Reagents and compositions for use in reactions catalysed by luciferase enzymes, and in particular for use in luciferase-based gene reporter assays are described. The invention also provides methods and compositions for, inter alia, increasing the sensitivity and/or improving the kinetics of luciferase-catalysed reactions.
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0. 16. A method for increasing the bioluminescent signal generated by a recombinant luciferase, the method comprising:
a) contacting the recombinant luciferase with a reagent composition comprising one or more of bromide anion, a chelator, an oxidizing agent, redox buffer, and a buffer with a ph greater than 8.0;
b) adding a substrate of the luciferase; and
c) detecting bioluminescence in the sample;
wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.
0. 22. A method for reducing the rate of decay of the bioluminescent signal generated by a recombinant luciferase, the method comprising:
a) contacting the recombinant luciferase with a reagent composition comprising one or more of bromide anion, a chelator, an oxidizing agent, redox buffer, and a buffer with a ph greater than 8.0;
b) adding a substrate of the luciferase; and
c) detecting bioluminescence in the sample;
wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.
0. 9. A method for determining the amount and/or activity of a recombinant luciferase in a cell or sample of cells, the method comprising:
a) contacting the cell or sample of cells with a reagent composition comprising a detergent and one or more of bromide anion, a chelator, an oxidizing agent, redox buffer, and a buffer with a ph greater than 8.0;
b) adding a substrate of the luciferase; and
c) detecting bioluminescence in the sample;
wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.
0. 20. A method for increasing the bioluminescent signal generated by a recombinant luciferase, the method comprising:
a) contacting the recombinant luciferase with a reagent composition that provides an environment that enables conversion of the luciferase into an active state, comprising one or more of bromide anion, a chelator, an oxidizing agent, redox buffer, and a buffer with a ph greater than 8.0;
b) adding a substrate of the luciferase; and
c) detecting bioluminescence in the sample;
wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.
0. 26. A method for reducing the rate of decay of the bioluminescent signal generated by a recombinant luciferase, the method comprising:
a) contacting the recombinant luciferase with a reagent composition that provides an environment that enables conversion of the luciferase into an active state, comprising one or more of bromide anion, a chelator, an oxidizing agent, redox buffer, and a buffer with a ph greater than 8.0;
b) adding a substrate of the luciferase; and
c) detecting bioluminescence in the sample;
wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.
0. 13. A method for determining the amount and/or activity of a recombinant luciferase in a cell or sample of cells, the method comprising:
a) contacting the cell or sample of cells with a reagent composition that lyses the cell or sample of cells and provides an environment that enables conversion of the luciferase into an active state, comprising a detergent and one or more of bromide anion, a chelator, an oxidizing agent, redox buffer, and a buffer with a ph greater than 8.0;
b) adding a substrate of the luciferase; and
c) detecting bioluminescence in the sample;
wherein the recombinant luciferase is a non-secreted variant or derivative of a luciferase that is secreted in its native form.
0. 1. A method for conducting a luciferase-based assay comprising,
expressing within a eukaryotic cell a luciferase that is secreted in its native form, wherein the luciferase lacks a functional signal peptide such that it is expressed intracellularly in the eukaryotic cell;
recovering the activity of the luciferase by contacting the luciferase with a reagent composition that provides an environment suitable for conversion of the luciferase into its active folded form, wherein the reagent composition comprises an agent selected from the group consisting of bromide anion, chelator, redox buffer, and buffer with a ph greater than 8.0;
contacting the luciferase with a substrate of the luciferase enzyme; and
detecting or measuring the bioluminescence.
0. 2. The method of
0. 3. The method of
0. 4. The method of
0. 5. A method for conducting a luciferase-based assay comprising,
expressing within a eukaryotic cell a luciferase that is secreted in its native form, wherein the luciferase lacks a functional signal peptide such that it is expressed intracellularly in the eukaryotic cell;
recovering the activity of the luciferase by contacting the luciferase with a reagent composition that provides an environment suitable for conversion of the luciferase into its active folded form, wherein the reagent composition comprises an oxidising agent;
contacting the luciferase with a substrate of the luciferase enzyme; and
detecting or measuring the bioluminescence.
0. 6. The method of
0. 7. The method of
0. 8. The method of
0. 10. The method of claim 9, wherein step (b) occurs simultaneously with, or following step (a).
0. 11. The method of claim 9, wherein the luciferase lacks a functional signal peptide.
0. 12. The method of claim 9, wherein the detergent is a non-ionic or zwitterionic detergent.
0. 14. The method of claim 13, wherein conversion of the luciferase into an active state means either the conversion from an inactive state to an active state, or the conversion from a partially active or less active state to a more active state.
0. 15. The method of claim 13, wherein the detergent is a non-ionic or zwitterionic detergent.
0. 17. The method of claim 16, wherein step (b) occurs simultaneously with, or following step (a).
0. 18. The method of claim 16, wherein the luciferase lacks a functional signal peptide.
0. 19. The method of claim 16, wherein the reagent composition further comprises a detergent selected from a non-ionic or zwitterionic detergent.
0. 21. The method of claim 20, wherein conversion of the luciferase into an active state means either the conversion from an inactive state to an active state, or the conversion from a partially active or less active state to a more active state.
0. 23. The method of claim 22, wherein step (b) occurs simultaneously with, or following step (a).
0. 24. The method of claim 22, wherein the luciferase lacks a functional signal peptide.
0. 25. The method of claim 22, wherein the reagent composition further comprises a detergent selected from a non-ionic or zwitterionic detergent.
0. 27. The method of claim 26, wherein conversion of the luciferase into an active state means either the conversion from an inactive state to an active state, or the conversion from a partially active or less active state to a more active state.
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The oxidising and reducing agents may be different compounds, or alternatively may be the oxidised and reduced forms of the same compound, e.g. the buffer may be comprised of a mixture of: R5—SH (reduced form) and R5—S—S—R5 (oxidised form) or may, for example be comprised of: R6—SH (reduced form) and R2—S—S—R2 (oxidised form).
One example of a suitable thiol for use in a redox buffer system is glutathione which is present as a thiol and a disulphide dimer. The glutathione redox buffer system uses the disulphide GSSG to provide oxidising equivalents and the monothiol GSH to generally catalyse disulphide bond isomerisation. The folding efficiency of an effective glutathione system generally has a solution potential similar to that of the endoplasmic reticulum (Esolution=−180 mV).
As generally described above, the GSH component of the GSH/GSSG buffer system can be replaced with other thiols. For example, the rate of in vitro folding of disulphide-containing proteins may be increased by utilising a small-molecule aromatic and aliphatic thiols. Monothiols with lower thiol pKa such as N-methylmercaptoacetamide (NMA) or 4-mercaptobenzoic acid form less stable disulphides and can be used at higher concentrations to give faster folding rates than glutathione. Generally, the leaving group ability of thiols is inversely correlated to the pKa of the thiol (Gough, J., D., J. Am. Chem. Soc., 2002, 124, 3885-3892; Gough, J., D., Bioorganic & Medicinal Chemistry Letters, 2005, 15, 777-781; Gough, J., G., Journal of Biotechnology, 2005, 115, 279-290; Gough, J., G., Journal of Biotechnology, 2006, 125, 39-47]. Examples of suitable small molecule aromatic thiols are shown below:
##STR00001##
Thiol
R
R1
R2
pKa
CH2COOH
H
H
6.6
CH2OH
H
H
6.5
COOH
H
H
6.95
SO3H
H
H
5.7
SO2NHCH2COOH
H
H
5.2
H
COOH
H
6.3
H
CH2COOH
H
6.7
H
H
COOH
8.3
H
H
CH2COOH
7.2
H
H
COOC2H4OC2H4OC2H4OH
6.9
The thiol concentration for aromatic thiols in redox buffer systems may vary considerably. The use of other aromatic thiols, such as 2,2′-[(4-mercaptophenyl)imino]bisethanol, in oxidation and thiol disulphide interchange reactions is known [DeCollo, T. V., J. Org. Chem., 2001, 66, 4244-4249].
Dithiols may also be used as a component of a redox buffer. In contrast to monothiols, dithiols can form cyclic disulphides and thus form less stable mixed disulphides. The addition of reduced dithiol (±)-trans-1,2-bis(mercaptoacetamido)cyclohexane (BMC or Vectrase-P) to a glutathione redox buffer may, for example, increase the rate and yield of folding of a protein. Moreover, using only covalent interactions, BMC can catalyse native disulphide bond formation, both in vitro and in vivo. Without wishing to be bound by theory, the second thiol of BMC may provide an intramolecular clock for substrate-induced thiol-disulphide exchange.
The oxidising agent may be an enzyme such as endoplasmic reticulum oxidoreductin 1 protein (Ero1p). Oxidative protein folding may involve both the oxidation of thiols and the isomerisation of non-native disulphide bonds. Accordingly, isomerase enzymes may also be used as part of a protein oxidising system. A suitable further component of an oxidative buffer is therefore protein disulphide isomerase (PDI) which has a role in catalysing the unscrambling of non-native disulphide bonds in proteins. Each PDI molecule has two active sites that contain the -Cys-Xaa-Xaa-Cys- sequence. Suitably, an enzymatic component of a redox buffer may contain the -Cys-Xaa-Xaa-Cys- sequence. Other enzymes that may be used, for example, in a redox buffer system include: thioredoxin, glutaredoxin and peroxiredoxin.
The isomerising component may be a mimic or an active fragment of an isomerase enzyme. Examples of active dithiol peptide fragments include the Cys-Xaa-Xaa-Cys tetrapeptide and the CysXaaCys tripeptide, wherein Xaa refers to any amino acid residue [Woycechowsky, K., J., Biochemistry, 2003, 42, 5387-5394]. For example, the tripeptide CysGlyCys (CGC) has been shown to have a disulphide reduction potential close to that of PDI. Another non-limiting example of a reducing agent suitable for use in a redox buffer for protein isomerisation and folding, and which is not a thiol derivative, is tris(2-carboxyethylphosphine) (TCEP) [Willis, M., S., Protein Science, 2005, 14, 1818-1826].
The oxidizing agent may be a mild oxidizing agent, suitable for oxidizing protein thiol groups, particularly cysteine thiol groups of the luciferase. The oxidizing agent may, itself, contain disulphide bridges and may be an amino acid derivative. The reducing agent may itself contain thiol groups and be an amino acid derivative.
Other suitable oxidising agents and methods include: molecular oxygen, metal ions, Bu3SnOMe/FeCl3, nitric oxide, halogens (e.g. bromine and iodine), sodium perbortate, borohydride exchange resin (BER)-transition metal salts system, a morpholine iodine complex, PCC, ammonium persulphate, KMnO4/CuSO4, H2O2, solvent free permanganate, PVP-N2O4 and cesium fluoride-Celite O2 system, 2,6-dicarboxypyridinium chlorochromate (2,6-DCPCC) [Tajbakhsh, M., Tetrahedron Letters, 45, 2004, 1889-1893]; dimethylsulphoxide [Shad, S., T., A., Tetrahedron Lett., 2003, 44, 6789; Sanz, R., Synthesis, 2002, 856; Karimi, B., Synthesis, 2002, 2513]; laser prepared copper nanoparticles have been demonstrated to oxidise thiols to disulphides [Chen, T-Y, J. Phys. Chem. B, 2002, 106, 9717-9722]; electrochemical methods for the formation of disulphides: Leite, S., L., S., Synth. Commun., 1990, 20, 393 and Do, Q., T., Tetrahedron Letters, 1997, 38(19), 3383-3384]; manganese nodules [Parida, K., M., Journal of Colloid and Interface Science; 1998, 197, 236-241]; oxidation by soluble polymeric MnO2 [Herszage, J., Environ. Sci. Technol., 2003, 37, 3332-3338]; molecular bromine on hydrated silica gel support [Ali, M., H., Tetrahedron Letters, 2002, 6271-6273]; oxidising polymers such as monochloro poly(styrenehydantoin) beads in water [Akdag, A., Tetrahedron Letters, 2006, 47, 3509-3510]; diamide; DTNB (5,5′-dithiobis(2-nitrobenzoic acid); hydrogen peroxide; N-methylmercaptoacetamide; sodium selenite (together with beta mercaptoethanol) [Rariy, R. V. & Klibanov A. M., (1997) Proc. Natl. Acad. Sci. USA 94: 13520-13523; Ferredoxin (reduced and oxidized); and Copper phenanthroline (Cu:phen) [Webb, T. I., Zang, Z., Lynch J. W. (2005) Proceedings of the Australian Physiological Society Vol 36: 44P].
A reagent composition of the invention may comprise one or more chelators. Suitable chelators include but are not limited to divalent metal chelators such as, for example, EDTA, CDTA and EGTA. The chelator may be present at any concentration but preferably at a concentration of between about 0.1 mM and about 50 mM, typically at a concentration of between about 0.1 mM and about 30 mM, more typically at a concentration of between about 0.1 mM and about 15 mM. The concentration may be at least about 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 7.5 mM, 10 mM, 12.5 mM or 15 mM.
Those skilled in the art will appreciate that where exemplary ranges or constituents are provided herein, these are not exhaustive but are merely illustrative of ranges and constituents that may be included in compositions of the invention in achieving rapid cell lysis or conversion of the luciferase to a more active state or conformation, enhanced bioluminescent signal intensity and/or prolongation of bioluminescent signal.
The findings, as exemplified herein, that bromide salts (including sodium bromide and potassium bromide) enhance activity of Gaussia and Metridia luciferases (in their native secreted form and as modified, intracellular enzymes) and that activity of both Gaussia luciferases and both Metridia luciferases is inhibited in the presence of detergent in a concentration dependent manner at detergent concentrations of less than 1% are particularly surprising in light of the previous dogma in the art that Gaussia and Metridia luciferase activity is sodium dependent and/or sensitive to cation concentration and is not inhibited by non-ionic detergents up to or above 2%.
Using reagent compositions of the invention, the bioluminescent signal generated by the luciferase can be prolonged during a “glow” phase that begins some minutes following addition of the luciferase substrate. For example, the bioluminescent signal can be prolonged during a phase that begins between about 3 minutes and about 15 minutes after addition of the substrate. Further, by way of example only, the bioluminescent signal generated by the luciferase may be such that starting from 10 minutes after initiation of the luciferase-catalysed reaction, the bioluminescent signal decays with a half-life of more than 20 minutes or more than 30 minutes.
The luciferase substrate may be a constituent of the reagent composition or may be independent. Where the luciferase substrate is not a constituent of the reagent composition, the substrate may be added to the sample containing luciferase either before, after or at the same time as addition of the reagent composition.
Typically luciferase-based reporter assay systems employ two buffers, a lysis buffer and an assay buffer (collectively referred to herein as “assay reagents”). The lysis buffer typically comprises components for lysing the cells containing the luciferase to be assayed while the assay buffer typically contains, inter alia, the substrate and any required cofactors for the luciferase reaction.
Reagent compositions of the present invention are typically in the form of cell lysis buffers. Advantageously, lysis buffers in accordance with the present invention effectively provide shorter lysis times than is possible with currently available buffers. Without wishing to be bound by theory, the present inventors suggest that these buffers provide a suitable environment for promoting or facilitating the folding or conversion of the luciferase to be assayed following cell lysis into an active state or conformation from an inactive state or conformation, or into a more active state or conformation from a less active state or conformation. Alternatively, reagent compositions of the invention may be in the form of a combined lysis and assay buffer such that only a single buffer composition is required to lyse cells, potentially directly within the medium in which the cells are cultured, and initiate the luciferase-catalysed reaction.
Reagent compositions in accordance with the present invention may typically be aqueous solutions, or alternatively may be in solid or dry form such as lyophilised. Whether aqueous or lyophilised, reagent compositions of the invention may be provided either comprising all constituents pre-mixed or as a combination of constituents to be mixed prior to use. The reagent composition may be used directly in an assay system for the determination of luciferase amount and/or activity, or may be reconstituted, dissolved, diluted or otherwise treated either chemically or physically such that the composition is capable of performing the desired function.
Reagent compositions of the present invention are applicable to determining the amount and/or activity of any luciferase in a sample. The luciferase may be a naturally occurring enzyme or modified enzyme. A naturally occurring luciferase may be derived from any one of a number of bioluminescent organisms, typically from the light organ thereof. Such organisms include but are not limited to bioluminescent bacteria, protozoa, coelenterates, molluscs, fish, flies, crustaceans and beetles. Conventionally, luciferases may be categorised according to the substrate utilised by the enzyme. One group of luciferases such as those of fireflies and click beetles utilise luciferin (D-luciferin). A second group of luciferases, such as those of the marine organisms Renilla, Gaussia, Pleuromamma, Metridia and Cypridina utilise coelenterazine. Other luciferases, such as Vargula luciferase, use a different substrate. The reagent compositions of the present invention are applicable to use with luciferases using luciferin or coelenterazine or any other substrates.
The reagent compositions of the invention are similarly applicable to use with either intracellular or secreted luciferases. Firefly and Renilla luciferases are intracellular in their natural state, whereas Gaussia and Metridia luciferases are secreted in their wild-type state. Gaussia luciferase is of particular interest as it has been shown to yield a bioluminescent signal strength higher than that achievable with Renilla luciferase and is the smallest known luciferase. Other secreted luciferases have also been shown to yield strong signal strength, for example Metridia longa luciferase.
The luciferase may be a variant or derivative of a naturally occurring luciferase. For example, the present invention finds particular application in reactions and assays involving modified, non-secreted forms of luciferases that are secreted in their native form. By way of example, a naturally secreted luciferase may be modified using standard molecular biological techniques by removal of signal sequences and/or fusion to an intracellular polypeptide such that the enzyme is no longer secreted but remains intracellular. Alternatively, or in addition, a number of other modifications well known to those in the art may be made, for example, to alter one or more amino acids in the luciferase polypeptide sequence to modulate expression and/or solubility of the enzyme in a cell culture system of choice. Such modulation may be an increase or decrease in expression and/or solubility, depending on the requirements of the particular application in which the luciferase, and the reagent compositions of the invention are to be employed. For example, it may be desirable to modify the luciferase by the introduction of one or more destabilising elements to destabilise the protein. Luciferases containing destabilising elements have shortened half-lives and are expressed at lower steady-state levels than luciferases which do not contain such elements. Suitable protein destabilising elements include PEST sequences (amino acid sequences enriched with the amino acids proline (P), glutamic acid (E), serine (S) and threonine (T)), a sequence encoding an intracellular protein degradation signal or degron, and ubiquitin. The enhanced sensitivity attained with reagent compositions of the present invention are particularly advantageous for use with destabilized luciferases given the lower steady state expression levels of such luciferases. Any suitable method for destabilising a protein is contemplated herein. Suitable methods are described, for example, in co-pending U.S. patent application Ser. No. 10/658,093 (the disclosure of which is incorporated herein by reference in its entirety). Expression of the luciferase may also be modified by, for example, the addition of sequences such as poly(A) tails, transcriptional or translational enhancers, and/or adapting codon usage in the encoding polynucleotide sequence for a particular expression system. For example, to optimise expression of the luciferase in insect cells or human cells, codon usage in the luciferase polynucleotide may be optimised for insect or human cells respectively. Approaches for codon usage adaptation and optimisation for different species are well known to those skilled in the art.
Further modifications that may be made to luciferase polypeptide or polynucleotide sequences are also well known to those skilled in the art. For example, restriction enzyme cleavage sites may be introduced into the polynucleotide, or the luciferase polypeptide may be fused or conjugated with a second polypeptide of different function such as a selectable marker (e.g. antibiotic resistance).
Those skilled in the art could readily predict using well know techniques such as computer modelling, those luciferases which are particularly amenable to use in accordance with the invention, as well as predict the modifications that may be made to a secreted luciferase the render the luciferase non-secreted. For example, luciferases that are secreted in their native form typically comprise cysteine residues that form disulphide bridges in the mature active conformation of the protein. The cysteine residues may be arranged in spacing patterns that are repeated within the amino acid sequence such that they can be predicted to form intramolecular disulphide bonds. Such luciferases typically show reduced activity when expressed intracellularly. Thus, those skilled in the art will appreciate that homologous luciferases sharing one or more of these characteristics of naturally secreted luciferases are particularly suitable for use in accordance with the present invention.
Suitable luciferases are readily obtainable by those skilled in the art using known techniques. The luciferase may be directly obtained from the light organ(s) of the bioluminescent organism. Alternatively the luciferase may be obtained from cultured cells, for example bacteria, yeast, insect cells or mammalian cells which have been transformed with nucleic acids encoding the luciferase.
When using secreted luciferases in in vitro reporter assays, a sample of the cell culture medium, rather than a cell lysate, is typically taken for measurement of luciferase activity. Whilst advantageous in some applications (e.g. repeated measurements from the same cells), secreted luciferases are not appropriate for other applications. In particular, the secreted luciferase accumulates in the cell culture medium such that rapid changes in gene expression can not be monitored accurately. Mutant, non-secreted forms of these luciferases (preferably containing destabilizing elements) overcome this limitation. One particular modified luciferase described herein is a modified Gaussia luciferase in which the 14 amino acid N-terminal signal peptide is deleted thereby generating a non-secreted luciferase. A second modified luciferase described herein is a modified Metridia luciferase in which the 17 amino acid N-terminal signal peptide is deleted thereby generating a non-secreted luciferase. Other secreted luciferases can be similarly modified for intracellular expression, particularly in eukaryotes, using a variety of methods well known to those skilled in the art.
In particular embodiments, reagent compositions of the present invention find application in reporter assay systems based on a luciferase, either intracellular or secreted, which utilises coelenterazine as a substrate or in dual luciferase reporter assays in which at least one of the luciferases utilises coelenterazine as a substrate and/or is secreted in its native form.
In accordance with the present invention, luciferase activity can be detected and measured by any one of a number of methods well known to those skilled in the art, including but not limited to using a luminometer, a scintillation counter, a photometer such as a photomultiplier photometer or photoemulsion film, or a charge-coupled device (CCD).
Reagent compositions of the invention provide improved kinetics of bioluminescence in luciferase reactions and offer advantages over the prior art. As such these compositions may be used in luciferase assays according to the invention, in preparing assay reagents and test kits according to the invention, and in standards and controls for assays and kits according to the invention. The present invention provides kits for carrying out assays of luciferase activity, such kits containing reagent compositions in accordance with the invention as described herein. Kits of the invention comprise, in one or more physical containers and typically packaged in a convenient form to facilitate use in luciferase assays, suitable quantifies of reagent compositions or constituents thereof for carrying out luciferase assays. Multiple reagent compositions, or various constituents of reagent compositions may be combined, for example in aqueous solution or lyophilised, in a single container or in multiple containers. Kits of the invention typically also comprise controls and standards to ensure the reliability and accuracy of assays carried out in accordance with the invention. Suitable controls and standards will be known to those skilled in the art.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
The present invention will now be described with reference to the following specific examples, which should not be construed as in any way limiting the scope of the invention.
Firefly luciferase and Renilla luciferase are the most commonly used luciferases in commercial luciferase-based reported systems. Although the secreted Gaussia luciferase and secreted Metridia luciferase provide higher sensitivity than firefly or Renilla luciferase, secreted luciferases accumulate in the culture medium and therefore are not ideal for reporter studies aimed at measuring temporal changes in gene expression.
To determine whether a mutant, non-secreted (and destabilized) version of a naturally secreted luciferase could provide the combined benefits of high signal strength and rapid response rates, the inventors cloned the Gaussia luciferase coding sequence into various RapidReporter plasmids (GeneStream) using a PCR strategy that deleted the 15 amino acid N-terminal signal peptide and fused the remaining Gaussia luciferase cDNA to N-terminal and C-terminal coding sequences that included sequences encoding protein destabilizing elements. The modified Gaussia luciferase is not secreted when expressed in mammalian cells. The precise intracellular location of the modified luciferase enzyme is assumed to be cytoplasmic. In the experiments described below, mammalian cells transfected with plasmids expressing either the native Gaussia luciferase (hereafter referred to as “secreted Gaussia luciferase”) or the modified Gaussia luciferase (hereafter referred to as “non-secreted Gaussia luciferase”) were lysed in the presence of a variety of lysis buffer compositions and bioluminescence measured.
To determine whether a second mutant, non-secreted (and destabilized) version of a naturally secreted luciferase could also provide the combined benefits of high signal strength and rapid response rates, the inventors cloned the Metridia luciferase coding sequence into various RapidReporter plasmids (GeneStream) using a PCR strategy that deleted the 17 amino acid N-terminal signal peptide and fused the remaining Metridia luciferase cDNA to N-terminal and C-terminal coding sequences that included sequences encoding protein destabilizing elements. The modified Metridia luciferase is not secreted when expressed in mammalian cells. The precise intracellular location of the modified luciferase enzyme is assumed to be cytoplasmic. In the experiments described below, mammalian cells transfected with plasmids expressing either the native Metridia luciferase (hereafter referred to as “secreted Metridia luciferase”) or the modified Metridia luciferase (hereafter referred to as “non-secreted Metridia luciferase”) were lysed in the presence of a variety of lysis buffer compositions and bioluminescence measured.
Using the non-secreted Gaussia or Metridia luciferases with commercially available buffers, the bioluminescent signal strength was very low, particularly in samples in which luciferase activity was measured shortly after cell lysis. Only after some hours of incubation in lysis buffer did the modified luciferase gain high activity.
Without wishing to be bound by theory, it is suggested that the non-secreted Gaussia and Metridia luciferases adopt a less active conformation intracellularly but can adopt an active conformation following cell lysis, albeit slowly. The inventors then tested a variety of modifications to the lysis buffer components in an attempt to develop a formulation enabling shorter incubation periods and in which the luminescent signal comprises a stronger flash phase (higher sensitivity) and a more stable glow phase (rate of decay of the signal is reduced); in particular providing an environment promoting the conversion of the luciferase from an inactive conformation to an active conformation following cell lysis.
HeLa cells were transiently transfected with a plasmid expressing native, secreted Gaussia luciferase and 24 hrs later the conditioned medium was collected. Wells of a 96-well plate were loaded with 20 ul of conditioned medium and assayed for luciferase activity by injecting 60 ul of an assay buffer comprising 26 uM Cz; pH 8.1 plus the salt as indicated in
Light output with NaBr was higher than with either NaCl or no salt. It can be seen from
HeLa cells stably expressing non-secreted and destabilized Gaussia luciferase were plated onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 30 ul of lysis buffer per well. The lysis buffer (LB) comprised; 25 mM Tris pH 7.8; 0.1% NP40; 1 mM EDTA plus NaBr at concentrations of (from left to right) 0 mM, 150 mM, 0 mM and 75 mM. Luciferase activity was assayed by injecting 30 ul of an assay buffer (AB) comprising 25 mM Tris pH 7.8; 40 uM Cz plus NaBr at concentrations of (from left to right) 0 mM, 0 mM, 150 mM and 75 mM. Thus, the final concentration of NaBr was 75 mM in all samples, except the no salt controls. Results were expressed as relative light units (RLU) (See
The data in
HeLa cells stably expressing non-secreted and destabilized Gaussia luciferase were plated onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ul of lysis buffer per well. The lysis buffer comprised; 25 mM Tris pH 8.1, 1 mM EDTA; 0.1% NP40; 63.4 uM Na-Oxalate; 5% Glycerol plus the concentration and type of salt indicated in
Both salts increased light output compared to no salt. However NaBr provided a superior enhancement to NaCl and enhancement was found to have some concentration dependency.
HeLa cells stably expressing either non-secreted and destabilized Gaussia or non-secreted and destabilized Metridia luciferase were plated onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ul of lysis buffer per well. The lysis buffer comprised; 25 mM Tris pH 8.1; 0.1% NP40; 63.4 uM Na-Oxalate; 5% Glycerol plus the concentration and type of salt indicated in
As was observed with non-secreted and destabilized Gaussia luciferase in Example 4, NaBr provided a superior enhancement to NaCl with non-secreted and destabilized Metridia luciferase. The enhancement was found to have some concentration dependency.
Luciferase assays were performed as described in Example 5 with either non-secreted and destabilized Gaussia luciferase or non-secreted and destabilized Metridia luciferase. Two experiments are shown as separate graphs, with results shown as relative luminescence in counts per second (CPS) (see
For both non-secreted Gaussia and non-secreted Metridia luciferase the signal strength is then lowest in the absence of salt. A higher signal strength was noted by including salt in either the lysis buffer or assay buffer. Furthermore, the bromide salt was superior to the chloride salt for both luciferases. Of the four treatment groups, the highest signal strength was seen when the bromide was included in the lysis buffer suggesting that in some circumstances it may be preferable to include the bromide in the lysis buffer rather than the assay buffer.
Luciferase assays were performed as described in Example 4. All salts were present at 150 mM in lysis buffer.
The highest level of signal enhancement (>5-fold) was achieved with NaBr, RbBr and KBr. A lower level of enhancement was seen with the corresponding Chloride salts and no enhancement was seen with iodide salts. These data demonstrate that, contrary to the existing literature, the anion and not the cation is important for achieving high luciferase activity. Moreover, they provide further evidence for the beneficial effect of bromide salts and demonstrate that a variety of different bromide salts can be used to achieve the desired effect (see
Luciferase assays were performed as described in Example 7, except using the indicated concentrations of salt and EDTA in the lysis buffer. Replicate samples were assayed for luciferase activity at 20, 30, 40 and 90 min after addition of lysis buffer. Results were expressed as a % of the light units obtained after 20 min lysis in the same lysis buffer (see
In the absence of salt and EDTA, a dramatic increase in luciferase activity was evident between 20 and 90 min after the onset of cell lysis. A far more stable (and therefore more desirable) level of luciferase activity was achieved by addition of either EDTA or NaBr alone. However, an additive beneficial effect was achieved by combining both components into the lysis buffer. It can be seen that addition of NaBr and EDTA enables the use of reduced lysis times.
Luciferase assays were performed as described in Example 8, except using lysis buffer containing 150 mM NaBr and the indicated concentration of EDTA. Two experiments are shown as separate graphs, with results shown as relative light units (RLU) (see
Luciferase assays were performed as described in Example 9, except using the indicated type and concentration of chelator and a 30 min lysis period. The results show that both EDTA and EGTA provide a concentration-dependent beneficial effect on signal intensity (see
Luciferase assays were performed with non-secreted and destabilized Metridia luciferase as described in Example 5, except the lysis buffer contained no salt and but contained the indicated type and concentration of chelators. Replicate luciferase assay were carried out after a 30 or 120 min lysis period. Results from the 30 min time point were expressed as a % of the light units obtained in the absence of chelator (see
Flasks of HeLa cells transiently expressing secreted Gaussia or secreted Metridia luciferases were incubated overnight. Aliquots of conditioned media were removed and diluted 1:10 into fresh medium (RPMI+10% fetal calf serum) with or without 5 mM EDTA. After 30 min, 80 ul aliquots were assayed for luciferase activity by injecting 20 ul medium containing 48 uM Cz. Results were expressed as the % of the light units obtained in the absence of EDTA (see
The data show that the benefits of addition of chelators noted with non-secreted Gaussia and non-secreted Metridia luciferase (see Examples 9, 10 and 11) do not apply to the (native) secreted versions of the same luciferases. In particular, there is a lack of any enhancement in samples that received the chelator only in the assay buffer (see
When expressed intracellularly, it has been shown that the secreted luciferases adopt a less active format such that the positive effect of chelator is pronounced with the intracellular versions that require refolding or other modifications in order to adopt the high activity state of the protein. This beneficial effect of chelator on luciferase activity has not previously been described.
HeLa cells expressing Firefly or Renilla luciferases were plated into 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ul of lysis buffer containing 25 mM Tris pH 8.1; 0.1% NP40; 63.4 uM Na-Oxalate; 5% Glycerol, 150 mM NaCl plus the concentration of EDTA in
The results indicate that the addition of EDTA to Renilla and firefly luciferases does not provide the same beneficial effect on signal intensity as is observed with non-secreted Gaussia or Metridia luciferases. In fact, the addition of EDTA clearly decreases the activity of Firefly luciferase.
HeLa cells expressing destabilised, non-secreted Gaussia luciferase were plated in equal aliquots onto 96-well plates and incubated overnight. Three experiments are shown as separate graphs (
HeLa cells expressing destabilised, non-secreted Gaussia or non-secreted Metridia luciferases were plated in equal aliquots onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ul of lysis buffer. The detergent type and concentration was varied as indicated. Luciferase assays were carried out by injecting 60 ul of assay buffer comprising 25 mM Tris pH 8.1; 1 mM EDTA, 2 mM Ascorbate; 24 uM Cz Two experiments are shown, in the first the lysis buffer also contained 5% glycerol, 64 uM sodium oxalate, 150 mM NaBr, 25 mM Tris pH 8.5, 5 mM EDTA, 0.6 mM reduced glutathione, 0.4 mM oxidised glutathione, 75 mM urea (v6) and luciferase activity was assayed at 40 min. In the second experiment, the lysis buffer also contained 2 5 mM Tris pH 8.1, 63.4 uM Na-oxalate, 150 mM NaBr, 5% Glycerol and the luciferase activity was assayed at 120 min. Results were expressed as the % of the light units obtained using 0.1% NP40 (see
The results from the first experiment (see
The results show that the detergents provide a concentration-dependent inhibition on signal intensity. This effect was most evident with the anionic detergents SDS and DOC. CHAPS, a zwitterionic detergent, provided the highest activity for non-secreted Metridia luciferase, notably, this detergent required a higher concentration to effectively lyse the cells. The remaining detergents, which are all non-ionic, performed well, with NP40 and Triton X100 providing the highest signal intensity.
Experiments with the cationic detergent CTAB (not shown) indicate that as with anionic detergent cationic detergents have a strong inhibitory effect on signal intensity.
HeLa cells expressing secreted Gaussia or secreted Metridia luciferase were plated in equal aliquots onto 96-well plates and incubated overnight. Medium was removed and diluted 1:10 into fresh medium containing the type and concentration of detergent indicated in
The results from both experiments indicate that, as with non-secreted Gaussia and non-secreted Metridia luciferases there is a concentration-dependent inhibitory effect of these detergents on the signal intensity derived from the secreted forms of these luciferases.
HeLa cells expressing destabilised, non-secreted Gaussia luciferase were plated in equal aliquots onto 96-well plates and incubated overnight. Two experiments are shown as separate graphs (see
The effect of detergent concentration on the activity of non-secreted Gaussia luciferase was determined as described in Example 17, except the experiment was also performed with cells expressing the secreted Gaussia. For such cells, the conditioned medium was used as a source of the secreted Gaussia protein, to which the detergent was added in the indicated concentrations. The results demonstrate that inhibition of luciferase activity by detergent occurs with both secreted and non-secreted Gaussia luciferase (see
Luciferase assays were performed as described in Example 9, except using lysis buffer with the indicated pH. EDTA was present at 1 mM except where indicated. Four experiments are shown as separate graphs (see
HeLa cells expressing destabilised, non-secreted Gaussia or non-secreted Metridia luciferases were plated in equal aliquots onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ul of lysis buffer. The lysis buffer comprised 63.4 uM Na-oxalate, 5% Glycerol, 150 mM NaBr and 25 mM Tris at the indicated pH. Luciferase assays were carried out at 30 min by injecting 60 ul of assay buffer comprising 25 mM Tris pH 7.75; 2 mM Ascorbate; 24 uM Cz. Results were expressed as the % of the light units obtained at pH 8.5 (see
The data demonstrates that the benefit of a lysis buffer with a higher pH, in terms of both signal intensity and the stability of the luciferase signal over time, is observed with both non-secreted Gaussia and Metridia luciferase. By contrast, in similar experiments conducted using firefly and Renilla luciferases suggest that there is no beneficial effect of high pH during cell lysis for these naturally intracellular luciferases (data not shown).
Luciferase assays were performed as described in Example 18, with EDTA at 5 mM and pH 8.5. The lysis buffer also contained the indicated concentrations of reduced (red) and oxidised (ox) glutathione. Replicate samples were assayed for Gaussia luciferase activity at 20 or 135 minutes after addition of lysis buffer. These data demonstrate that the presence within the lysis buffer of oxidised glutathione, and more preferably a mixture of reduced and oxidised glutathione, increases the rate at which the luciferase acquires its maximum activity during the cell lysis step (see
Luciferase assays were performed as described in Example 21, using the indicated total amounts of glutathione and ratio of reduced:oxidized. Replicate samples were assayed for Gaussia activity at 20 or 60 minutes after addition of lysis buffer. These data demonstrate that at all concentrations and ratios tested, the presence of glutathione in lysis buffer increases the rate at which the luciferase acquires its maximum activity during the cell lysis step. At the higher concentrations of reduced glutathione, a somewhat reduced signal was seen at 60 minutes (see
HeLa cells transiently expressing either non-secreted Gaussia luciferase or non-secreted Metridia luciferase were plated onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ul of lysis buffer per well, comprising 25 mM Tris pH 8.1, 63.4 uM Na-oxalate, 0.1% NP40, 5% Glycerol and either no glutathione or a combination of 0.6 mM reduced glutathione and 0.4 mM oxidised glutathione. Replicate samples were assayed for luciferase activity at 30 min after addition of lysis buffer by injecting 60 ul of an assay buffer comprising 25 mM Tris pH 7.75, 0.6 mM reduced glutathione, 0.4 mM oxidised glutathione, 1 mM EDTA, 2 mM Ascorbate, 24 uM Cz Results were expressed as the % of the light units obtained in the absence of glutathione for the same luciferase (see
The data demonstrates that at the ratio tested, the presence of glutathione in the lysis and assay buffer also increases the signal of both non-secreted Gaussia and non-secreted Metridia luciferases.
Luciferase assays were performed as described in Example 22 except using a single concentration and ratio of additive (glutathione; 1.2 mM reduced, 0.8 mM oxidised) in either the lysis buffer and/or assay buffer or neither. Two experiments are shown as separate graphs (see
Luciferase assays were performed as described in Example 23 with the glutathione in both lysis buffer and assay buffer. Additionally, the lysis buffer contained the indicated concentrations of urea. Replicate samples were assayed for Gaussia luciferase activity at 15 or 60 minutes after addition of lysis buffer and the 60 min data were expressed as a % of the signal at 15 mins. All lysis buffers showed an increase in signal during this time. However, the increase was less pronounced with 50-100 mM urea suggesting that maximum activity can be reached sooner under these conditions (see
HeLa cells expressing destabilised, non-secreted Gaussia luciferase were plated in equal aliquots onto 96-well plates and incubated overnight. The same assay buffer was used for all samples. Replicate samples were assayed for luciferase activity at 20 or 120 minutes after addition of lysis buffer containing 5% glycerol, 64 uM sodium oxalate, 0.1% NP40, 150 mM NaBr plus either 25 mM Tris pH 8.1, 1 mM EDTA (v3) or 25 mM Tris pH 8.5, 5 mM EDTA, 0.6 mM reduced glutathione, 0.4 mM oxidised glutathione, 75 mM urea (v6). Whereas v3 lysis buffer achieved only ˜50% maximal activity after 20 mins lysis, the v6 lysis buffer achieved 100% activity within 20 mins (see
HeLa cells expressing destabilised, non-secreted Metridia luciferase were plated in equal aliquots onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ul of lysis buffer containing 5% glycerol, 63.4 uM sodium oxalate, 0.1% NP40, plus 25 mM Tris at the indicated pH, the indicated salt at 150 mM and, where indicated, 1 mM EDTA or 0.6 mM reduced glutathione and 0.4 mM oxidised glutathione (GSH/GSSG) Replicate samples were assayed for luciferase activity at 30 or 120 min after addition of lysis buffer by injecting 60 ul of assay buffer containing 25 mM Tris pH 7.75, 2 mM ascorbate, 24 uM Cz. Results were expressed as the % of the light units obtained at 30 min lysis (see
The data show that with NaCl as the only additive, only a small portion of maximum activity is attained within the first 30 min of lysis. An increase of more than 1200% occurs between 30 and 120 min in this treatment group. Treatment groups comprising either a redox buffer (GSH/GSSG) or a chelator (EDTA) performed considerably better as indicated by the smaller increase in activity beyond 30 min lysis. In each case, NaBr performed better than NaCl demonstrating the additive benefit of combining bromide with redox buffer or chelator. The benefits of a combination of bromide, redox buffer, chelator and high pH can be clearly observed as the increase in activity between 30 and 120 min was less pronounced.
Collectively, these data demonstrate the cumulative benefit of combining multiple different components of the invention. Additionally, this data shows that the addition of bromide does not only improve signal strength (see Example 6 and
HeLa cell expressing non-secreted Gaussia or Renilla luciferases were plated into 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ul of GeneStream's v6 (as Example 26) lysis buffer or Promega's passive lysis buffer (PLB) per well. Luciferase activity was assayed by injecting 60 l of assay buffer comprising 25 mM Tris pH 7.75; 0.6 mM reduced glutathione, 0.4 mM oxidised glutathione, 1 mM EDTA, 2 mM Ascorbate, 24 uM Cz (see
The results in
HeLa cells expressing destabilised, non-secreted Gaussia luciferase were plated in equal aliquots onto 96-well plates and incubated overnight (see
The method of Example 29 was followed except that the kinetics of light emission were measured. The data show that compared to a prior art “glow” buffer, the compositions that are the subject of the present invention provide a more stable glow signal and higher sensitivity (see
HeLa cells expressing destabilised, non-secreted Gaussia and non-secreted Metridia luciferase were plated in equal aliquots onto 96-well plates and incubated overnight. Medium was removed and the cells lysed in 20 ul of lysis buffer. Replicate samples were assayed for luciferase activity at 20, 30, 40, 60, 90 or 120 min after addition of lysis buffer. The lysis buffers used were GeneStream's v3 and v6 (as Example 26), Promega's passive lysis buffer (PLB) and a generic standard lysis buffer (STD-LB) containing 25 mM Tis pH 7.7; 1% Triton X100; 10% glycerol. Replicate samples were assayed for luciferase activity by injecting 60 ul of assay buffer containing 25 mM Tris pH 7.75; 1 mM EDTA, 2 mM ascorbate; 0.66 mM reduced glutathione, 0.4 mM oxidised glutathione, 24 uM Cz. Results were expressed as the % of the light units obtained using v6 lysis buffer (see
The data in
Luciferase-based gene reporter assay kits according to the invention are provided in 2-parts designed for 20 ul lysis buffer and 60 ul assay buffer per well of a 96-well plate.
Lysis Buffer:
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification individually or collectively, and any and all combinations of any two or more of said steps or features.
Daly, John Michael, Leu, Marco Peter
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