WO2001059149A2 - Peptide biosensors for anthrax protease - Google Patents
Peptide biosensors for anthrax protease Download PDFInfo
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- WO2001059149A2 WO2001059149A2 PCT/US2001/004253 US0104253W WO0159149A2 WO 2001059149 A2 WO2001059149 A2 WO 2001059149A2 US 0104253 W US0104253 W US 0104253W WO 0159149 A2 WO0159149 A2 WO 0159149A2
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- protease biosensor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
- G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/32—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Bacillus (G)
Definitions
- LF lethal factor
- FRET FRET will only occur if the donor and acceptor are physically constrained, for example attached to the same peptide Upon physical separation of the donor and acceptor, which might occur if the peptide were to be cleaved by a protease, the donor and acceptor would diffuse away from one another and FRET would not occur.
- This pnnciple has been used to design FRET-based biosensors and assays for detecting proteolytic enzymes (Knight, C G (1995) Methods Enzymol 248, 18-34)
- FRET-based biosensors can be measured in the presence of the products and reactants, and thus no separation steps are necessary, as is sometimes the case with fluorescent substrates
- the FRET measurement is a ratio-based measurement, so the result is intensity independent (as long as it is within the range of detectabi ty)
- the present invention provides FRET-based protease biosensors, and kits containing them, for detecting the presence of the anthrax protease, as well as methods for using the protease biosensors to detect the presence of Bacillus anthracis m a test sample
- the present protease biosensors and assays provide a significant improvement over previous biosensors and assays for detecting the presence of Bacillus anthracis in a sample, by significantly improving both the speed and efficiency of the assay, and by detecting live, virulent strains of Bacillus anthracis Therefore, the biosensors of the present invention will have fewer false positives, which is desirable for a sensor to be used m a potentially hazardous situation
- Figure 4A shows that both trypsm and LF efficiently cleaved the substrate, with trypsin cleavage occur ⁇ ng at a slightly higher rate
- the present invention provides FRET-based protease biosensors that can be used to detect the presence of Bacillus anthracis lethal factor in a test sample
- the protease biosensor compnses a) a peptide compnsmg an ammo acid sequence of the general formula I
- X-Pro-Y-Z-W wherein X is a sequence of between 5 and 13 ammo acids, wherein at least one ammo acid is selected from the group consisting of Arg, Lys, or His, and wherein none are Asp or Glu, Y is selected from the group consisting of hydrophobic amino acids or Gly,
- Z is selected from the group consisting of uncharged amino acids
- W is a sequence of between 2 and 10 ammo acids of anv type, wherein a cleavage site for lethal factor is between the Pro and Y residues; b) a fluorescent donor molecule attached to the peptide that is capable of participating in fluorescence resonance energy transfer; and c) an acceptor molecule attached to the peptide that has an absorption spectrum overlapping the emission spectrum of the donor molecule, wherein the fluorescence donor molecule and the acceptor molecule are attached to amino acid residues that are on opposite sides of the cleavage site.
- Fluorescence resonance energy transfer occurs between the two fluorophores in the absence of a protease that cleaves the peptide
- cleavage of the peptide results in separation of the two fluorophores and loss of FRET
- the fluorescent donor typically is excited by incident radiation at a particular wavelength which it then re-emits at a different (longer) wavelength.
- the acceptor When the donor fluorophore is held in close proximity to the acceptor molecule, the acceptor absorbs the light that would have been emitted by the fluorescent donor, thereby quenching the fluorescence signal of the donor molecule. Cleavage of the peptide joining the fluorescent donor and the acceptor results in separation of the two molecules, release of the quenching effect and an increase in intensity of emission spectrum of the donor molecule.
- the sequence of the peptide portion of the biosensor is denved from the native EK 1 recognition site for lethal factor.
- KKKPTPIQLN SEQ ID NO: l
- MEK2 RRKPVLPA TI; SEQ ID NO. 14
- MEK6 SEQ ID NO: 15
- the recognition sites further require an uncharged ammo acid following the hydrophobic residue, and at least one positively charged amino acid (and no negative!) charged amino acid, such as Asp and Glu) within the 5 ammo acids to the N-terminal side of the proline residue.
- Other residues in the sequence provide appropnate spacing between the cntical residues or between the donor and acceptor, and thus their composition is not cntical, and can include any natural or unnatural amino acid.
- the range of optimal lengths for the peptide backbone of the biosensor depends on several factors including the secondary structure of the peptide (which would alter the through-space distance between the donor and acceptor), the spectral overlap between the donor and acceptor (a greater overlap generally allows longer peptide length), and the flexibility of the attachment of the peptide to the dye. For example, if a linker is used to attach the dye to the peptide and the linker is not flexible (i.e.: can not rotate about the axis of the bond(s) between the peptide and the dye), then the alignment of the dye molecule could be optimal and FRET would be very efficient, or the alignment could be very poor and there would be no FRET.
- the peptide portion of the protease biosensor is between 10 and 18 amino acids in length, and more preferably is between 10 and 15 ammo acids in length.
- "Y" is selected from the group consisting of He, Tyr, Val, Leu, Ala, Phe, and Gly.
- "Z" is selected from the group consisting of He, Tyr, Val, Leu, Ala, Phe, Gly, Gin, Asn, Ser, Thr, Trp, Pro, Met, and He.
- the acceptor molecule is fluorescent, which permits detection of the fluorescence emission of both the donor and acceptor, thus providing the ability to determine a ratio of the fluorescence of the donor and acceptor molecules.
- the peptide comprises an amino acid sequence of general formula II:
- Rl-R2-R3-R4-R5-Pro-R6-R7-R8-R9 wherein Rl, R2, R3, R4, and R5 can be any amino acid residue with the proviso that at least one is selected from the group consisting of Arg, Lys, and His, and with the further proviso that none are Asp or Glu;
- R6 comprises any hydrophobic amino acid or Gly;
- R7 comprises any uncharged amino acid except Cys; and
- R8 and R9 can be any amino acid;
- R2, R3, R4, and R5 are selected from the group consisting of Arg, Lys, and His, and even more prefened that Rl, R2, and R3 are selected from the group consisting of Arg, Lys, and
- R6 is selected from the group consisting of He, Tyr,
- R7 is selected from the group consisting of He, Tyr, Val,
- R9 or the C-terminal residue is selected to allow for site specific labeling of the peptide.
- the peptide is selected from the group consisting of Cys, Lys, and N- methylaminooxy amino acid; wherein if R9 or the C-terminal residue is Lys, then none of Rl ,
- R2, R3, R4, and R5 are Lys.
- a recent reference discloses the use of the unnatural amino acid N-methylammooxy ammo acid for incorporation into a peptide sequence and labeling by a suitably reactive dye, including but not limited to a succmimide ester of rhodamme, fluorescein, and Cy3 (Amersham Pharmacia) (Bark et al , J Am Chem Soc 2000 122(15).3567-3573 )
- an amine- reactive dye can be used to selectively label the R9 position
- Such amine-reactn e dyes include but are not limited to isothiocyanate or succmimide ester denvatives of commercially available dyes including but not limited to most ALEXA FLUOR® dyes (Molecular Probes, Eugene OR), as well as fluorescein, rhodamme, eosin, and Cy3 Other methods for specifically incorporating dyes
- R9 or the C-terminal residue of general formula I is Cys and is used to link ALEXA-FLUOR® 546 maleimide
- the acceptor molecule compnses fluorescein
- the peptide portion of the biosensor compnses a sequence of general formula III:
- Rl-R2-R3-R4-R5-Pro-R6-R7-R8-R9-R10-Rl l wherein Rl is selected from the group consisting of Arg, Lys, Ser, and His, R2 is selected from the group consisting of Arg, Lys, His, and Gin,
- R3 is selected from the group consisting of Arg, Lys, His, Pro, and Gin,
- R4 is selected from the group consisting of Pro, Val, and Arg,
- R5 is selected from the group consisting of Thr. His, Arg. Leu. and Asn.
- R6 is selected from the group consisting of He, Tyr. Val. Ala, Leu, Gly, and Phe,
- R7 is selected from the group consisting of Gin, Tyr, Leu, and Pro
- R8 is selected from the group consisting of Leu, He, and Thr
- R9 is selected from the group consisting of Asn, He, and Pro
- RIO is absent or is selected from the group consisting of Pro and Lys
- Rl 1 is absent or is selected from the group consisting of Cys, Lys, and N- methylaminooxy ammo acid, wherein if Rl 1 is Lys, then none of R l , R2.
- the peptide component of the protease biosensor compnses an ammo acid sequence selected from the group consisting of a) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro (SEQ ID NO 2), b) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:3), c) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Tyr-Leu-Asn-Pro-Cys (SEQ ID NO:4); d) Met-Pro-Lys-Lys-Lys-Pro-His-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:5), e) Met-Pro-Lys-Lys-Pro-His-Pro
- the peptide component of the protease biosensor comprises an amino acid sequence selected from the group consisting of: a) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro (SEQ ID NO:2); b) Met-Pro-Lys-Lys-Lys-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:3 ); c) Met-Pro-His-His-His-Pro-Thr-Pro-Ile-Gln-Leu-Asn-Pro-Cys (SEQ ID NO:9); d) Met-Pro-His-His-His-His-Pro-Thr-Pro-Ile-Tyr-Leu-Asn-Pro-Cys (SEQ ID NO: 10); e) Arg-Arg-Lys-Pro-Val-Leu-Pro-Ala
- the peptide component of the protease biosensor consists essentially of an amino acid sequence selected from the group consisting of SEQ ID NO: 1
- the peptide component of the protease biosensor consists essentially of an amino acid sequence of the one of the cited general formulas.
- the peptides of the instant invention may be synthesized by any conventional method, including, but not limited to, those set forth in J. M. Stewart and J. D. Young, Solid Phase Peptide Svnthesis, 2nd ed , Pierce Chemical Co , Rockford, 111 ( 1984) and J Meienhofer, Hormonal Proteins and Peptides, Vol 2, Academic Press, New York, ( 1973) for solid phase synthesis and E Schroder and K Lubke, The Peptides, Vol 1 , Academic Press, New York ( 1965) for solution synthesis
- the disclosures of the foregoing treatises are incorporated bv reference herein
- these methods involve the sequential addition of protected amino acids to a growing peptide chain (U S Patent No 5,693,616, herein incorporated by reference in its entirety) Normally, either the ammo or carboxyl group of the first amino acid and any reactive side chain group are protected This protected ammo acid is then either attached to an inert solid support, or utilized in solution, and the next ammo acid m the sequence, also suitably protected, is added under conditions amenable to formation of the amide linkage
- the fluorophores can be added dunng the solid-phase svnthesis reaction, or as a later step in aqueous phase, as is known to those of skill in the art After all the desired amino acids have been linked in the proper sequence, protecting groups and any solid support are removed to afford the crude polypeptide The polypeptide is desalted and punfied, preferably chromatographically, to yield the final product
- peptides are synthesized according to standard solid-phase methodologies, such as may be performed on an Applied Biosystems Model 430A peptide synthesizer (Applied Biosystems, Foster City, Calif), according to manufacturer's instructions Other methods of synthesizing peptides or peptidomimetics. either by solid phase methodologies or in liquid phase, are well known to those skilled m the art
- the peptide portion of the protease biosensor can be produced v ia standard recombinant DNA technology
- a DNA sequence encoding the desired amino acid sequence is cloned into an appropnate expression vector and used to transform a host cell so that the cell expresses the encoded peptide sequence.
- Methods of cloning, expression, and purification of recombinant peptides are well known to those of skill m the art. See. for example, Sambrook, et al, Molecular Cloning: A Laboratory Manual (2nd Ed., Vols. 1-3, Cold Spring Harbor Laboratory (1989)), Methods m Enzymology, Vol. 152.
- the present invention provides isolated polynucleotide sequences that encode the peptide portion of the biosensors disclosed herein.
- the polypeptide sequence is selected from the group consisting of;
- NAA(A/G) (SEQ ID NO:29), which encodes SEQ ID NO: 15
- the codon (T/C)TN when the codon (T/C)TN is specified, it encodes leucine, when the codon (A/T)(G/C)N is specified, it encodes senne, and when the codon (A/C)GN is specified, it encodes argmine.
- the polynucleotide sequence is preferably double stranded and cloned into an expression vector for recombinant expression and punfication of the peptide, followed by attachment of the fluorophore as descnbed above.
- the polynucleotide sequence preferably encodes one or more additional ammo-termmal amino acids for attaching a fluorophore.
- the polynucleotide may also encode one or more additional carboxy-termmal ammo acids for attaching a fluorophore
- cells are provided that ha ⁇ e been transfected with the expression vectors and that express the peptide portion oi the biosensor
- the donor and acceptor fluorophores are attached to the peptide by any of a number of means w ell known to those of skill m the art
- the fluorophores are linked directly from a reactive site on the fluorophore to a reactive group on the peptide such as a terminal amino or carboxyl group, or to a reactive group on an am o acid side chain such as a sulfhydryl, an amino, or a carboxyl moiety
- Suitable linkers are well known to those of skill in the art and include, but are not limited to, isothiocyanate, succmimide ester, maleimide, lodoacetamide, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers
- Choice of an acceptor molecule depends on the abov e as well as availabi lity of suitably reactive denvatives of the acceptor and its solubility (more soluble fluorophores are prefened)
- labeling of the FRET-based peptide biosensors of the present invention with both fluorescein and eosin resulted in a non-soluble product
- the sensor itself must be soluble m aqueous (preferably salme) solution
- the position of the donor and acceptor fluorophores on the peptide mav vary, but the ⁇ must be on opposite sides of the peptide cleavage site and must not interfere with protease activity It is possible that the particular attachment of each d ⁇ e may result in poor alignment with respect to each other, resulting in low efficiency of energy transfer in spite of good overlap
- the use of soluble fluorophores with freelv-rotating linkers (as discussed above) between the peptide and the dye make this
- R9 in general formula II or Rl 1 in general formula III is Cys and is used to link a thiol-specific fluorophore, such as ALEXA-FLUOR® 546 maleimide, BODIPY® 530/550 lodoacetamide, eos ⁇ n-5-male ⁇ m ⁇ de. and QSYTM-7 maleimide (all available from Molecular Probes, Eugene, OR)
- a thiol-specific fluorophore such as ALEXA-FLUOR® 546 maleimide, BODIPY® 530/550 lodoacetamide, eos ⁇ n-5-male ⁇ m ⁇ de. and QSYTM-7 maleimide (all available from Molecular Probes, Eugene, OR)
- R9 in general formula II or Rl 1 in general formula III is Cys and is used to link ALEXA-FLUOR® 546 maleimide
- the acceptor molecule compnses fluorescein
- the protease biosensors of the present invention mav be obtained m solution or linked to a solid support
- a solid support can compnse any solid matenal that does not dissolve in or react with any of the components present in the solutions utilized for assaying for protease activity, and that provides a functional group for attachment of the biosensor
- Solid support matenals are well known to those of skill in the art and include, but are not limited to silica, controlled pore glass (CPG), polystyrene, polystyrene/latex, carboxyl modified teflon, dextran, denvatized polysacchandes such as agar beanng amino, carboxyl or sulfhydryl groups, and vanous plastics such as polyethylene and acrylic
- the solid supports may be denvatized with functional groups (e g hydroxyls, amines, carboxyls, esters, and sulfhydryls) to provide reactive sites for the attachment of linkers or the direct attachment
- a solution-phase method for detecting the presence of lethal factor from Bacillus anthracis in a test sample compnsing a) providing one or more protease biosensors as disclosed above, b) contacting the protease b ⁇ osensor(s) with the test sample, and c) measu ⁇ ng fluorescence resonance energy transfer from the protease biosensor, wherem an increase m the emission spectra of the donor molecule indicates the presence of lethal factor from Bacillus anthracis in the test sample
- fluorescence resonance energy transfer is detected by measunng the ratio of emission from the donor and the acceptor, where the ratio of donor acceptor emission intensity increases in the presence of the lethal factor
- a cell-based method for detecting the presence of lethal factor from Bacillus anthracis in a test sample comprising a) providing one or more protease biosensors as disclosed above, b) loading the protease b ⁇ osensor(s) into cells to be analyzed, c) contacting the cells with the test sample, d) measunng cell-based fluorescence resonance energy transfer from the protease biosensor, wherem an increase in the emission intensity of the donor indicates the presence ot lethal factor from Bacillus anthracis in the test sample
- the contacting of the protease biosensor or the cells is earned out in the presence of generalized protease inhibitors that do not interfere with lethal factor activity, including but not limited to AEBSF (4-(2- am ⁇ noethyl)benzenesulfonylfluonde), aprotmm, bestatin, E-64, leupeptm, and pepstatin A
- AEBSF 4-(2- am ⁇ noethyl)benzenesulfonylfluonde
- aprotmm bestatin
- E-64 leupeptm
- leupeptm leupeptm
- pepstatin A pepstatin A
- the test sample compnses an environmental sample, including but not limited to water, soil , and air samples Alternatively, the test sample may compnse any type of cell culture fluid
- the peptide biosensor may be loaded into cells and function as an intracellular indicator of protease activity Such loading can be accomplished by techniques including, but not limited to, tnton-mediated loading, scrape loading, synnge-loadmg, and micromjection
- a fluorescent acceptor dye is preferable because it provides a signal regardless of whether it is mtact or cleaved, and it enables ratio imaging
- the methods can further be used to identify the presence of Bacillus anthracis lethal factor m anv biological tissue or fluid, including but not
- the measurements can be made in a high-throughput mode in solution or in a cell without subcellular resolution by any method known in the art including but not limited to fluorescence spectroscopy, flow cytometry, fluorescence microscopy, and b> use of real-time PCR monitors (Fisher Scientific, Roche)
- the cell screening system described m U S Patent Senal Nos 5,989,835 and 6, 103.479 could be used, both references incorporated by reference herein in their entirety
- the measurements can be made m a high content mode with subcellular resolution by any method known in the art, such as descnbed in U S Patent Senal Nos 5,989,835 and 6,103,479
- the real-time reporting of the proteolytic events make this type of biosensor amenable to kinetic assays
- the method can be used to detect lethal factor from a test sample, or to serve as the basis for a drug screening program to identify candidate compounds that interfere with the effect of lethal factor on a cell
- the method can be used to
- the protease biosensors of the present invention can be used as part of a combined high throughput (HT)-h ⁇ gh content (HC) cell-based screen
- the protease biosensor would serve as an HT component, allowing the user to screen cells in a high-throughput mode for the presence or absence of fluorescence emission (in the green channel m the examples below)
- a non-fluorescent acceptor dye erythrosin or QSY-7 m this example
- the sequence of the peptide is MPKKKPTPIQLNPC (SEQ ID NO:3).
- the peptide was synthesized by a custom synthesis facility (BioPeptide, San Diego, CA) with carboxyfluorescein specifically attached to the N-terminus dunng solid phase synthesis.
- the peptide was resuspended at a concentration of approx. 10 mg/ml by weight in 0.1% acetic acid.
- Fluorescence Spectra Solutions of dye or peptide with a maximal absorbance of approximately 0.1 were generally used to collect fluorescence data. Spectra were recorded in a K2 Multifrequency Phase Fluorometer (ISS. Champaign, IL). Temperature control for kinetics experiments was provided by a circulating water bath providing heat to the cuvette chamber The cuvette chamber was also equipped with a stirnng mechanism Excitation and emission spectra were recorded for charactenzation of the dual-labeled peptide and trypsin- treated peptide For some kinetics expenments involving proteases, the fluorometer was set up to record the emission at two fixed wavelengths at a set interval over a two-hour period In other cases, a CYTOFLUOR® plate reader (Perseptive Biosystems, Inc ) was used to measure fluorescence intensity at two different emission wavelengths
- Tnton-based loading the following procedure was used On the day before they were to be loaded, BHK cells were seeded into 6-well dishes at 1 x 10' per well The cells were approx.
- the cells were treated with a solution of 0.001% Tnton X-100 in Hank's Balanced Salt Solution for 4 minutes
- the saline was replaced with complete medium containing vanous concentrations of dual-labeled peptide, with or without additional Tnton X-100
- the cells were incubated for 1 5 hours, then the peptide solutions were removed and complete medium was added to the wells Cells were observed under a fluorescence microscope both immediately after peptide removal and after a 3.5 hour recovery penod in the incubator Images were acquired using an Axiovert 25 microscope and QED Imaging software. Images were saved as TIFF files and imported into Adobe Photoshop.
- the peptide was not taken up by cells by simply placing it in the extracellular medium, indicating that the chemical properties of the peptide do not allow for transfer through the plasma membrane
- this method did not work for loading peptide, this may be due to the small size of the peptide which is not conducive to forming peptide-hposome aggregates, as well as the isoelectnc point of the peptide (8 5) which would make it neutral at the pH of the aggregation step (anionic proteins are prefened)
- anthrax LF (provided by Dr Stephen Leppla, National Institutes of Health), or 0 l ⁇ M anthrax
- the peptide was cleaved by anthrax LF, no cleavage of the peptide by trypsin or chymotrypsm occurred ( Figure 4B). The rate of cleavage of the peptide by anthrax LF was unaffected by the inhibitors.
- the peptide is a detector of anthrax LF Nonspecific zinc metalloproteases, such as thermolysin. would also not be inhibited in the reaction and could cleave the peptide.
- Thermolysin is a bactenal protease from Bacillus thermoproteolyticus rokko. and not a known human toxm Non-limiting examples of such variations to the sensor structure and their expected effects are listed in the table below
- protease sensor for detecting anthrax protease is an improvement over cunent methods for detection of Bacillus anthracis employing PCR, which does not discriminate among live versus dead, or virulent vs. non-virulent strains of anthrax Because the protease (lethal factor) is only expressed when the bactena are viable and expressing the appropriate virulence factors, this sensor detects the most dangerous strains of this potential biological warfare agent. Therefore, a sensor of this type will have fewer false positives, which is desirable for a sensor to be used in a potentially hazardous situation.
Abstract
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Priority Applications (4)
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EP01937142A EP1354201A2 (en) | 2000-02-11 | 2001-02-09 | Peptide biosensors for anthrax protease |
US10/182,303 US7410769B2 (en) | 2000-02-11 | 2001-02-09 | Peptide biosensors for anthrax protease |
CA002398583A CA2398583A1 (en) | 2000-02-11 | 2001-02-09 | Peptide biosensors for anthrax protease |
AU2001262906A AU2001262906A1 (en) | 2000-02-11 | 2001-02-09 | Peptide biosensors for anthrax protease |
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US18201100P | 2000-02-11 | 2000-02-11 | |
US60/182,011 | 2000-02-11 |
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002010433A2 (en) * | 2000-05-03 | 2002-02-07 | Expressive Constructs, Inc. | Method and device for detecting bacterial contamination |
WO2003073066A2 (en) * | 2002-02-25 | 2003-09-04 | Merck & Co., Inc. | Reagents and methods for assaying bacillus anthracis lethal factor protease |
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WO2010059051A2 (en) * | 2008-11-20 | 2010-05-27 | Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno | Rapid fret based pathogen diagnosis |
WO2010059051A3 (en) * | 2008-11-20 | 2010-09-30 | Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno | Rapid fret-based diagnosis of bacterial pathogen |
US8852883B2 (en) | 2008-11-20 | 2014-10-07 | Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno | Rapid FRET-based diagnosis of bacterial pathogens |
Also Published As
Publication number | Publication date |
---|---|
US7410769B2 (en) | 2008-08-12 |
US20020076741A1 (en) | 2002-06-20 |
WO2001059149A3 (en) | 2003-08-21 |
CA2398583A1 (en) | 2001-08-16 |
AU2001262906A1 (en) | 2001-08-20 |
US20030166028A1 (en) | 2003-09-04 |
EP1354201A2 (en) | 2003-10-22 |
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