, two primer sets (set “1” and set “2”) of 2 primers each (ie primers “la / lb” and “2a / 2b”) were designed, namely: 101 2782 22

Primer la: 5 "-AATTCG A ACC ATCT C3TTC AAGGACGACGGC AACT AC-3" 5 Primer lb: 5 "-TAGGCCTTGATCCCGTTCTTCTGCTTGTCGGCCGTGATA-3"

Primer 2a: 5 "-CATATGGATCTTCGCGAACTTCAAGACrcGCC ACAACATCGA 10 GACGGCGGCGTG-3"

Primer 2b: 5 '- AAC AGCTACT GGT AC AGCTCGTCC AT GCC4 7GAGTGATCCCGG-3' 15 These sequences are also shown in SEQ IDs no. 3 through 6 respectively. These primer sets are designed to substitute amino acids in the egfp gene used as starting product. of plasmid pEGFP (Clontech) by PCR. The base (s) that result in the mutations are shown in bold and italics and are as follows (from 5 'end): primer 1a: F99S; primer lb: M153T; primer 2a: V163A; 1167T; 20 S175G; primer 2b: K238Q; L231H. The underlined bases represent the restriction sites in these primers, and are (again from the 5 'end): primer 1a: ZfcfBI; primer lb: SM; primer 2a: NdeI; Nrul; primer 2b: Alwl.

The nucleotide sequences (determined by BaseClear) of the two PCR products obtained with these 25 primer sets are shown in SEQ ID NO 7 (obtained with primer set 1) and SEQ ID NO 8 (obtained with primer set 2). The nucleotide sequences of the primers used form the 5 "and 3" ends of these PCR phagments.

As "target" sequences for primer set 1 and 2, the "middle" and 3 'portion of egfp from pEGFP respectively. A PCR reaction, including a so-called "hot" start, was then performed with each primer set using the GeneAmp XL PCR kit (PE applied Biosystems). The PCR products obtained with primer sets 1 and 2 were then cloned into vector pCR®2.1 using the 101 2782 23 “Original TA Cloning Kit” (Invitrogen). The obtained ligation products were transformed by electroporation into E.coli "ElectroMAX DH10B ™ cells" (GibcoBRL life technologies). Following the transformation protocol provided by "GibcoBRL life technologies". The base sequence of the PCR cloned in pCR®2.1 5 products were determined by BaseClear (Leiden) and are listed in SEQ ID no 7 and 8 ..

The amplified portions of egfp were found to be 100% identical to the "targef" sequences of the egfp gene in pEGFP, except for the sequences of the primers used with the mutations. then reconstructed by a three-point ligation ligating the following three fragments: 1) an unmodified 5 'portion of the egfp from pEGFP.

2) the modified "middle" part of egfp (obtained with primer set 1).

3) the modified 3 'part of egfp (obtained with primer set 2).

The unmodified 5 'portion of egfp was obtained from pEGFP by digestion with the restriction enzymes Asel and HihPl. A fragment of about 0.47 kb was isolated. The second fragment was obtained from the pCR®2.1 vector with the modified "middle" part of egfp. After digestion thereof with the restriction enzymes 5siBI and Stul, an approximately 0.2 kb fragment was isolated. The third fragment was obtained by digesting the pCR®2.1 vector with the modified 3 'part of egfp. For this purpose, the restriction enzymes Ndel and Nruï were used, allowing a fragment (including the pCR®2.1 vector) of over 4.1 kb to be isolated. All fragments were obtained by standard gel electrophoresis and isolation techniques. For isolation of the fragments, the “QiaexII gel extraction kit” (Qiagen) was used. After ligation of the three fragments, the ligation mixture was transformed by electroporation into E.coli "ElectroMAX DH10B ™" cells (GibcoBRL life technologies). Following the transformation protocol provided by "GibcoBRL life technologies". using a “Leica MZ FLIII” fluorescence stereo microscope, clear fluorescent colonies were observed, proving that ligation of the fragments resulted in a fully open reading frame, also restriction analyzes performed on plasmid DNA isolated from fluorescent colonies.

1012782 24

The plasmid thus obtained, called pKG1620, which contains the gfp gene of the invention, called smegfp, is schematically shown in Figure 2. The total sequence of pKG1620 is derived from the published vector pCR®2.1 (base 438-4346) , the published vector pEGFP (base 4347-4816) and the sequences as determined by BaseClear of the PCR products obtained with primer set 1 (base 1-202) and 2 (base 203-437).

In pKG1620, the gfp gene of the invention is fused to an exemplary lac gene (in addition, the / ac promoter is also used in pKG1620). It will be clear to the skilled person that this lac gene can be replaced by one or more other genes of interest.

The smegfp thus obtained showed a dual excitation peak around 395 and 490 nm and an emission peak around 510 nm. Very good filter sets for the Leica MZ FLIK ”fluorescence stereo microscope for use with smegfp according to the invention are, for example, for filter set“ GFP2 ”an excitation filter and“ Barrier ”filter 15 of 480/40 and 510 nm respectively and for filter set“ GFP3 ”a excitation filter and “Barrier” filter of 470/40 and 525/50 nm respectively.

Cultivation of colonies with this new construct, called pKG1620, from plate to liquid medium was also no problem. The phenomenon in GFP work in which E. coli colonies sometimes do not want to "catch on" is usually explained by the toxic effect of GPF aggregation in the cell. Thus, the observations just described may indicate an improved soluble GFP in E. coli.

Example 2: Expression of efp fusion.

In this example, the gfp gene of Example 1, called smegfp, was fused in frame 25 at the carboxy-terminal end of another open reading frame. The smEGFP still appeared to fluoresce well as a fusion protein.

101 2782 25 TABLE 1

GFP l.wildtype 2.Anderso 3.Davis 4.Haseloff 5.Clontech ó.Héim 7. Heim UITV

n gfp-Gen wt -gfp g / p-Vexl smRSgfp mg / p5-ER cgfp w7 w2 smeg / p exitation 395 (470) ~ 4Ö5 "49Ö 395 & 470 490 433 (453) 333 (453) 395 & (max) nm 490 emission 5ÏÖ 51Ö ~ 5ÏÖ 5ÏÖ TlÖ 475 (501) 180 lïÖ (max) nm cryptic yes yes no no no yes yes no intron _ _ _ - x 17 ~ _ _ _ _ _ _ _ _ _ _ _ __ _ _ - 'rr _ _ _ _ _ J: "v __ - _ _ _ _ - ^

"Ï53" M T T T T

_ __ _ __ _ _ _ _ _ - _; : x __ _ _ _ _ _ _ __ - j _ _; :: “K _ - - __ - - mod. none none 237-447 237-540 none none none integer codons (b.p) a) letters indicate amino acid (one letter code) 5 b) all positions not indicated are equal to wild type 101 2782 26 c) wavelengths are in nm; wavelengths in brackets indicate minor peak.

d) for all variants, the chromophore is S, C or T65 / Y66 / G67 1. Wild type described by Prasher et al., 1992 5 2. Anderson et al, 1996

3. Davis / Vierstra; additional N-terminal sequence: MKTNLFLFLIFSLLLSLSSAEF (F

instead of M1) / additional C-terminal sequence: HDEL

4. Haseloff 5. Clontech Manual, Ine.

10 6. Heim & Tsien, 1996 7. Heim & Tsien, 1996

Invention: 1 extra amino acid (V) after M1.

101 2782 27 T A B E L 1 (continued)

GFP 8. Heim 9. 10. Mitra ll.Cormac I2.Clontec 13. Pang l4.Quantu UITV

Clontech khmg / p-Ge n p4-l cbfp bfp g / pmut2 cyfp S65Tpg / p pQB125 sm egfp exitation 505 (395) "38Ö ~ 38 (j ~ 48Ö" 5Ï3 "490" 475 395 & 490 (max) nm emission TÏ5 ~ 44Ö ~ 44Ö TfÖ ~ 527 ~ 51Ö ÜiÖ ^ ÏÖ (max) nm cryptic no unknown yes no no yes no intron _ _ _ _ _ _ - _ _ _ _ _ - _ _ _ _ _ _ - _ _ _ _ _; _ _ - _ _ __ _ _ __; ___ _ -: "t __;" a __ _ - __ _ - __

"2Ö3 T

_; :: ~ __ "238" One o ~ mod. no fault. unknown. no unknown. whole 504-708 whole codons (b.p) a) letters indicate amino acid (one letter code) 1012782 28 b) all positions not indicated are equal to the wild type c) wavelengths are in nm; wavelengths in brackets indicate minor peak.

d) for all variants, the chromophore is S, C or T65 / Y66 / G67 58. Heim & Tsien, 1996 9. Clontech Manual 10. Mitraetal., 1996 11. Cormack et al., 12. Clontech Manual 1013. Pang et al., 1996: "ST-LS1" intron between base pairs 379-380 14. Quantum Biotechnologies Manual; 1 extra amino acid (V) after M1.

Invention: 1 extra amino acid (V) after M1.

101 2782 29

SEQUENCE LISTING

<110> Keygene N.V.

5 <120> Improved GFP variant, usable as marking, and GFP construct <130> BO 42497 10 <140> <141> <160> 7 15 <170> Patent Ver. 2.1

<210> 1 <211> 720 <212> DNA

20 <213> Artificial Sequence <220> <223> Description of Artificial Sequence: sme-gfp 25 <220> <221> misc_feature <222> (4) .. (6) <223> inserted codon GTG compared to wild-type gfp sequence 30 <400> 1 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 35 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg aaccatctct 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca cggccgacaa gcagaagaac 480 40 gggatcaagg cgaacttcaa gactcgccac aacatcgagg acggcggcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc CGCC gggatc actcatggca tggacgagct gtaccagtag 720 45

<210> 2 <211> 239 <212> PRT

<213> Artificial Sequence 50 <220> <223> Description of Artificial Sequence: sme-GFP <220>

55 <221> UNSURE

<222> (2) <223> inserted Valine compared to wild-type GFP sequence <400> 2 60 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val lie Leu 1012782 1 5 .. 10 15 30

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 5

Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 10 50 55 60

Leu Thr Tyr Gly Val Gin Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 15 Gin His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gin Glu 85 .90 95

Arg Thr lie Ser Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 20

Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125

Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 25 130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gin Lys Asn 145 150 155 160 30 Gly Ile Lys Ala Asn Phe Lys Thr Arg His Asn Ile Glu Asp Gly Gly 165 170 175

Val Gin Leu Ala Asp His Tyr Gin Gin Asn Thr Pro Ile Gly Asp Gly 180 185 190 35

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gin Ser Ala Leu 195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 40 210 215 220

Val Thr Ala Ala Gly lie Thr His Gly Met Asp Glu Leu Tyr Gin 225 230 235 45

<210> 3 <211> 22 <212> PRT

50 <213> Artificial Sequence <220> <223> Description of Artificial Sequence: N-terminal signal sequence 55 <400> 3

With Lys Thr Asn Leu Phe Leu Phe Leu Ile Phe Ser Leu Leu Leu Ser 15 10 15 60 Leu Ser Ser Ala Glu Phe 20 101 2782 31 <210> 4 5 <211> 37

<212> DNA

<213> Artificial Sequence <220> 10 <223> Description of Artificial Sequence: primer la <400> 4 aattcgaacc atctctttca aggacgacgg caactac 37 15

<210> 5 <211> 39 <212> DNA

<213> Artificial Sequence 20 <220> <223> Description of Artificial Sequence: primer lb <400> 5 25 taggccttga tcccgttctt ctgcttgtcg gccgtgata 39

<210> 6 <211> 54 30 <212> DNA

<213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 2a 35 <400> 6 catatggatc ttcgcgaact tcaagactcg ccacaacatc gagacggcgg cgtg 54 40 <210> 7

<211> 43 <212> DNA

<213> Artificial Sequence 45 <220> <223> Description of Artificial Sequence: primer 2b <400> 7 aacagctact ggtacagctc gtccatgcca tgagtgatcc egg 43 50 101 2782

Claims (12)

1. A variant of the g / p gene, or the nucleus gene sequence or genetic construct encoding this variant, comprising, with respect to the sequence of the wild type: - at least one mutation in at least one of the codons encoding amino acid positions 99, 153 and / or 163 such that the protein encoded by the variant gene exhibits improved solubility compared to the wild type GFP; 10. at least one mutation in at least one of the codons encoding amino acid positions 65, 66 and / or 67 such that the excitation spectrum of the protein encoded by the variant gene is shifted to the red or blue end of the spectrum, compared with the wild type GFP; at least one mutation in at least one of the codons encoding 15 amino acid positions 163,167 and / or 175 such that the protein encoded by the variant gene exhibits improved "dual excitatiori" characteristics compared to the wild type GFP, and preferably also - at least one mutation in the codon encoding amino acid position 64 such that the protein encoded by the variant gene exhibits enhanced fluorescence compared to the wild type GFP, and equivalent or analogous sequences thereto, as well as fragments thereof. A variant, nucleic acid sequence or genetic construct according to claim 1, comprising, with respect to the wild type sequence: 25. at least one mutation in each of the codons encoding amino acid positions 99, 153 and / or 163, such that encoded protein exhibits improved solubility over the wild type GFP; - at least one mutation in at least one of the codons encoding amino acid positions 65,66 and / or 67, and preferably in the codon encoding amino acid position 65, such that the excitation spectrum of the encoded protein is shifted to the red end of the spectrum, compared to the wild type GFP; 1012782 - at least one mutation in each of the codons encoding amino acid positions 163,167 and / or 175 such that the encoded protein exhibits improved dual excitation characteristics compared to the wild type GFP; and - at least one mutation in the codon encoding amino acid position 64 such that the encoded protein exhibits enhanced fluorescence compared to the wild type GFP; and equivalent or analogous sequences thereto, as well as fragments thereof. The variant, nucleic acid sequence or genetic construct according to claim 1 or 10 2, wherein the mutations at positions 65,66 and / or 67 are further such that the encoded protein also exhibits enhanced fluorescence (ie emission) compared to the wild type GFP especially at wavelengths between 450 and 550 nm. The variant, nucleic acid sequence or genetic construct according to any one of claims 1 to 3, wherein the mutations in the codons at positions 64, 65, 99, 153, 163, 167 and 175 comprise one or more substitions of one or more nucleotides such that the mutant codon encodes for another amino acid as present at the appropriate position in the wild type GFP. 20 The variant, nucleic acid sequence or genetic construct according to any one of claims 1 to 4, wherein the mutations encode the following amino acid substitutions, relative to the wild type: Amino acid position Wild type Variant of the invention ~ 64 F L or M 65. C, T, G or A 66 67 99. S 153. Great A 163. A 167. T 175. G Nucleic acid sequence or genetic construct according to any one of claims 1 to 5, further comprising one or more known per se elements of nucleic acid sequences or genetic constructs, such as promoters or other control elements, enhancers, integration factors, signal sequences, and the like. Nucleic acid sequence or genetic construct according to any one of claims 1-6, further comprising at least one further sequence encoding a gene, a desired property, a protein or a polypeptide, the protein or polypeptide optionally as a fusion with the β. variant of the invention is expressed. A variant of the GFP protein, as encoded by a gfp variant according to any one of claims 1-7, or as obtainable by expression of a nucleic acid sequence or genetic construct according to any one of claims 1-7. 9. Use of a gfp variant, a nucleic acid sequence or a genetic construct according to claims 1-7, and / or of a variant of the GFP protein according to claim 8, as a label or marker in (micro) biological, biochemical and / or molecular biological techniques. 1012782 Use of a g / p variant, a nucleic acid sequence or a genetic construct according to claims 1-7, and / or a variant of the GFP protein according to claim 8, as a genetic marker or expression marker in vivo, in particular in plants, humans, animals or in cells or tissues thereof, or in cell lines derived therefrom. Use according to claim 10, comprising transforming a host organism or a cell, part or tissue thereof, with a nucleic acid sequence according to any one of claims 1-7; and / or comprising expressing in the host organism a gjp variant according to any one of claims 1-7. 12. Organism, such as a plant, an animal, a human, a bacteria, an alga, a yeast (cell) or other micro-organisms, as well as tissues or cells of these organisms, or cell lines derived therefrom, which have a sequence or construct according to any one of claims 1-15., and / or expressing a GFP variant according to claim 8. 1012782