WO2000071565A9 - Fluorescent protein indicators - Google Patents
Fluorescent protein indicatorsInfo
- Publication number
- WO2000071565A9 WO2000071565A9 PCT/US2000/013684 US0013684W WO0071565A9 WO 2000071565 A9 WO2000071565 A9 WO 2000071565A9 US 0013684 W US0013684 W US 0013684W WO 0071565 A9 WO0071565 A9 WO 0071565A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- fluorescent protein
- polypeptide
- moiety
- receptor
- nucleic acid
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
Definitions
- the invention relates generally to fluorescent proteins and more particularly to compositions and methods for measuring the response of a sensor polypeptide to an environmental (e g , biological, chemical, electrical or physiological) parameter
- Fluorescent Ca 2+ indicators such as fura-2, ⁇ ndo-1 , fluo-3, and Calcium-Green have been the mainstay of intracellular Ca "+ measurement and imaging (see, for example, U S Pat No 4,603,209 and U S Pat No 5,049,673) These relatively low molecular weight indicators can suffer from many technical problems relating to ester loading, leakage of the dyes from the cell, compartmentation in organelles, and perturbation of the indicators by cellular constituents
- the Ca "+ - ⁇ nd ⁇ cat ⁇ ng photoprotein aequo ⁇ n is targetable, the photoresponse to Ca" is low since it is chemiluminescent Moreover, aequo ⁇ ns need to incorporate exogenous coelenterazine
- CaM calmoduhn
- cyclic AMP can be detected by fluorescence resonance energy transfer between separately labeled proteins that associate with each other but are not covalently attached to each other See, U S Pat No 5,439,797
- GFP Green Fluorescent Protein
- FRET fluorescence resonance energy transfer
- a sensor polypeptide when a sensor polypeptide is inserted into an Aeqitot e ⁇ -related fluorescent protein (e g , Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP) or Cyan Fluorescent Protein (CFP)) to form a construct, interaction of the sensor polypeptide with a biological, chemical, electrical or physiological parameter, for example, results in a change in fluorescence of the fluorescent protein
- a biological, chemical, electrical or physiological parameter for example, results in a change in fluorescence of the fluorescent protein
- Such constructs are useful in measuring interactions of a sensor polypeptides with environmental stimuli in vitro or in vivo or in measuring particular characteristics of a cell (e g , redox potential, intracellular ion concentration)
- These constructs rely on the responsiveness of a sensor polypeptide inserted within a GFP- sensor-related protein itself to influence the actual fluorescence of the fluorophore and not the interaction of tandem fluorescent molecules
- the present invention provides an isolated nucleic acid sequence which encodes a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide
- the fluorescent protein moiety can be any fluorescent protein, for example, an Aequorea-related fluorescent protein moiety More specifically, the Aequore ⁇ -rdated fluorescent protein can be, for example, a GFP, CFP or YFP moiety
- the sensor polypeptide may be any polypeptide moiety, for example, a moiety that undergoes a conformational change upon interaction with a molecule, oxidation-reduction, or changes in electrical or chemical potential
- the indicator may further include a linker moiety, linking the N- and C-terminal amino acids of the sensor polypeptide to the fluorescent protein
- the present invention provides an expression vector having expression control sequences operatively linked to a nucleic acid sequence coding for the expression of a fluorescent indicator
- the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide.
- the present invention provides a host cell transfected with an expression vector having an expression control sequence operatively linked to a sequence coding for the expression of a fluorescent indicator
- the host cell can be any host cell capable of transfection and expression of the indicator such as, for example, a prokaryote (e g , E Coli), a eukaryotic cell (e g , a yeast cell) or a mammalian cell
- the present invention provides a fluorescent indicator polypeptide, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide
- the present invention provides a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide the responsiveness resulting in protonation or deprotonation of the chromophore of the fluorescent protein moiety.
- the present invention provides a method for detecting the presence of a environmental parameter in a sample, by contacting the sample with a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical, or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide, and detecting a change in fluorescence wherein a change is indicative of the presence of a parameter which affects the sensor polypeptide.
- the invention provides an isolated nucleic acid sequence encoding a circularly pe ⁇ nuted fluorescent protein and the polypeptide encoded thereby, having a linker moiety linking the amino-terminal and carboxy- terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein.
- the invention provides an expression vector comprising expression control sequences operatively linked to a nucleic acid sequence coding for the expression of a fluorescent indicator, the indicator having a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy- terminal amino acids of the fluorescent protein.
- the invention provides a host cell containing the expression vector
- the invention provides a method of producing a nucleic acid sequence encoding a fluorescent indicator, by linking a nucleic acid sequence encoding a linker moiety to the 5' nucleotide of a polynucleotide encoding a fluorescent protein, circularizing the polynucleotide with the nucleic acid sequence encoding the linker sequence, and cleaving the circularized polynucleotide with a nuclease, wherein cleavage linearizes the circularized polynucleotide.
- the invention provides a method of producing a circularly permuted fluorescent protein by expressing a nucleic acid sequence encoding a linker moiety linking the amino-te ⁇ ninal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein.
- FIG. 1 shows the designs of a calmoduhn or a Z ⁇ f268 insertion into a fluorescent indicator of the present invention
- FIG. 2A shows pH effects on a calmoduhn insertion indicator.
- FIG 2B shows titration curves for a calmoduhn insertion indicator
- FIG 3 further shows the effects of calcium concentration on fluorescence of a fluorescent indicator of the invention containing a sensor polypeptide of calmoduhn
- FIG 4 show the effect of Zn concentration on fluorescence of a fluorescent indicator of the invention containing a sensor polypeptide of inserted Zif polypeptide
- FIG 5 shows the overall design of a circularly permuted polypeptide
- FIG 6 shows the result of random digestion of a circularly permuted nucleic acid sequences
- FIG 7 shows insertions into GFP
- FIG 8 shows a schematic diagram of potential insertional sites and sites for circular permutations in GFP
- the present invention relates to polynucleotides encoding fluorescent indicators, fluorescent indicators having a sensor polypeptide, and methods of producing and using the same
- the inventors have discovered a number of sites in Aequot ea-rzlal d fluorescent protein moieties that are tolerant to insertions and rearrangements
- the inventors have additionally discovered that the insertion of sensory polypeptides into such sites results in polypeptides that are useful in detection of chemical, biological, electrical or physiological parameters, for example
- Such insertion sites include, for example, one or more amino acids between residues 128 - 148, residues 155-160, residues 168-176 or residues 227-229 of the fluorescent protein moiety
- Other positions which may tolerate insertions include, but are not limited to, residues 49-50, 78-79, 1 16-1 17, 134-135, 140-141 , 157- 158, 172- 173, 194-195, 189-190 and 213-214 (see Abedi et al , Nucleic Acids Research, 26(2) 623-630 (1998), the disclosure of which is incorporated herein) More specifically, the insertion is a Y 145
- a sensor polypeptide when inserted into an Aequoi e ⁇ -related fluorescent protein (e g , GFP, YFP or CFP) that provides a response related to an interaction with a biological, chemical, electrical or physiological parameter, the responsiveness results in a change in fluorescence of the fluorescent protein
- an Aequoi e ⁇ -related fluorescent protein e g , GFP, YFP or CFP
- Such constructs are useful in measuring interactions of a sensor polypeptides with environmental stimuli in vitro or in vivo
- These new constructs rely on, for example, detectable changes within a GFP-sensor-related protein itself to influence the actual fluorescence of the fluorophore and not the interaction of tandem fluorescent molecules For example, when calmoduhn is inserted into YFP at position Y145, interaction of calmoduhn with its ligand (e g ; calcium) results in a change in the brightness of the fluorescent protein of between 2-8 fold
- the indicators of the present invention are advantageous due to their reduced size as compared to the FRET-based sensors described above
- the reduced size has importance in allowing the indicator to measure chemical, biological, electrical or physiological interactions with the sensor polypeptide in, for example, subcellular compartments previously inaccessible to the larger, FRET-based sensors
- the maximal change in fluorescence intensity observed in the present indicators e g , up to 8 fold increase
- those in the cameleons e g , FRET-based sensors
- the invention provides polynucleotides and nucleic acid sequences encoding fluorescent indicators having a fluorescent protein moiety and a sensor polypeptide, or fragments thereof, inserted in operable association into the fluorescent protein moiety, in which the sensor polypeptide is responsive to an environmental parameter (e g , a chemical, a biological, a electrical, or a physiological parameter) Accordingly, the responsiveness of the sensor polypeptide causes a change in fluorescence of the fluorescent indicator The degree of change in the fluorescence of the indicator is sensitive to pH
- "operatively inserted” or “operably inserted” is meant between two amino acids of a polypeptide or two nucleotides of a nucleic acid sequence Accordingly, insertion excludes hgating or attaching a polypeptide to the last terminal amino acid or nucleotide in a sequence
- a "detectable change” or “responsiveness” means any response of a polypeptide to a chemical, biological, electrical, or physiological parameter or stimuli
- a response includes small changes, for example, a shift in the orientation of an amino acid or peptide fragment of the sensor polypeptide as well as, for example, a change in the primary, secondary, or tertiary structure of a polypeptide, including for example, changes in protonation, electrical and chemical potential and or conformation
- Conformation is the three-dimensional arrangement of the primary, secondary and tertiary structure of a molecule including side groups in the molecule, a change in conformation occurs when the three-dimensional structure of a molecule changes Examples of conformational changes include a shift from -helix to a -sheet or a shift from -sheet to a -helix It is understood that detectable changes need not be a conformational change, so long as the fluorescence of the fluorescent protein moiety is altered
- Fragments are a portion of a naturally occurring sensor protein which can exist in at least two different states or conformations Fragments can have the same or substantially the same amino acid sequence as the naturally occurring protein "Substantially the same” means that an amino acid sequence is largely, but not entirely, the same, but retains a functional activity of the sequence to which it is related In general two amino acid sequences are substantially the same” or “substantially homologous” if they are at least 85% identical Fragments which have different three dimensional structures as the naturally occurring protein are also included
- responsive as used herein is intended to encompass any response of a polypeptide which is related to an interaction of a chemical, biological, electrical, or physiological parameter with a sensor polypeptide (e g , conformational change in a voltage-gated ion channel (e g , Shaker) in detection of membrane voltage across a biological membrane, phosphorylation of a hormone receptor resulting in a conformational change in the receptor upon hormone stimulation)
- a sensor polypeptide e
- the fluorescent protein contains a second fluorescent protein and a sensor moiety within the insert
- utilization of FRET based techniques to analyze or detect changes in chemical, biological or electrical parameters may be performed
- binding of an analyte such as calcium to a sensor polypeptide such as calmoduhn would change the distance or angular orientation of the two fluorescent protein chromophores relative to each other and thereby modulate FRET
- a circularly permuted fluorescent protein may be tandemly or insertionally fused via a sensor moiety to a second fluorescent protein (which itself may optionally be a circular permutation) so that the FRET between the two fluorescent proteins changes in response to chemical, biological, or electrical parameters
- Classes of sensor polypeptides which may be used in the compositions and methods of the invention include, but are not limited to, channel proteins, receptors, enzymes, and G-proteins
- Channel polypeptides useful with the invention include, but are not limited to voltage-gated ion channels including the potassium, sodium, chloride, G-protein- responsive, and calcium channels
- a "channel polypeptide” is typically a polypeptide embedded in the cell membrane which is part of a structure that determines what particle sizes and or charges are allowed to diffuse into the cell
- Channel polypeptides include the "voltage-gated ion channels", which are proteins imbedded in a cell membrane that serve as a crossing point for the regulated transfer of a specific ion or group of ions across the membrane
- Shaker potassium channels or dihydropu ⁇ dine receptors from skeletal muscle may be advantageously used in the present invention
- Table 1 Several ion channel polypeptides of use with the invention are listed in Table 1
- Channels also include those activated by intracellular signals such as those where the signal is by binding of ligand such as calcium, cyclic nucleotides, G- proteins, phosphoinositols, arachidonic acid, for example, and those where the signal is by a covalent modification such as phosphorylation, enzymatic cleavage, oxidation/reduction, and acetylation, for example
- ligand such as calcium, cyclic nucleotides, G- proteins, phosphoinositols, arachidonic acid, for example
- Channel proteins also include those activated by extracellular ligands (e g , lonotropic receptors) These can be activated by acetylchohne, biogenic amines, amino acids, and ATP, for example
- a "receptor polypeptide” is a polypeptide found on a cell, often on a membrane, that can combine with a specific type of molecule, e g , a ligand, which alters a function of the cell
- Receptor polypeptides of use with the invention include, but are not limited to, the growth factor receptors, hormone receptors, cytokine receptors, chemokine receptors, neurotransmitter receptors, gand-gated channels, and steroid receptors Specifically polypeptides encoding insulin-like growth factor, insulin, somatostatin, glucagon, interleukins, e g , IL-2, transforming growth factors (TGF- ⁇ , TGF- ⁇ ), platelet-dem ed growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), fibroblast growth factor (FGF), interferon- ⁇ (IFN- ⁇ ), and GM-CSF receptors are of use with the invention
- Enzyme is a polypeptide that acts as a catalyst, which speeds the rate at which biochemical reactions proceed do not alter the direction or nature of the reaction.
- Enzyme polypeptides useful in the invention include, but are not limited to, protein kinases, catalyses, amidase, phosphatases, guanylyl and adenylyl cyclases, and poxygenases
- Polypeptides encoding the se ⁇ ne/threonine protein kinases are of use with the invention.
- Several genes encoding human enzymes of use with the invention are listed in Table 3 TABLE 3 ENZYMES
- the responsiveness of the sensor polypeptide (e g , a change in conformation or state) that occurs in response to interaction of a sensor polypeptide with a chemical, biological, electrical or physiological parameter will, as discovered by the inventors, cause a change in fluorescence of the fluorescence indicator.
- the change can be the result of an alteration in the environment, structure, protonation or ohgome ⁇ zation status of the fluorescent indicator or chromophore.
- the molecular component responsible for a conformational change is known for many enzymes (e g , Blostien, R., et ⁇ l ( 1997) J. Biol.
- optical properties (e g , fluorescence) of the indicator which can be altered in response to the conformational change in the sensor polypeptide include, but are not limited to, changes in the excitation or emission spectrum, quantum yield, extinction coefficient, excited life-time and degree of self-quenching for example.
- the cause of the changes in these parameters may include but are not limited to changes in the environment, changes in the rotational or vibrational freedom of the sensor, changes in the angle of the sensor with respect to the exciting light or the optical detector apparatus, changes in the protonation or deprotonation of amino acids or side groups associated with a chromophore or changes in distance or dipole orientation between sensors on associated responsive polypeptides
- insertion of a peptide or protein in place of tyros ⁇ ne-145 (Y145) in mutants of GFP increases the sensitivity of fluorescence to quenching by acidic pH
- a detectable change e g , a change in conformation
- such interactions change the fluorescence via a shift in the acid sensitivity
- a calcium sensing protein replaces residue Y145 in a yellow mutant of GFP
- calcium binding increases the fluorescence by up to 8-fold, depending on the pH at which the measurement is made
- Other sites for insertion into GFP or GFP-mutants are allowable where circular permutation is tolerated, as discussed more fully below
- the sensor polypeptide is operably inserted into an optically active polypeptide (e g , a fluorescent protein moiety)
- an optically active polypeptide e g , a fluorescent protein moiety
- a protein-based "optically active polypeptide” is a polypeptide which contains a means for emitting light Fluorescence is one optical property of an optically active polypeptide which can be used as the means of detecting the responsiveness of the sensor or responsive polypeptide of the fluorescent indicator or circularly permuted fluorescent proteins of the invention
- fluorescent property refers to the molar extinction coefficient at an appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation spectrum or emission spectrum, the excitation wavelength maximum and emission wavelength maximum, the ratio of excitation amplitudes at two different wavelengths, the ratio of emission amplitudes at two different wavelengths, the excited state lifetime, or the fluorescence amsotropy
- a measurable difference in any one of these properties between the active and inactive states suffices for the utility of the fluorescent protein
- excitation radiation from an excitation source having a first wavelength, passes through excitation optics The excitation optics cause the excitation radiation to excite the sample
- fluorescent proteins in the sample emit radiation which has a wavelength that is different from the excitation wavelength.
- the device can include a temperature controller to maintain the sample at a specific temperature while it is being scanned
- a multi-axis translation stage moves a microtiter plate holding a plurality of samples in order to position different wells to be exposed
- the multi-axis translation stage, temperature controller, auto-focusing feature, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer
- the computer also can transform the data collected during the assay into another format for presentation
- Other means of measuring fluorescence can also be used with the invention
- any fluorescent protein can be used in the invention, including proteins that fluoresce due to intramolecular rearrangements or the addition of cofactors that promote fluorescence.
- green fluorescent proteins of cnida ⁇ ans which act as their energy-transfer acceptors in bioluminescence, are suitable fluorescent proteins for use in the fluorescent indicators
- a green fluorescent protein (“GFP”) is a protein that emits green light
- BFP blue fluorescent protein
- YFP yellow fluorescent protein
- CFP cyan fluorescent protein
- GFPs have been isolated from the Pacific Northwest jellyfish, Aequorea victoria, the sea pansy, Renilla reniformis, and Phiahdium gregarium See, Ward, W W , et al ,
- Suitable pairs for example a blue-shifted GFP mutant P4-3 (Y66H/Y145F) and an improved green mutant S65T can respectively serve as a donor and an acceptor for fluorescence resonance energy transfer (FRET) See, Tsien, R.Y., et al , Trends Cell Biol. 3.242-245 (1993)
- FRET fluorescence resonance energy transfer
- a fluorescent protein is an " e ⁇ u ⁇ re ⁇ -related fluorescent protein" if any contiguous sequence of 150 amino acids of the fluorescent protein has at least 85% sequence identity with an amino acid sequence, either contiguous or non-contiguous, from the wild type Aequorea green fluorescent protein.
- a fluorescent protein is an Aequorea-related fluorescent protein if any contiguous sequence of 200 amino acids of the fluorescent protein has at least 95%o sequence identity with an amino acid sequence, either contiguous or noncontiguous, from the wild type Aequorea green fluorescent protein.
- any continuous sequence of the circularly permuted sequence which has identity to an Aequorea- related fluorescent protein, as described above, whether further N- or C- terminal than the comparison sequence is considered related.
- the fluorescent protein can be related to Renilla or Phialidium wild-type fluorescent proteins using the same standards.
- WlBl included the following mutations: F64L;S65T; Y66W; F99S; and V163A.
- fluorescent proteins can be used in the fluorescent indicators, such as, for example, yellow fluorescent protein from Vibrio ⁇ scheri strain Y-1 , Peridinin- chlorophyll a binding protein from the dinoflagellate Symbiodimum sp phycobihproteins from marine cyanobacte ⁇ a such as Synechococcus, e g , phycoerythrin and phycocyanin, or oat phytochromes from oat reconstructed with phycoerythrobihn
- These fluorescent proteins have been described in Baldwin, T O , et al , Biochemistry 29 5509-5515 ( 1990), Morris, B J , et al , Plant Molecular Biology, 24 673-677 (1994), and Wilbanks, S M , et al , J Biol Chem 268 1226- 1235 (1993), and Li et al , Biochemistry 34 7923-7930 (1995), Murphy, J T , & Laga ⁇ as, J
- the fluorescent indicators can be produced as chimeric proteins by recombinant DNA technology
- Recombinant production of fluorescent proteins involves expressing nucleic acids having sequences that encode the proteins
- Nucleic acids encoding fluorescent proteins can be obtained by methods known in the art
- Mutant versions of fluorescent proteins can be made by site-specific mutagenesis of other nucleic acids encoding fluorescent proteins, or by random mutagenesis caused by increasing the error rate of PCR of the original polynucleotide with 0 1 mM MnCl 2 and un
- the sensor polypeptide is operably inserted to an optically active polypeptide, which responds (e g , a conformation change)to, for example, a cell signaling event
- an optically active polypeptide which responds (e g , a conformation change)to, for example, a cell signaling event
- Cell signaling events that occur in vivo can be of very short duration
- the optically active polypeptides of the invention allow measurement of the optical parameter, such as fluorescence, which is altered in response to the cell signal, over the same time period that the event actually occurs Alternatively, the response can be measured after the event occurs (over a longer time period) as the response that occurs in an optically active polypeptide can be of a longer duration than the cell signaling event itself
- the invention provides isolated nucleic acid sequences which encode fluorescent indicator polypeptides having operatively inserted therein a sensor polypeptide, or fragment thereof, which normally exists in one state e g , conformational shape or charge, prior to an interaction with a chemical, biological, electrical or physiological parameter at which time it undergoes a response during or after the interaction of the chemical, biological, electrical or physiological parameter with the sensor polypeptide
- Polynucleotide or “nucleic acid sequence” refers to a polymeric form of nucleotides at least 10 bases in length
- isolated nucleic acid sequence is meant a polynucleotide that is no longer immediately contiguous with both of the coding sequences with which it was immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived
- the term therefore includes, for example, a recombinant DNA which is inco ⁇ orated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryotic or eukaryotic cell or organism, or which exists as a separate molecule (e g a cDNA) independent of other sequences
- the nucleotides of the invention can be ⁇ bonucleotides, deoxy ⁇ bonucleotides, or modified forms of either nucleotide
- the term includes single and double stranded forms of DNA
- Nucleic acid sequences which encode a fluorescent indicator of the invention, wherein the indicator includes a sensor polypeptide, or fragment thereof, which normally has two or more states or conformational arrangements, and which undergoes a response during interaction with a chemical, biological, electrical or physiological parameter can be operatively linked to expression control sequences
- “Operatively linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner
- An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences
- expression control sequences refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked
- Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence
- expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start cod
- promoter minimal sequence sufficient to direct transcription
- promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue- specific, or inducible by external signals or agents, such elements may be located in the 5' or 3' regions of the gene Both constitutive and inducible promoters, are included in the invention (see e g , Bitter et al , 1987, Methods in Enzymology 153:516-544).
- inducible promoters such as pL of bacte ⁇ ophage , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used
- promoters derived from the genome of mammalian cells e g , metallothionein promoter
- mammalian viruses e g , the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter, CMV promoter
- Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences of the invention.
- fluorescent protein and “fluorescent protein moiety” are used interchangeably and refer to any protein capable of emitting light when excited with appropriate electromagnetic radiation, and which has an amino acid sequence that is either natural or engineered and is derived from the amino acid sequence of an Aequorea-re ated fluorescent protein
- fluorescent indicator refers to a fluorescent protein having a sensor polypeptide whose emitted light vanes with the response state or conformation of the sensor polypeptide upon interaction with a chemical, biological, electrical or physiological parameter
- the term also refers to a fluorescent protein whose amino acid sequence has been circularly permuted
- the fluorescent indicators of the invention are also sensitive to pH in the range of about 5 to about 10
- the invention provides, for example, a functional engineered fluorescent protein indicator whose amino acid sequence is substantially identical to the 238 amino acid Aequoi ea victoi la green fluorescence protein (SEQ ID NO 3)
- the invention also includes functional polypeptide fragments of a fluorescent indicator
- the term "functional polypeptide fragment” refers to a polypeptide which possesses biological function or activity which is identified through a defined functional assay
- the term "functional fragments of a functional engineered fluorescent protein” refers to fragments of a functional engineered protein that retain a function of the engineered fluorescent protein, e g , the ability to fluoresce in a manner which is dependent upon interactions of a chemical, biological, electrical or physiological parameter with a sensor polypeptide over the pH range 5 to 10
- substantially identical or “substantially homologous” is meant a protein or polypeptide that retains the activity of a functional engineered fluorescent indicator, or nucleic acid sequence or polynucleotide encoding the same, and which exhibits at least 80%, preferably 85%, more preferably 90%, and most preferably 95%) homology to a reference amino acid or nucleic acid sequence
- the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids
- the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 1 10 nucleotides
- the reference amino acid sequence or nucleic acid sequence is considered homologous if the reference amino acid sequence is 80-95% homologous to any portion of the amino acid or nucleic acid sequence in question For example, where a circularly per
- substantially identical is meant an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one amino acid for another of the same class (e g , vahne for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the protein (e.g., assayed as described herein).
- such a sequence is at least 85%>, more preferably 90%, more preferably 95%o, more preferably 98%>, and most preferably 99%o identical at the amino acid sequence to one of the sequences of EGFP (SEQ ID NO:4), EYFP (SEQ ID NO 5), ECFP (SEQ ID NO 6), EYFP-V68L/Q69K (SEQ ID NO 7), YFP H 148G (SEQ ID NO- 8), or YFP H 148Q (SEQ ID NO-9).
- EGFP EYFP
- EYFP-V68L/Q69K SEQ ID NO 7
- YFP H 148G SEQ ID NO- 8
- YFP H 148Q SEQ ID NO-9
- sequence analysis software e.g.,
- the amino acid sequence of the protein includes one of the following sets of substitutions in the amino acid sequence of the Aequorea green fluorescent protein (SEQ ID NO 3) F64L/S65T/H231 L, referred to herein as EGFP (SEQ ID NO 4), S65G/S72A/T203Y/H231 L, referred to herein as EYFP (SEQ ID NO 5), S65G/V68
- ECFP K26R/F64L/S65T/Y66W/N146I/M153TN163A/N164H/H231L
- ECFP SEQ ID NO 6
- the amino acid sequences of EGFP, EYFP, ECFP, and EYFP-V68L/Q69K are shown in Tables 5-8, respectively
- the numbering of the amino acids conforms to that in native Aequorea GFP
- the first serine is amino acid number 2 even if a vahne (amino acid no la) has been inserted to optimize ⁇ bosome initiation
- F64L corresponds to a substitution of leucine for phenylalanine in the 64th amino acid following the initiating methionine
- the amino acid sequence of the protein is based on the sequence of the wild-type A equora green fluorescent protein, but includes the substitution H 148G (SEQ ID NO:8) or H 148Q (SEQ ID NO:9).
- these substitutions can be present along with other substitutions, e.g., the proteins can include the substitutions S65GN68L/S72 A/H 148G/Q80R T203 Y (SEQ ID ⁇ O:8), which is referred to herein as the "YFP H 148G mutant," S65G/V68L/S72A/H 148Q/Q80R T203Y, which is referred to herein as the "YFP H 148Q mutant" (SEQ ID NO:9), as well as EYFP-H 148G (SEQ ID NO: 10) and EFP-H148Q (SEQ ID NO: 1 1).
- the amino acid sequences of these mutants are shown in Tables 9-12, respectively. Table 9. Amino Acid Sequence of
- EYFP-H 148Q (SEQ ID NO: 11) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTG KLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSQNVYIMA DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSA LSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
- the protein or polypeptide is substantially purified.
- substantially pure protein or polypeptide is meant an functional engineered fluorescent polypeptide which has been separated from components which naturally accompany it.
- the protein or polypeptide is substantially pure when it is at least 60%), by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
- the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, of the protein.
- a substantially pure protein may be obtained, for example, by extraction from a natural source (e.g., a plant cell); by expression of a recombinant nucleic acid encoding a functional engineered fluorescent protein, or by chemically synthesizing the protein Purity can be measured by any appropriate method, e g , those described in column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis
- substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in E Colt or other prokaryotes
- the invention also provides polynucleotides encoding the functional engineered fluorescent protein described herein
- polynucleotides include DNA, cDNA, and RNA sequences which encode functional engineered fluorescent proteins It is understood that all polynucleotides encoding functional engineered fluorescent proteins are also included herein, as long as they encode a protein or polypeptide whose fluorescent emission intensity changes as pH vanes between 5 and 10
- Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides For example, the polynucleotide may be subjected to site-directed mutagenesis
- the polynucleotides of the invention include sequences that are degenerate as a result of the genetic code Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of the functional engineered fluorescent protein or derivative is functionally unchanged
- a polynucleotide sequence encoding a functional engineered fluorescent protein that includes one of the following sets of substitutions in the amino acid sequence of the Aequorea green fluorescent protein (S ⁇ Q ID NO 3) S65G/S72A/T203Y /H231L, S65G/V68L/Q69K/S72A/T203Y/H231L, or K26R/F64L/S65T/Y66W/N146I/ M153T/V163A/N 164H/H231L
- the DNA sequences encoding ⁇ GFP, ⁇ YFP, ⁇ CFP, ⁇ YFP-V68L/Q69K, YFP H 148G, and YFP H 148Q are those shown in Table 13-20 (SEQ ID NOs 12 to 19), respectively
- the nucleic acid encoding functional engineered fluorescent proteins may be chosen to reflect the codon choice in the native A.
- nucleic acids encoding a target functional engineered protein to be expressed in a human cell may have use a codon choice that is optimized for mammals, or especially humans.
- the nucleic acid sequences encoding the fluorescent indicator or circularly permuted fluorescent protein of the invention may be inserted into a recombinant expression vector
- recombinant expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the nucleic acid sequences encoding the chimeric peptides of the invention.
- the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells.
- Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al, Gene,56: 125, 1987), the pMSXND expression vector, or adeno or vaccinia viral vectors for expression in mammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521 , 1988), baculovirus-derived vectors for expression in insect cells, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV.
- the nucleic acid sequences encoding a fluorescent indicator or circularly permuted fluorescent protein of the invention can also include a localization sequence to direct the indicator to particular cellular sites by fusion to appropriate organellar targeting signals or localized host proteins.
- a polynucleotide encoding a localization sequence, or signal sequence can be ligated or fused at the 5' terminus of a polynucleotide encoding the fluorescence indicator such that the signal peptide is located at the amino terminal end of the resulting chimeric polynucleotide/polypeptide. In the case of eukaryotes, the signal peptide is believed to function to transport the chimeric polypeptide across the endoplasmic reticulum.
- the secretory protein is then transported through the Golgi apparatus, into secretory vesicles and into the extracellular space or, preferably, the external environment.
- Signal peptides which can be utilized according to the invention include pre-pro peptides which contain a proteolytic enzyme recognition site. Other signal peptides with similar properties to those described herein are known to those skilled in the art, or can be readily ascertained without undue experimentation.
- the localization sequence can be a nuclear localization sequence, an endoplasmic reticulum localization sequence, a peroxisome localization sequence, a mitochondrial localization sequence, or a localized protein. Localization sequences can be targeting sequences which are described, for example, in "Protein Targeting", Chapter 35 of Stryer, L., Biochemistry (4th ed.), W.H. Freeman, 1995. The localization sequence can also be a localized protein.
- Some important localization sequences include those targeting the nucleus (KKKRK) (SE Q ID NO:20), mitochondrion (amino terminal MLRTSSLFTRRVQPSLFRNILRLQST (SEQ ID NO.21)), endoplasmic reticulum (KDEL (SEQ ID NO:22) at C-terminus, assuming a signal sequence present at N-terminus), peroxisome (SKF at C-terminus), synapses (S/TDV or fusion to GAP 43, kinesin and tau) prenylation or insertion into plasma membrane (CaaX (SEQ ID NO 23), CC, CXC, or CCXX (SEQ ID NO:24) at C- terminus), cytoplasmic side of plasma membrane (chimeric to SNAP-25), or the Golgi apparatus (chimeric to funn)
- the construction of expression vectors and the expression of genes in transfected cells involves the use of molecular cloning techniques also well known in the art. Sambrook
- agents which induce a sensor polypeptide include agents that contain any of the amino acid sequences in Table 21 , or a portion thereof with the proviso that the parameter must bind to a calmoduhn sensor polypeptide
- the parameter can be a subsequence of a calmoduhn-binding domain.
- the moieties listed in Table 21 are recognized by the sensor polypeptide CaM. See, for example, C ⁇ vici, A. & Ikura, M Annu Rev. Biophys. Biomol. Struct. 24-84-1 16 (1995).
- the parameter can be modified to enhance the response of the fluorescent indicator to the parameter.
- Other parameter are known in the art for other sensor polypeptides.
- AC adenylyl cyclase
- BBMHCI brush-border myosin heavy chain-I
- CaMKJI calmoduhn kinase II
- CBP2 calmoduhn binding peptide-2
- GIP gastrin inhibitory peptide
- HIV-1 gpl60 human immunodeficiency virus envelope glycoprotein 160
- HSP heat-shock protein
- MARCKS myristoylated alaminte-rich C kinase substrate
- MHC myosin heavy chain
- NOS nitric oxide synthase
- PDE phosphodiesterase
- PFK phosphofructokinase
- PhK phosphorylase kinase
- sk- smMLCK
- VIP vasoactive intestinal peptide.
- the length of the linker moiety is chosen to optimize the kinetics and specificity of responsiveness of the sensor polypeptide induced by the interaction of the chemical, biological, electrical or physiological parameter with the sensor polypeptide.
- the linker moiety should be long enough and flexible enough to allow the sensor polypeptide to freely interact and respond to a particular parameter.
- the linker moiety is, preferably, a peptide moiety.
- the preferred linker moiety is a peptide between about one and 30 amino acid residues in length, preferably between about two and 15 amino acid residues.
- One preferred linker moiety is a -Gly-Gly- linker.
- the linker moiety can include flexible spacer amino acid sequences, such as those known in single-chain antibody research.
- the linker moiety can be GGGGS (SEQ ID NO:56)(GGGGS)n, GKSSGSGSESKS (SEQ ID NO:57), GSTSGSGKSSEGKG (SEQ ID NO:58), GSTSGSGKSSEGSGSTKG (SEQ ID NO:59) GSTSGSGKSSEGKG (SEQ ID NO:60), GSTSGKPGSGEGSTKG (SEQ ID NO:61), EGKSSGSGSESKEF (SEQ ID NO:62), GGTGEL (SEQ ID NO: 1), FKTRHN (SEQ ID NO:2), or GGTGGS
- any of a number of suitable transcription and translation elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see, e.g., Bitter, et al, Methods in Enzymology 153:516-544, 1987). These elements are well known to one of skill in the art.
- a number of expression vectors may be advantageously selected depending upon the intended use. For example, when large quantities of a protein of the invention is desired, vectors which direct the expression of high levels of chimeric protein products that are readily purified may be desirable. Those which are engineered to contain a cleavage site to aid in protein recovery are preferred.
- yeast a number of vectors containing constitutive or inducible promoters may be used.
- Current Protocols in Molecular Biology Vol. 2, Ed. Ausubel, et al, Greene Publish. Assoc. & Wiley Interscience, Ch. 13, 1988; Grant, et al, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp.516-544, 1987; Glover, DNA Cloning, Vol. II, IRL Press, Wash., D.C, Ch.
- yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol. l 1, A Practical Approach, Ed. DM Glover, IRL Press, Wash., D.C, 1986).
- vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome.
- An alternative expression system which could be used to express the proteins of the invention is an insect system.
- Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
- the virus grows in Spodoptera frugiperda cells.
- the sequence encoding a protein of the invention may be cloned into non-essential regions (for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
- Another alternative expression system includes plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing a fluorescent indicator or circularly permuted fluorescent protein.
- recombinant virus expression vectors e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
- plasmid expression vectors e.g., Ti plasmid
- transformation is meant a permanent or transient genetic change induced in a cell following incorporation of new DNA (i.e. DNA exogenous to the cell).
- new DNA i.e. DNA exogenous to the cell.
- a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell
- transformed cell is meant a cell into which (or into an ancestor of which has been introduced), by means of recombinant DNA techniques, a DNA molecule encoding a fluorescent indicator or circularly permuted fluorescent protein having an optically active polypeptide having operatively inserted therein a sensor polypeptide, or fragment thereof, which normally has two or more states, and which is affected by a chemical, biological, electrical or physiological parameter
- Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art Where the host is prokaryotic, such as E Coh, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCL method by procedures well known in the art Alternatively, MgCl or RbCl can be used Transformation can also be performed after forming a protoplast of the host cell or by electroporation
- Eukaryotic cells can also be cotransfected with DNA sequences encoding the chimeric polypeptide of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene
- a eukaryotic viral vector such as simian virus 40 (SV40) adenovirus, vaccinia virus, or bovine papilloma virus
- SV40 simian virus 40
- Eukaryotic systems and preferably mammalian expression systems, allow for proper post-translational modifications of expressed mammalian proteins to occur Eukaryotic cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation. and, secretion of the gene product should be used as host cells for the expression of fluorescent indicator
- host cell lines may include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and WI38
- Mammalian cell systems which utilize recombinant viruses or viral elements to direct expression may be engineered
- the nucleic acid sequences encoding a fluorescent indicator or circularly permuted fluorescent protein of the invention may be gated to an adenovirus transcription/translation control complex, e g , the late promoter and tripartite leader sequence
- This nucleic acid sequence may then be inserted in the adenovirus genome by in vitro or in vivo recombination Insertion in a non-essential region of the viral genome (e g , region E l or E3) will result in a recombinant virus that is viable and capable of expressing the fluorescent indicator in infected hosts (e g , see Logan & Shenk, Proc Natl Acad Sci USA, 81 3655-3659, 1984)
- the vaccinia virus 7 5K promoter may be used (e g , see, Mackett, et al ,
- vectors can be used for stable expression by including a selectable marker in the plasmid, such as the neo gene
- the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the fluorescent indicator gene in host cells (Cone & Mulligan, Proc Natl Acad Sci USA, 81 6349-6353, 1984)
- High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothiomne IIA promoter and heat shock promoters For long-term, high-yield production of recombinant proteins, stable expression is preferred.
- host cells can be transformed with the cDNA encoding a fluorescent indicator or circularly permuted fluorescent protein of the invention controlled by appropriate expression control elements (e g , promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc ), and a selectable marker.
- expression control elements e g , promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc
- the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
- engineered cells may be allowed to grow for 1 -2 days in an enriched media, and then are switched to a selective media.
- a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al , Cell, 1 1 : 223, 1977), hypoxanthine- guanine phosphonbosyltransferase (Szybalska & Szybalski, Proc. Natl Acad. Sci.
- adenine phosphonbosyltransferase genes can be employed in tk-, hgprt- or aprt- cells respectively
- antimetabohte resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al , Proc. Natl Acad Sci USA, 77: 3567, 1980; O ⁇ are, et al, Proc. Natl. Acad. Sci.
- trpB which allows cells to utilize indole in place of tryptophan
- hisD which allows cells to utilize histinol in place of histidine
- ODC ornithine decarboxylase
- a fluorescent indicator or circularly permuted fluorescent protein of the invention can be produced by expression of nucleic acid encoding the protein in prokaryotes. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors encoding a chimeric protein of the invention.
- a primary advantage of the optically active polypeptides of the invention is that they are prepared by normal protein biosynthesis, thus avoiding organic synthesis and the requirement for customized unnatural amino acid analogs.
- the constructs can be expressed in E Coli in large scale for in vitro assays. Purification from bacteria is simplified when the sequences include tags for one-step purification by nickel-chelate chromatography.
- the construct can also contain a tag to simplify isolation of the fluorescent indicator.
- a polyhistidine tag of, e.g., six histidine residues, can be incorporated at the amino terminal end of the fluorescent protein.
- the polyhistidine tag allows convenient isolation of the protein in a single step by nickel- chelate chromatography.
- the substrates can be expressed directly in a desired host cell for assays in situ.
- Techniques for the isolation and purification of either microbially or eukaryotically expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies or antigen.
- nucleic acid sequence can also function as the concentration or available parameter in another embodiment of the invention.
- a response may result from the interaction of a nucleic acid sequence with a sensor polypeptide comprising a DNA binding protein motif.
- the invention features a method for determining the presence of a chemical, biological, electrical or physiological parameter, by contacting the sample with a fluorescent indicator or circularly permuted fluorescent protein of the invention; exciting the indicator or protein; and measuring the amount of an optical property of the indicator or protein in the presence and absence of a parameter, such that a change in the optical property is indicative of an affect of the parameter on the indicator or protein
- a series of standards, with known levels of activity can be used to generate a standard curve
- the optical event such as intensity of fluorescence, that occurs following exposure of the sample to the fluorescent indicator or protein is measured, and the amount of the optical property is then compared to the standard curve.
- a standard with a known level of activity, can be used to generate a standard curve, or to provide reference standards
- the optical event, such as fluorescence, that occurs following exposure of the sample to the fluorescent indicator or protein is measured, and the amount of the optical property (e g , fluorescence) is then compared to the standard in order to generate a relative measure of the affect of the sample on the fluorescent indicator
- the invention features a method for determining if a cell exhibits an activity, which includes transfectmg the cell with a nucleic acid encoding a fluorescent indicator or circularly permuted fluorescent protein of the invention, exciting the fluorescent indicator or circularly permuted fluorescent protein; and measuring the amount of an optical property in the presence of the activity and in the absence of the activity, such that a change in the optical property is indicative of activity
- the optical property is calibrated against standard measurements to yield an absolute amount of protein activity
- the invention additionally, features methods for determining transient changes in a chemical, biological, electrical or physiological parameter, by contacting the sample with a fluorescent indicator or circularly permuted fluorescent protein of the invention and measuring a change in the optical property of the indicator over time
- a cell containing such a nucleic acid sequence is a composition provided by the present invention
- the invention can be used in screening assays to determine whether a compound (e g , a drug, a chemical or a biologic) alters the activity of a particular protein, i e , the sensor polypeptide (e g , ligand binding to a receptor)
- the assay is performed on a sample containing the chimeric protein in vitro
- a sample containing a known amount of activity, such as an enzymatic activity is mixed with a fluorescent indicator substrate of the invention, with the co- factors required for activity, and with a test compound The amount of the enzyme activity in the sample is
- the ability of a compound to alter the activity of a particular protein (i e , a sensor polypeptide) in vivo is determined
- a particular protein i e , a sensor polypeptide
- cells transfected with a expression vector encoding a substrate of the invention are exposed to different amounts of the test compound, and the effect on the optical parameter, such as fluorescence, in each cell can be determined Typically, the difference is calibrated against standard measurements to yield an absolute amount of protein activity
- This provides a method for screening for compounds which affect cellular events (e g , receptor-hgand binding, protein-protein interactions or protein kinase activation)
- any measurable change between activity in the presence of the test compound as compared with the activity in the absence of the test compound is indicative of activity
- kits for determining the presence of an activity in a sample may contain a container containing a chimeric protein comprising an optically active polypeptide having operatively inserted therein a sensor polypeptide, or fragment thereof, which is affected by a change in a parameter or the environment, wherein optical properties of the sensor are altered in response to the change
- a kit of the invention contains an isolated nucleic acid sequence which encodes a chimeric protein comprising an optically active polypeptide having operatively insereted therein a sensor polypeptide, or fragment thereof, which is affected by a change in a parameter or the environment, wherein optical properties of the sensor are altered in response to the change
- the nucleic acid sequence of the later kit may be contained in a host cell, preferably stably transfected The cell could optionally be transiently transfected Thus, the cell acts as an indicator kit in itself
- the present invention relates to transgenic animals that have cells that express an optically active polypeptide having operatively inserted therein a sensor polypeptide, or fragment thereof, which normally is capable of existing in two or more states, and which causes a change in the optical properties of the optically active polypeptide upon environmental conditions or parameters
- Transgenic animals expressing high levels of the tagged transgene may be obtained, for example, by over-expression of the transgene with an enhanced promoter and/or with high copy numbers of the transgene
- the transgenic animal may be heterozygous or homozygous for an ablated or disrupted endogenous indicator gene
- non-human animals comprise any non-human animal having nucleic acid sequence which encodes a fluorescent indicator or circularly permuted fluorescent protein of the invention
- non-human animals include vertebrates such as rodents, non-human primates, sheep, dog, cow, pig, amphibians, reptiles and fish
- Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse
- the "transgenic non-human animals” of the invention are produced by introducing "transgenes" into the germhne of the non-human animal Embryonal target cells at various developmental stages can be used to introduce transgenes Different methods are used depending on the stage of development of the embryonal target cell The zygote is the best target for micro- mjection In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of 1 -2 pi of DNA solution.
- zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be inco ⁇ orated into the host gene before the first cleavage (Bnnster et al , Proc. Natl. Acad. Sci. USA 82 4438-4442, 1985). As a consequence, all cells of the transgenic non-human animal will carry the inco ⁇ orated transgene This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50%> of the germ cells will harbor the transgene. Microinjection of zygotes is the preferred method for inco ⁇ orating transgenes in practicing the invention.
- transgenic is used to describe an animal which includes exogenous genetic material within all of its cells
- a "transgenic” animal can be produced by cross-breeding two chimeric animals which include exogenous genetic material within cells used in reproduction Twenty-five percent of the resulting offspring will be transgenic / e., animals which include the exogenous genetic material within all of their cells in both alleles. 50% of the resulting animals will include the exogenous genetic material within one allele and 25% will include no exogenous genetic matenal.
- Viral infection can also be used to introduce transgene into a non-human animal (e.g., retroviral, adenoviral or any other RNA or DNA viral vectors).
- the developing non-human embryo can be cultured in vitro to the blastocyst stage. Dunng this time, the blastomeres can be targets for retro viral infection (Jaenich, R , Proc Natl. Acad. Sci USA 73: 1260-1264, 1976). Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan, et al. (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spnng Harbor, N.Y.).
- the viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner, et al , Proc. Natl. Acad. Sci. USA 82:6927-6931 , 1985; Van der Putten, et al, Proc. Natl. Acad. Sci USA 82:6148-6152, 1985). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart, et al, EMBO J.
- infection can be performed at a later stage Virus or virus-producing cells can be injected into the blastocoele (D Jahner et al , Nature 298 623-628, 1982)
- Most of the founders will be mosaic for the transgene since inco ⁇ oration occurs only in a subset of the cells which formed the transgenic nonhuman animal
- the founder may contain various retro viral insertions of the transgene at different positions in the genome which generally will segregate in the offspring
- ES embryonal stem cell
- Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retro virus-mediated transduction Such transformed ES cells can thereafter be combined with blastocysts from a nonhuman animal The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
- Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retro virus-mediated transduction
- Such transformed ES cells can thereafter be combined with blastocysts from a nonhuman animal The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal.
- Heterologous refers to a nucleic acid sequence that either originates from another species or is modified from either its original form or the form primarily expressed in the cell
- Transgene means any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism (i e , either stably integrated or as a stable extrachromosomal element) which develops from that cell
- a transgene may include a gene which is partly or entirely heterologous (i e , foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism Included within this definition is a transgene created by the providing of an RNA sequence which is transcribed into DNA and then inco ⁇ orated into the genome
- the transgenes of the invention include DNA sequences which encode the fluorescent indicator or circularly permuted fluorescent protein of the invention which may be expressed in a transgenic non-human animal
- transgenic as used herein additionally includes any organism whose genome has been altered by in vitro manipulation of the early embryo or fertilized egg or by any transgenic technology to induce a specific gene knockout
- gene knockout refers to the
- Sites in, for example, GFP mutants which can tolerate insertions of sensor polypeptides can be identified by generating mutant proteins by manipulating the DNA sequence such that a vanety of different insertions are produced and screening the mutants by flow cytometry for mutants which retain fluorescence
- Such insertions may include replacement of certain amino acids, as well as the addition of a new sequence without a corresponding deletion or replacement in the sequence of the fluorescent protein Variants identified in this fashion reveal sites which can tolerate insertions while retaining fluorescence
- circularly permutation techniques are also useful in identifying sites in fluorescent proteins which are capable of tolerating insertions while retain the ability to fluoresce
- Such techniques include are exemplified herein as well as known to those of skill in the art (see, for example, Graf et al , Proc Natl. Acad. Sci USA, 93 : 1 1591 - 1 1596 (October 1996), the disclosure of which is inco ⁇ orated herein)
- the original N- and C-terminal amino acids of a fluorescent protein are engineered to be linked by a linker moiety.
- linker moieties include those described above, as well as other easily ascertain by one skilled in the art. This is typically performed at the nucleic acid level resulting in a polynucleotide sequence wherein the 5' codon encoding the N-terminal amino acid is linked to the 3' codon encoding the C-terminal amino acid, resulting in a circularized fluorescent protein nucleic acid sequence
- the circularized sequence is then cleaved with a nuclease to create a linear polynucleotide sequence, the cleavage site corresponding to an amino acid in of the fluorescent protein
- the cleavage of the circularized sequence is either random or specific depending on the desired product, nuclease, and desired sequence.
- linearized polynucleotide which contains sequence homologous to the starting fluorescent protein sequence, is cloned into an expression vector and expressed
- the expressed polypeptide sequence is then screened, for example by flow cytometry, for polypeptides retaining the ability to fluoresce. Accordingly, polypeptides which retain the ability to fluorescence correspondingly, via identification of the cleavage site, identify amino acids which can tolerate insertions without destroying the ability of the fluorescent protein to fluoresce
- Bacteria used in this study were BL21(DE3) Gold cells from Stratagene Transformation was performed by electroporating cells suspended in 10%> glycerol directly with a hgation mixture (0.1cm cuvette, 12 5 kV/cm, 200 ⁇ , 25uFd)
- a hgation mixture 0.1cm cuvette, 12 5 kV/cm, 200 ⁇ , 25uFd
- cells were grown in LB containing 100 mg/L Ampicilhn to an OD 6 oo of 0.6, at which time they were induced with ImM Isopropylthiogalactoside Bacteria were allowed to express recombinant protein for 6 hours at room temperature and then overnight at 4°C The bacteria were then pelleted by centrifugation, resuspended in 50mM Tns, 300mM NaCl and lysed with a French Press The bacterial lysates were centrifuged at 30,000xg for 30 minutes, and the supernatants were in
- Yellow GFP mutants with peptide insertions replacing Y145 were made by performing two separate polymerase chain reactions (PCRs).
- the first PCR amplified the N terminal piece of YFP to include a 5' BamHI site and 3' replacement of Y 145 with the hexapeptide linker GGTGEL (coded for by DNA containing Kpnl and Sacl restriction sites for subsequent cloning).
- the second PCR amplified the C terminal piece of YFP to include the 5' linker (GGTGEL) replacing Y'145 and a 3' EcoRI site
- GGTGEL 5' linker
- N and C terminal YFP primers were combined, amplified with N and C terminal YFP primers to yield a full length cDNA containing the insertion
- the full length cDNA was restricted with BamH I and EcoRI, ligated and cloned into the BamHI and EcoRI sites of pRSET B (Invitrogen) to yield the plasmid pYFPins.
- Protein pH titrations were carried out in 125mM KCl, 20mM NaCl, 0 5mM CaCl 2 , 0 5mM MgCl 2 , and 50mM buffer Buffers were chosen to span a wide pH range, and included citrate (pH4-5), MES (pH 5 5-6 5), HEPES (pH 7-8 15), glycine (pH 8 8-10 7), and phosphate (pHl 1 3-13 2)
- citrate pH4-5
- MES pH 5 5-6 5
- HEPES pH 7-8 15
- glycine pH 8 8-10 7
- phosphate pHl 1 3-13 2
- a weakly buffered protein solution was mixed with an equal volume of the corresponding buffer solution and analyzed for total fluorescence in triplicate on a microplate fluorescence reader using a 482+/- 10 nm excitation filter and a 532+/- 14 nm emission filter (Figure 2a)
- Zinc titrations of YFP-zinc finger insertions were done in 50mM MOPS, pH 7 0 A fluorescence emission spectrum was taken of the protein in buffer containing 50uM EDTA, and then small aliquots of ZnCl 2 were added, and subsequent spectra were recorded ( Figure 4)
- Titration curves were generated by averaging the three intensity values for each pH (for microplate data) or be integrating the total emission intensity (for full spectra), plotting these data versus analyte concentration, and fitting a sigmoidal curve to the data
- YFP containing calmoduhn replacing Y145 show an pH-sensitive increase in fluorescence intensity on calcium binding, with an apparent Kd of fluorescence of 7 ⁇ M
- the increase in fluorescence observed at constant pH reflects a shift of 1 pKa unit between the calcium-free and calcium-bound states of the protein, as observed in pH titrations done in the presence of or absence of free calcium (Figure 2)
- the absorbance of this protein changes from a predominantly ultraviolet, non-fluorescent band to a predominantly blue, fluorescent band on calcium binding at constant pH Since other studies suggest that the non-fluorescent, ultraviolet- absorbing band represents the protonated chromophore and the fluorescent, blue- absorbing band represents the deprotonated chromophore, these data show that calcium, binding alters the equilibrium between protonated and deprotonated chromophore states, i e changes the pKa of fluorescence
- YFPs containing a zinc-finger motif derived from z ⁇ f268 also increase in fluorescence on binding zinc
- the change in fluorescence for Zn-sensing YFPs is substantially less than that of Calmoduhn- YFPs described above
- Zn finger motifs contain cysteine residues in close proximity, they can be prone to oxidation, which would prevent zinc binding
- the change of inserted proteins on substrate binding is responsible for the change in pKa and therefore the change in fluorescence
- zinc-finger motifs adopt a non-optimal conformation on binding zinc, leaving the chromophore still vulnerable to protonation
- each of the three reasons listed above concerning the performance of YFP-Calmoduhn Insertions could in principle also apply analogously to the zinc finger insertions
- the zinc-finger inserted YFP reported here is just a first generation prototype of what will likely become a powerful new class of indicators
- Circular Permutations of GFP mutants with a new N terminus at Y 145 were made by performing two separate PCRs
- the first PCR amplified the C terminal piece of a GFP mutant (N terminal in the final permuted gene) to have a 5' BamHI site, the mutation Y145M, and a 3' hexapeptide linker (GGTGGS) containing a Kpnl site
- the second PCR amplified the N terminal piece of the GFP mutant (C terminal in the final permuted gene) to have a 5' hexapeptide linker (GGTGGS) containing a Kpnl site, and a 3' EcoRI site
- the first PCR product was digested with sequentially with BamH I and Kpnl
- the second PCR product was digested sequentially with EcoRI and Kpnl
- the fragments were purified by agarose gel electrophoresis
- the N and C terminal PCR fragments were then clone
- CFP cDNA was then ligated to the YC3.1 *2 ⁇ CFP fragment, and the DNA was transformed into BL21(DE3) Gold cells as described above.
- Random Circular Permutations were based on the method of Graf, et al. with major modifications because their original conditions were found to be unsuccessful when applied to GFP cDNAs. Through extensive testing, it was found that circular permutation required 1) reducing the concentration of DNA used when circularizing the gene from 300 ⁇ g/mL to 5 ⁇ g/mL, 2) increasing the amount of DNAse used to relinearize the fragment from 1 U/mg DNA to 100 U/mg DNA, 3) changing the temperature of DNAse incubation from 16 degrees to 22 degrees Celsius, and switching the DNA repair enzyme used from T4 to T7 DNA polymerase. Accordingly, the method of Graf et al, was substantially modified as follows.
- An expression vector for the random circular permutations was made by ligating an oligonucleotide containing a 5' EcoRV site and three downstream stop codons in each reading frame between the BamHI and EcoRI sites of pRSET B.
- This vector (“pRSET triple stop”) was digested with EcoRV, treated with Alkaline Phosphatase, and purified by agarose gel electrophoresis.
- the circularly permuted GFP gene described above was amplified by PCR with primers that created a final PCR amplicon starting and ending at an Xhol site (ctcgag) coding for residues L141 and E142.
- the PCR product was digested with Xhol and cloned into the Xhol site of pBluescript.
- This plasmid was amplified in bacteria, purified with a Qiagen maxi- prep, digested with Xhol , and the ⁇ 730bp fragment was agarose gel-purified to yield a linearized GFP gene.
- the linear fragment was circularized at a concentration of 5 ⁇ g/mL with 8000 U/mL T4 DNA ligase (New England Biolabs) overnight at 16°C. After ethanol precipitation, the DNA was digested with DNAse (100 U/mg DNA, Pharmacia) for 15 minutes at room temperature in 50mM T ⁇ s HCl, pH7.5 and ImM MnC12. Digestion was stopped by phenol extraction, then subsequent phenol/chloroform/Isoamyl Alcohol and Chloroform Isoamyl alcohol extractions.
- the linear, repaired, randomly permuted DNA library was purified by agarose gel electrophoresis, ligated blunt into the pRSET triple stop expression vector, and electro-transformed into BL21 bacteria
- LB/agar plates containing ampicilhn usually displayed a few thousand colonies per plate and were screened by digital imaging of fluorescence.
- the plates were illuminated with a 150 W xenon arc lamp through a 450-490 nm bandpass filter and a pair of fiber optic light guides (Oriel Instruments) positioned to illuminate the top surface of the agar as evenly as possible.
- the emitted fluorescence was selected by a 510-550 nm bandpass filter and focused by a f/1.2 camera lens (Nikon) onto a cooled charge-coupled-device camera (Photometries). Digital images from the camera were analyzed with Metafluor software (Universal Imaging Co ⁇ ).
- GFP forms a fluorescent circularly permuted protein when its native N and C termini are connected with the hexapeptide linker GGTGGS and new N termini are formed at E142, Y143, Y145, H148, D155, H169, E172, D173, A227, or 1229 (See Table 22).
- the permuted protein with the N terminus at Y 145 was made and studied for the Cyan, Green, and Yellow mutants of GFP (cpCFP, cpGFP, cpYFP). In each case, the protein had a higher pKa of fluorescence than its native counte ⁇ art, although the fluorescence spectra were similar. This suggests that interrupting GFP and its mutants at Y 145 generally increases the chromophore's sensitivity to protonation, which is in agreement with the results obtained from the GFP insertions described above.
- Starting Amino Acid is the first amino acid for which is not coded for by the expression vector (which may also have been mutated), e g. E142M means that the GFP starts at Position 142, but the glutamate residue has been changed to methionine by the cloning process.
- N 144LSE means that the GFP sequence ends with asparagine at position 144, but is appended by the C terminal tnpeptide LSE.
- N144 means that the protein simply ends with asparagine at position 144 with no addition peptide.
- cpGFP mutants Another possible use of cpGFP mutants is in making novel insertions of GFP into other proteins for use as biosensors GFP, because its termini are close in space, can be inserted into other proteins, but only rarely to date has it been shown to sense a conformational change in such a construct
- cpGFP mutants When cpGFP mutants are inserted into a protein, they are topologically similar to the GFP insertion constructs described above, and they might reasonably be expected to have similar sensing properties as GFP insertions (Figure 7)
- GFP mutants with peptide insertions replacing Y145 were made by performing two separate polymerase chain reactions (PCRs)
- the first PCR amplified the N-terminal piece of GFP to include a 5' BAMHI site and 3' replacement of Y145 wit the hexapeptide linker GGTGEL (coded for by DNA containing Kpnl and Sacl restriction sites for subsequent cloning)
- the second PCR amplified the C-terminal piece of GFP to include the 5' linker (GGTGEL) replacing Y145 and a 3' EcoRI site
- GGTGEL 5' linker replacing Y145 and a 3' EcoRI site
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AU52751/00A AU5275100A (en) | 1999-05-21 | 2000-05-17 | Fluorescent protein indicators |
US09/999,745 US7060793B2 (en) | 1999-05-21 | 2001-10-23 | Circularly permuted fluorescent protein indicators |
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US09/316,920 US6699687B1 (en) | 1999-05-21 | 1999-05-21 | Circularly permuted fluorescent protein indicators |
US09/316,919 US6469154B1 (en) | 1999-05-21 | 1999-05-21 | Fluorescent protein indicators |
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US6900304B2 (en) * | 1996-01-31 | 2005-05-31 | The Regents Of The University Of California | Emission ratiometric indicators of phosphorylation |
US8669074B2 (en) | 1996-01-31 | 2014-03-11 | The Regents Of The University Of California | Chimeric phosphorylation indicator |
EP1132397A4 (en) | 1998-11-11 | 2002-10-23 | Mitsubishi Chem Corp | Sensor protein and utilization thereof |
US8148110B2 (en) | 1999-03-15 | 2012-04-03 | The Board Of Trustees Of The Leland Stanford Junior University | Detection of molecular interactions by β-lactamase reporter fragment complementation |
WO2001009177A2 (en) * | 1999-07-29 | 2001-02-08 | Whitehead Institute For Biomedical Research | Affinity fluorescent proteins and uses thereof |
JP3650815B2 (en) * | 2000-11-22 | 2005-05-25 | 大学共同利用機関法人自然科学研究機構 | Method for producing biosensor protein capable of controlling fluorescence characteristics of green fluorescent protein, and biosensor protein produced by the method |
JP2002253261A (en) * | 2001-03-05 | 2002-09-10 | Inst Of Physical & Chemical Res | Fluorescent protein |
US7176345B2 (en) | 2001-03-20 | 2007-02-13 | Dana-Farber Cancer Institute, Inc. | Transgenic animals expressing light-emitting fusion proteins and diagnostic and therapeutic methods therefor |
US7919274B2 (en) * | 2001-03-20 | 2011-04-05 | Dana-Farber Cancer Institute, Inc. | Light-emitting fusion proteins and diagnostic and therapeutic methods therefor |
US7166463B2 (en) | 2001-11-16 | 2007-01-23 | The Regents Of The University Of Colorado | Nucleic acids encoding modified olfactory cyclic nucleotide gated ion channels |
DE10257354A1 (en) * | 2002-12-09 | 2004-07-08 | Bayer Healthcare Ag | Isolated fluorescent protein CGFP and its use |
CA2445420A1 (en) * | 2003-07-29 | 2005-01-29 | Invitrogen Corporation | Kinase and phosphatase assays |
US7619059B2 (en) * | 2003-07-29 | 2009-11-17 | Life Technologies Corporation | Bimolecular optical probes |
EP1686379A4 (en) * | 2003-10-15 | 2007-11-21 | Riken | Fluorescence indicator utilizing fret |
EP1793867A4 (en) | 2004-09-17 | 2011-04-13 | Univ Utah Res Found | Imaging reporters of transgene expression |
US20080178309A1 (en) * | 2005-03-25 | 2008-07-24 | Belousov Vsevolod V | Fluorescent Indicators of Hydrogen Peroxide and Methods for Using Same |
US20100143942A1 (en) * | 2006-09-21 | 2010-06-10 | Leiden University | Method of detection |
EP2069785A1 (en) * | 2006-09-21 | 2009-06-17 | Leiden University | Immobilisation of fluorescent proteins |
GB0625678D0 (en) * | 2006-12-21 | 2007-01-31 | Lux Biotechnology Ltd | Composition and method for detection of demineralisation |
KR20100065294A (en) * | 2007-08-03 | 2010-06-16 | 국립대학법인 홋가이도 다이가쿠 | Fluorescent protein with deep blue color |
WO2009131951A2 (en) | 2008-04-21 | 2009-10-29 | Institute For Oneworld Health | Compounds, compositions and methods comprising isoxazole derivatives |
US8207205B2 (en) | 2008-04-21 | 2012-06-26 | Institute For Oneworld Health | Compounds, compositions and methods comprising oxadiazole derivatives |
EP2141179B1 (en) | 2008-07-04 | 2010-12-01 | Technische Universität Dresden | A fluorescence based reporter construct for the direct detection of TGF-beta receptor activation and modulators thereof |
US8343976B2 (en) | 2009-04-20 | 2013-01-01 | Institute For Oneworld Health | Compounds, compositions and methods comprising pyrazole derivatives |
CN102128938B (en) * | 2010-12-20 | 2013-06-19 | 长春理工大学 | Anabolic steroids and polycyclic aromatic hydrocarbon high-efficiency bioluminescence sensor and construction method thereof |
WO2015190083A1 (en) * | 2014-06-11 | 2015-12-17 | 国立研究開発法人科学技術振興機構 | Calcium reporter gene |
CN106489828A (en) * | 2016-10-17 | 2017-03-15 | 广西大学 | The method for building up of oxidative stress model in the infection induced Mice Body of PRRSV |
GB201700317D0 (en) | 2017-01-09 | 2017-02-22 | Calcivis Ltd | Detection device |
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