WO1999012036A1 - Immobilisation reversible de fractions marquees a l'arginine sur une surface de silicate - Google Patents

Immobilisation reversible de fractions marquees a l'arginine sur une surface de silicate Download PDF

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Publication number
WO1999012036A1
WO1999012036A1 PCT/US1998/018531 US9818531W WO9912036A1 WO 1999012036 A1 WO1999012036 A1 WO 1999012036A1 US 9818531 W US9818531 W US 9818531W WO 9912036 A1 WO9912036 A1 WO 9912036A1
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arginine
tag
layered silicate
protein
mica
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PCT/US1998/018531
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English (en)
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James A. Spudich
Steffen Nock
Peter Wagner
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Stanford University
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Application filed by Stanford University filed Critical Stanford University
Priority to AU92225/98A priority Critical patent/AU9222598A/en
Priority to US09/486,480 priority patent/US6960457B1/en
Publication of WO1999012036A1 publication Critical patent/WO1999012036A1/fr
Priority to US10/850,207 priority patent/US20050283003A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand

Definitions

  • the immobilization of functional proteins on flat surfaces is of crucial importance for studying their interaction with ligands and examining their structure by means of electron and scanning probe microscopy and other biophysical techniques requiring a solid interface.
  • Targeting proteins at specific sites and anisotropically immobilizing them on a surface while preserving their functionality is a major precondition to facilitate biochemical recognition and interaction, to present selected sites for structural investigation, to induce two-dimensional crystallization, and to develop new biosensors and supramolecular assemblies.
  • two methods have been used to attach (immobilize) proteins to a solid surface. In the first method, the peptide is applied to the surface in solution, which is then evaporated off, leaving the peptide dried to the surface.
  • the solid surface is first passively coated with a large protein, such as an immunoglobulin or bovine serum albumin. Then, a hetero-bifunctional cross-linking agent, such as SPDP or glutaraldehyde, is attached to the protein and used to capture peptide from solution.
  • a hetero-bifunctional cross-linking agent such as SPDP or glutaraldehyde
  • SPDP amino acid sequence polymer
  • glutaraldehyde glutaraldehyde
  • IMAC Interleukin Affinity Chromatography
  • Mica with the ideal structure KAl 2 [AlSi 3 O ⁇ 0 ](OH,F) 2 , refers to a group of layered aluminosilicate minerals whose crystals exhibit a large degree of basal cleavage, allowing them to be split into very thin atomically flat sheets. Due to its flatness and hydrophilic surface, mica has been established as a standard substrate for electron and scanning probe microscopy applications (see e.g., Zahn et al. (1993) J. Mol.
  • N-dodecylpyridinium chloride (1996) J. Colloid Interface Sci. 181: 476- 489) and N-dodecylpyridinium chloride (NDP) (Sheldenet al. (1993) J. Colloid Interface Sci., 157: 318-327) are known to hydrophobize negatively charged minerals. Exchange with bivalent cations has been used to mediate binding of DNA for SPM studies (Hansma et al. (1995) Biophys. J. 68: 1672-1677). Similarly, 2,2'-azobisisobutyramidine hydrochloride (ALB A) has been used as an azo initiator for the polymerization of styrene directly on the mica surface (Shelden et al. (1993) J. Colloid Interface Sci., 157: 318-327; Shelden et al. (1994) Polymer, 35: 1571-1575).
  • ALB A 2,2'-azobisisobutyramidine hydrochlor
  • This invention provides methods and materials for the controlled (oriented) attachment of virtually any moiety to an atomically smooth surface.
  • the invention is premised, in part, on the surprising discovery that arginine, more preferably polyarginine molecules show a highly specific interaction with the surfaces of layered silicates mediated, at least in part, by a cation exchange with the silicate surface. Unlike previously described cation exchange systems, binding of the arginine tag is highly resistant to physiologically relevant (compatible) concentrations of sodium and other ions.
  • this invention provides methods of attaching a moiety to a layered silicate surface.
  • the methods involve covalently attaching the moiety to an arginine tag and contacting the arginine tag with the layered silicate surface.
  • the arginine tag can comprise at least two argine residues (or arginine residue analogs) and preferably comprises from about two to about 100 arginine residues.
  • the arginine tag can be an arginine homopolymer consisting only of arginine residues or it can be a heteropolymer comprising arginine residues and other moieties (e.g., other amino acids).
  • the arginine residues can occur in one or more stretches having at least 2, preferably at least 4, more preferably at least 6, and most preferably at least 10 contiguous arginine residues.
  • One preferred layered silicate is mica (e.g., a muscovite mica).
  • the method can further comprise contacting the layered silicate with a solution containing a sodium salt in a concentration (e.g., 1 mM - 200 or even 300 mM Na ⁇ ) sufficient to remove molecules bound to the layered silicate by non-specific ion exchange.
  • the moiety can be virtually any object, or article of manufacture including, but not limited to biological moieties such as cells, tissues, organelles, and various biomolecules (e.g., proteins, nucleic acids, lipids, glycoproteins, polysaccharides, and the like). Proteins and nucleic acids are particularly preferred moieties.
  • a protein can be chemically conjugated to the arginine tag or fused to the amino or carboxyl terminus of the arginine tag. Where the protein is fused, the protein can be recombinantly expressed as a fusion protein with the arginine tag.
  • Particularly preferred proteins include DNA binding proteins, molecular motors, an actin filament, a microtubule, a myosin filament, an actin binding protein, and a myosin filament binding protein.
  • this invention provides a surface functionalized for the attachment of organic molecules where the functionalization is compatible with physiological sodium salt concentrations (e.g., a the concentration of NaCl in human blood).
  • the surface can comprise a layered silicate contacted with any of the arginine tag molecules described herein.
  • the arginine tag can be directly functionalized or covalently joined to a molecule selected from the group consisting of a protein, an antibody, a DNA binding protein, a molecular motor, an actin filament, a microrubule, a myosin filament, an actin filament binding protein, a myosin filament binding protein, a cell surface receptor, a growth factor, a hormone, and a nucleic acid.
  • the arginine tag is fused to the amino or carboxyl terminus of a polypeptide. The fusion can be chemical created or recombinantly expressed.
  • this invention provides methods of orienting a polypeptide on a layered silicate surface (e.g., a mica surface).
  • the methods involve providing a polypeptide covalently linked to an arginine tag; and contacting the arginine tag with the layered silicate surface. Any of the argimne tags described herein are suitable.
  • the methods can additionally involve contacting the surface with a sodium salt in a concentration sufficient to remove molecules bound to the layered silicate by ion exchange.
  • Suitable polypeptides can include molecular motors, actin filaments, microtubules, myosin filaments actin filament binding proteins, myosin filament binding proteins, and the like.
  • this invention provides a surface bearing anisotropically oriented proteins.
  • the surface can comprise a mica surface contacted with a plurality of proteins, each protein covalently attached to the surface through an arginine tag.
  • the arginine tag can include any of the arginine tags described herein.
  • Preferred polypeptides include molecular motors, actin filaments, microtubules, myosin filaments, actin filament binding proteins, myosin filament binding proteins, and the like.'
  • This invention also provides methods of purifying a target molecule from a heterogeneous mixture of molecules.
  • the methods involve providing a target molecule attached to an arginine tag and then contacting the target molecule with the surface of a layered silicate whereby the target molecule binds to the layered silicate surface.
  • the arginine tag can include any of the arginine tags described herein.
  • the purification methods can additionally involve contacting the layered silicate surface with a sodium salt and/or with a potassium salt, an argine, or a polyarginine in a concentration sufficient to release the target molecule.
  • the contacting of the mixture with the layered silicate can involve flowing the heterogeneous mixture over one or more layered silicate (e.g., mica surfaces) and/or combining the layered silicate (e.g., mica) the heterogeneous mixture.
  • the methods can also involve removing (e.g., via centrifugation) the layered silicate from the heterogeneous mixture.
  • the methods can also involve contacting the layered silicate with a c a potassium salt, an arginine, or a poly-arginine.
  • the target molecule is any of the fusion polypeptdides described herein.
  • the layered silicate used in the purification can include a mica powder or a mica flake.
  • Preferred devices comprise a vessel such as a chromatography column or any other vessel equipped with an inlet and an outlet port.
  • the vessels contain a bed of layered silicate.
  • the layered silicate can include a powdered layered silicate (e.g., powdered mica) and/or a mica flake.
  • the ports are compatible with the frit of a syringe.
  • isolated refers to material which is substantially or essentially free from components which normally accompany it as found in its native state.
  • nucleic acid refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
  • polypeptide peptide
  • protein protein
  • amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • arginine or "arginine residue” as used herein refers to natural, synthetic, or modified arginine amino acids.
  • An arginine can also include arginine analogs that offer the same or similar functionality as natural arginine with respect to their ability to be incorporated into a polypeptide and to interact with a layered silicate.
  • nucleic acid encoding or “nucleic acid sequence encoding” refers to a nucleic acid that directs the expression of a specific protein or peptide.
  • the nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein.
  • the nucleic acid sequences include both full-length nucleic acid sequences as well as shorter sequences derived from the full- length sequences. It is understood that a particular nucleic acid sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.
  • the nucleic acid includes both the sense and antisense strands as either individual single strands or in the duplex form.
  • immunoassay is an assay that utilizes an antibody to specifically bind an analyte.
  • the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the analyte.
  • molecular motor refers to a molecule that utilizes chemical energy to produce mechanical force and drives the motile properties of muscle or the cytoskeleton.
  • Layered silicates are a group of laminated silica minerals that include, but are not limited to vermiculite, montmorillonite, bentonite, hectorite, fluorohectorite, hydroxyl hectorite, muscovite boron fluorophlogopite, hydroxyl boron phlogopite, and the like.
  • fusion protein refers to a protein (polypeptide) composed of two polypeptides that, while typically unjoined in their native state native state, typically are joined by their respective amino and carboxyl termini through a peptide linkage to form a single continuous polypeptide. It will be appreciated that the two polypeptide components can be directly joined or joined through a peptide linker/spacer.
  • phrases "compatible with physiological sodium salt concentrations” or compatible with “salt concentrations” are used herein to indicate that the bond formed between a arginine tag and a layered silicate is substantially stable to physiological sodium salt concentrations or to salt concentrations of a particular strength.
  • substantially stable us used herein to indicate that at least 50%, preferably at least about 80%, more preferably at least about 90%, and most preferably at least about 95%, 98%, or even 99% of a moiety attached to a layerd silicate surface, remains attached to the layered silicate when contacted with the stated salt solution.
  • normal physiological conditions is used herein to refer to conditions that are typical inside a living organism or a cell. While it is recognized that some organs or organisms provide extreme conditions, the intra-organismal and intra- cellular environment normally varies around pH 7 (i.e. from pH 6.5 to pH 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C and below 50°C.
  • physiological salt concentrations refer to the concentrations of a particular salt typically found within an organism. Again, it will be recognized that that the concentrations of various salts depends on the organ, organism, cell, or cellular compartment used as a reference. Nevertheless, the concentrations of various salts vary between generally well known limits and average concentrations are represented by the salt concentrations provided in standard ringers solutions.
  • One measure of a physiological concentration of a sodium salt is the concentration of sodium chloride in human blood.
  • moiety when referred to in the context of attaching a moiety to a surface is used to refer to essentially any composition or molecule that is to be attached to the surface.
  • the moiety can include macroscopic compositions (e.g., an article of manufacture, a bead, etc.) and microscopic compositions, including for example, a biological molecule (biomolecule), an organelle, a cell, a tissue, virtually any naturally occurring natural or synthetic material that is chemically compatible with arginine tag.
  • the arg-tag attached moieties are biological molecules including, but not limited to, proteins, carbohydrates, lipids, and nucleic acids.
  • Particularly preferred biological molecules include antibodies, DNA binding proteins, molecular motors, actin filaments, myosin filaments, microtubules, actin filament binding proteins, and the like.
  • Figure 1 schematically illustrates a protein immobilized to the mica surface via its Arg-tag (not drawn to scale).
  • the muscovite mica structure is shown in the inset.
  • Figure 2 schematically illustrates three different GFP variants.
  • the N- and C- terminally added sequences are shown in the one letter amino acid code.
  • the hexaarginine tag is marked in bold letters and the GFP is shown as a gray bar.
  • Figure 3 indicates stepwise elution of immobilized protein as a function of consecutive washes of the same surface with increasing NaCl concentration in the wash buffer followed by a 100 mM Arg wash.
  • the values for GFPR ⁇ , GFPH 6 R 6 and GFPH 6 are shown in black, light gray and dark gray, respectively.
  • Figure 4 shows the dependency of the elution of immobilized protein on MgCl 2 (A), KCl (B) and Arg (C) concentration.
  • GFPR6 is shown in black and GFPH6R6 is shown in light gray.
  • Figure 5 shows XPS-survey spectra of (A) freshly cleaved mica and (B) GFPR6 immobilized to mica. The binding strength of bound GFPR6 and GFPH6 to mica after washing with increasing amounts of NaCl and KCl is shown in (C) as monitored by the XPS Nls narrow scans (arbitrary units and normalized to the Si Is signal). The dashed line means no photoelectron counts.
  • This invention provides novel materials and methods for the attachment of proteins, or other moieties, to solid surfaces, in particular to the surfaces of layered silicates.
  • the methods and materials are particularly advantageous in that the attachment is easily reversed (undone) and yet stable to physiologically relevant concentrations of ions such as K + , Na + , Mg 2+ , Ca 2+ and the like.
  • the precise point of attachment of the protein (or other moiety) can be predetermined, it is possible to prepare proteins bound to surfaces in which critical binding, recognition, or reactive sites are free (or attached) to the surface and thus able to participate in various reactions.
  • the precise attachment permits the proteins to be uniformly oriented on the surface.
  • layered silicates e.g., mica
  • bound proteins, or other moieties are not hidden or masked from reactive agents by surface irregularities.
  • This invention is premised, in part, on the discovery that an arginine tag (e.g., a single arginine or a series of arginine molecules covalently linked together) will interact with the surface of layered silicates (i.e., negatively charged layered silicates such as mica) and form a highly specific interaction with the layered silicate surface.
  • layered silicates i.e., negatively charged layered silicates such as mica
  • the binding reaction can be used to purify (isolate) a protein, or other arg-tag labeled, moiety from a heterologous collection of molecules.
  • the arginine- or polyarginine-tag (referred to herein as an arginine-tag or arg-tag) can be utilized to attach virtually any moiety to the surface of a layered silicate.
  • the attachment method essentially involves attaching the arg-tag to the moiety it is desired to attach to the layered silicate surface and contacting the arg-tag to that surface. Contacting is preferably under conditions (e.g., salt, temperature and pH) in which the arg- tag participates in a binding reaction (e.g., an ion exchange reaction) with the surface whereby the arg-tag becomes bound to the surface.
  • a binding reaction e.g., an ion exchange reaction
  • the arg-tag can be bound to the layered silicate surface first and then reacted with the moiety it is desired to attach to the surface.
  • the arg-tag can be attached to the moiety it is desired to attach to the surface first and then contacted with the layered silicate surface, or the arg-tag can be attached to the moiety and contacted with the surface essentially simultaneously (e.g., in a single reaction).
  • Binding of the arg-tag to the surface or of the arg-tag and its attached moiety to the surface can essentially functionalize the surface for subsequent reactions.
  • the amino acid or polypeptide provides a free amino and a free carboxyl terminus suitable for subsequent reaction (e.g., to attach another molecule).
  • the arginine tag itself can be derivatized with a functional group to attach other molecules and/or, the arg-tag can be used to anchor a second molecule (e.g., a linker) which itself bears one or more reactive sites (e.g., -SH, -NH , -COOH, -OH, etc.) capable participating in reactions with other molecules.
  • Suitable linkers such as maleimide and others are well known to those of skill in the art.
  • a single moiety is attached to a single arg-tag.
  • the moiety can include macroscopic compositions (e.g., an article of manufacture, a bead, etc.) and microscopic compositions, including for example, a biological molecule (biomolecule), an organelle, a cell, a tissue, virtually any naturally occurring natural or synthetic material that is chemically compatible with arginine tag.
  • the arg-tag attached moieties are biological molecules including, but not limited to, proteins, carbohydrates, lipids, and nucleic acids.
  • biological molecules include antibodies, DNA binding proteins, molecular motors, actin filaments, myosin filaments, microtubules, actin filament binding proteins, microtubule binding proteins, nucleic acids, ribozymes, lectins, enzymes, ligands, receptors, growth factors, cytokines, and the like.
  • the arginine tag comprises one or more arginine molecules.
  • the argine tag comprises at least two arginine molecules, and hence, may be referred to as a polyarginine tag.
  • the length of the arginine tag has no theoretical upper limit and the arginine tag, in principle, can be hundreds, thousands, or even tens of thousands of residues in length.
  • the tag size there are practical and commercial limitations to the tag size. For example, where the arg-tag size interferes with the bound moiety, where the addition of additional residues does not significant improve binding, or where the arg-tag is of a length that renders it difficult to synthesize, express or purify.
  • the arg-tag will comprise less than about 1000 arginine residues, preferably less than about 500 arginine residues, more preferably less than about 100 residues, still more preferably less than about 50 arginine residues, and still most preferably less than about 30 or about 20 arginine residues.
  • the arg-tag comprises at least two arginine residues, more preferably at least about 4 arginine residues, and most preferably at least about 6, 8, 10, or even 15 arginine residues.
  • preferred arg-tags include about 1 to about 100 arginine residues, more preferably about 4 to about 50 arginine residues and most preferably about 6 to about 40 arginine residues.
  • the arginine residues comprising the arg-tag need not be contiguous.
  • the arg-tag may contain amino acid residues interspersed between the arginine residues.
  • the arginine tag may comprise individual arginine residues separated by one or more other amino acid residues, or alternatively, may comprise stretches of two or more contiguous argimne residues interspersed with one or more other amino acid residues.
  • the arg-tag need not even be a peptide, but rather may consist of arginine or polyarginine residues joined by linker molecules (e.g., straight or branched-chain carbon linkers, or peptide nucleic acids, etc.). However, in a preferred embodiment, particularly where the arg-tag is recombinantly expressed, a polypeptide arg-tag is preferred.
  • an arg-tag of relatively small size or one in which the concentration of guanidino groups (number of guanidino groups per Da of argine-tag) is high.
  • an arginine-tag consisting entirely of arginine residues is preferred.
  • Such an arginine-tag may be referred to as a homoarginine tag.
  • the arginine tags of this invention are not limited to naturally occurring arginine residues.
  • the arginine residues can be chemically modified according to any of a number of means well known to those of skill in the art as long as the guanidino group(s) are maintained in a conformation that permit contact with the silicate surface.
  • the arg-tag can comprise one or more non-amino acid arginine analogues containing guanidio groups.
  • the arginine can be decarboxylated or the ⁇ -amino group can be modified by any of a number of well known reactions (e.g., acetylation).
  • the secondary amine can be substituted (e.g., with methylene or other groups).
  • the arginine tag is attached to a polymer (e.g., a biopolymer such as a polypeptide, or nucleic acid) the arginine tag is attached either at the end of the polymer or at one or more locations within the polymer.
  • the arginine tag can be attached to the amino or carboxyl terminal or to the amino or carboxyl terminal amino acids through their respective side chains.
  • the arginine tag be inserted within the polypeptide.
  • the arginine tag is preferably located in an internal domain in which its presence does not interfere with the property of the polypeptide it is desired to exploit.
  • the myosin molecule may be attached to the silicate surface by an internal rather than a terminal arginine tag.
  • an attachment tag in a myosin is in loop 2 which is at the actin binding face of the molecule opposite to the tail (see, e.g., Spudich (1994) Nature, 372: 515-518).
  • the arginine tag can be attached to a terminal or be placed internally in nucleic acids, polysaccharides, glycoproteins, and the like.
  • the arginine tag(s) are placed in site(s) selected so as to leave domains having the desired activity free from the surface.
  • an arginine-tag need not be the only tag on the subject moiety.
  • One or more additional tags may be present in addition to the arginine tag(s).
  • the use of multiple tags will permit detection, immobilization, and/or detection under different conditions (e.g., salt, pH, etc.). Virtually any tag and/or label may be additionally be present.
  • Such tags are well known to those of skill in the art.
  • One example of a multiply tagged moiety is a polypeptide tagged both with a polyhistidine and a polyarginine as shown in Example 1.
  • the methods of this invention means for the attachment of virtually any moiety to a layered silicate surface.
  • Layered silicates such as mica
  • Silicate composite materials include least one mica and a structurally compatible species. Methods of preparing silicate composites are well known to those of skill in the art (see, e.g., U.S. Pat. Nos. 4,239,519, 4,297,139, and 4,339,540).
  • micas are those that can be fractured to produce a smooth surface, more preferably an atomically smooth surface. Due to its extreme flatness (smoothness at an atomic scale) and hydrophilic surface, mica has been established as a standard substrate for electron and scanning probe microscopy applications (see e.g., Zahn et al. (1993) J. Mol. Biol, 229: 579-584; Yang et al. (1994) FEBSLett. 338: 89-92; Guckenberger et al. (1994) Science 266: 1538-1540; Mueller et al. (1996) Biophys. J. 70: 1796-1802).
  • the layered silicates are micas with the structure KAl 2 [AlSi 3 O ⁇ 0 ](OH,F) 2 , which include a group of layered aluminosilicate minerals whose crystals exhibit a large degree of basal cleavage, allowing them to be split into very thin atomically flat sheets.
  • arginine tag can be covalently attached to the arginine tag according to any of a variety of methods well known to those of skill in the art.
  • the arginine tag can be recombinantly expressed or chemically synthesized and then chemically conjugated to the moiety.
  • the moiety is a polypeptide, a nucleic acid or a peptide nucleic acid
  • the arg-tag can be synthesized as a part of the process of chemically synthesizing the polypeptide, nucleic acid or peptide nucleic acid.
  • the arg-tag can be recombinantly expressed as a fusion protein with the polypeptide moiety.
  • the arg-tag is chemically conjugated to the moiety (e.g., polypeptide) it is desired to attach to the layered silicate surface.
  • the moiety e.g., polypeptide
  • Means of chemically conjugating molecules are well known to those of skill in the art.
  • polypeptides typically contain variety of functional groups; e.g., carboxylic acid (COOH) or free amine (-NH 2 ) groups, which are available for reaction with a suitable functional group on the arg-tag or on a linker attached to the arg tag to bind the polypeptide thereto.
  • functional groups e.g., carboxylic acid (COOH) or free amine (-NH 2 ) groups, which are available for reaction with a suitable functional group on the arg-tag or on a linker attached to the arg tag to bind the polypeptide thereto.
  • the arg-tag and/or moiety may be derivatized to expose or attach additional reactive functional groups.
  • the derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical
  • a "linker”, as used herein, is a molecule that is used to join the arg-tag to the moiety.
  • a preferred linker is capable of forming covalent bonds to both the arg-tag and to the moiety molecule.
  • Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.
  • the linkers may be joined to the constituent amino acids through the amino acid side groups (e.g., through a disulfide linkage to cysteine).
  • the linkers will be joined to the alpha carbon amino and carboxyl groups of the terminal amino acids.
  • a bifunctional linker having one functional group reactive with a group on a an arg-tag, and another group reactive with a group on the moiety that is to be attached may be used to form the desired arg-tag conjugate.
  • derivatization may involve chemical treatment of the arg-tag and/or the moiety that is to be attached. For example, where a glycoprotein (e.g., an antibody) is to be attached to the arg tag, glycol cleavage of the sugar moiety of a the glycoprotein using periodate will generate free aldehyde groups.
  • the free aldehyde groups glycoprotein may be reacted with free amine or hydrazine groups on an arg-tag to bind the agent thereto (see, e.g., U.S. Patent No. 4,671,958).
  • Procedures for generation of free sulfhydryl groups on polypeptides, such as antibodies or antibody fragments, are also known (see, e.g., U.S. Pat. No. 4,659,839).
  • Many procedures and linker molecules for attachment of various compounds including radionuclide metal chelates, toxins and drugs to proteins such as antibodies are known and can be easily modified for attachment to an arginine tag (see, e.g., European Patent Application No. 188,256; U.S. Patent Nos.
  • conjugates comprising linkages which are cleavable in the vicinity of the target site may be used when the arginine-tag is to be released at the target site.
  • Cleaving may be accomplished by enzymatic activity or particular chemical conditions.
  • the arg-tag labeled molecule can be chemically synthesized de novo.
  • the moiety is a polypeptide and the arg-tag and moiety are relatively short (i.e., less than about 50 amino acids) they may be synthesized using standard chemical peptide synthesis techniques. Where both molecules are relatively short the chimeric molecule may be synthesized as a single contiguous polypeptide.
  • the arg-tag and the polypeptide moiety may be synthesized separately and then fused by condensation of the amino terminus of one molecule with the carboxyl terminus of the other molecule thereby forming a peptide bond.
  • the arg-tag and polypeptide moiety may each be condensed with one end of a peptide spacer molecule thereby forming a contiguous fusion protein.
  • Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is the preferred method for the chemical synthesis of the polypeptides of this invention. Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp.
  • the peptide nucleic acid can be synthesized first and the the free terminus used as an initiation point for arginine-tag synthesis, or conversely, the arginine tag can be synthesized first and the free terminus used as an initiation point for peptide nucleic acid synthesis.
  • a peptide nucleic acid refers to nucleotides attached to each other through a peptide backbone. Methods of synthesizing peptide nucleic acids can be found in U.S. Patents 5,539,083 and 5,539,082.
  • the arginine- tagged nucleic acid can also be synthesized de novo.
  • Methods of nucleic acid synthesis include, but are not limited to the phosphoramidite method described by Beaucage and Carruthers (1981) Tetrahedron Lett. 22:1859-1862, or the triester method according to Matteucci et al. (1981) J. Am. Chem. Soc, 103:3185.
  • Synthesis of the arg-tag labeled nucleic acid involves either first synthesizing the arg-tag and then utilizing the free terminus of the arg tag as an initiation point for oligonucleotide synthesis, or conversely, first synthesizing the oligonucleotide and then using the free oligonucleotide as an initiation point for peptide synthesis.
  • Compatible oligonucleotide/peptide chemistries are well known to those of ordinary skill in the art.
  • the arginine tagged fusion proteins of the present invention are synthesized using recombinant DNA methodology. Generally this involves creating a DNA sequence that encodes the an argimne tag fused to either the amino or carboxyl terminus of a polypeptide it is desired to attach to the silicate surface.
  • the fusion can be direct or can involve a peptide linker that provides spacing between the arginine tag and the polypeptide or that adjusts reading frame, etc.
  • the nucleic acid encoding the fusion protein is placed in an expression cassette under the control of a particular promoter, a host cell is transf ⁇ cted with the expression cassette, the fusion protein is expressed in the host cell, isolated, and if required, renatured.
  • the arginine-tag fusion protein expression cassettes can be constructed according to ordinary methods well known to those of skill in the art. Construction of these cassettes is exemplified in Example 1.
  • constructs can all be created using standard amplification and cloning methodologies well known to those of skill in the art. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in
  • prokaryotic expression systems may be used to express arg-tag polypeptide fusion proteins. Examples include, but are not limited to, E. coli, Bacillus, Streptomyces, and the like.
  • Prokaryotic expression plasmids preferably contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator.
  • regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway as described by Yanofsky (1984) J. Bacteriol, 158: 1018-1024 and the leftward promoter of phage lambda (P) as described by Herskowitz, et al. (1980), Ann. Rev. Genet., 14: 399-445.
  • the inclusion of selection markers in DNA vectors transformed in E. coli is also useful.
  • markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. See Sambrook et al. for details concerning selection markers for use in E. coli.
  • the polypeptides produced by prokaryote cells may not necessarily fold properly.
  • the expressed polypeptides may first be denatured and then renatured. This can be accomplished by solubilizing the bacterially produced proteins in a chaotropic agent such as guanidine HC1 and reducing all the cysteine residues with a reducing agent such as beta-mercaptoethanol. The polypeptides are then renatured, either by slow dialysis or by gel filtration (see, e.g., U.S. Patent No. 4,511,503).
  • polyarginine tagged fusion proteins may also be expressed in these eukaryotic systems.
  • yeast expression systems are described in Wahleithner et al. (1991) Proc. Natl. Acad. Sci. USA 88:10387-10391, Murphy and Lagarias (1997) Photochem. Photobiol, 65: 750-758, and Wu et al. (1996) Proc. Natl. Acad. Sci., USA, 93: 8989-8994. Further examples of yeast expression are described below. A number of yeast expression plasmids like YEp6, YEpl3, YEp4 can be used as vectors. A gene of interest can be fused to any of the promoters in various yeast vectors. The above-mentioned plasmids have been fully described in the literature (Botstein et al.
  • the polypeptides can be isolated from yeast by lysing the cells and applying standard protein isolation techniques, or the arginine-tag purification techniques described herein, to the lysates.
  • the monitoring of the purification process can be accomplished by using spectroscopic techniques, or by using Western blot techniques or radioimmunoassays, or other standard immunoassay techniques.
  • the arginine-tag fusion polypeptides of this invention can also be expressed in plants or plant tissues.
  • Plant tissue includes differentiated and undifferentiated tissues of plants including, but not limited to, roots, shoots, leaves, pollen, seeds, tumor tissue, such as crown galls, and various forms of aggregations of plant cells in culture, such as embryos and calli.
  • the plant tissue may be in plants, cuttings, or in organ, tissue, or cell culture.
  • the recombinant DNA molecule encoding the arginine-tag fusion polypeptide under the control of promoter sequences may be introduced into plant tissue by any means known to the art.
  • the technique used for a given plant species or specific type of plant tissue depends on the known successful techniques.
  • the various DNA constructs described above may be introduced into the genome of the desired plant by a variety of conventional techniques.
  • the DNA construct may be introduced directly into the genomic DNA of the plant cell using polyethylene glycol precipitation (Paszkowski et al. (1984) Embo J. 3: 2717-2722) electroporation and microinjection of plant cell protoplasts (Fromm et al. (1985) Proc. Natl. Acad. Sci.
  • the DNA constructs can be introduced into plant tissue using ballistic methods, such as DNA particle bombardment (Klein et al. (1987) Nature 327: 70-73).
  • the DNA constructs may be combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
  • the virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker gene(s) (if present) into the plant cell DNA when the cell is infected by the bacteria.
  • Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus expresses the desired fusion protein.
  • Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium. Plant regeneration from cultured protoplasts is described in Evans et al. (1983) pp. 124-176 In: Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, MacMillan Publishing Company, New York; and Binding, Regeneration of Plants, Plant Protoplasts, pp. 21-73; CRC Press, Boca Raton, 1985. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. (1987) Ann. Rev. of Plant Phys. 38 : 467-486.
  • the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • Illustrative of cell cultures useful for the production of the arginine-tag fusion polypeptides of this invention are cells of insect or mammalian origin. Mammalian cell systems often will be in the form of monolayers of cells although mammalian cell suspensions may also be used. Illustrative examples of mammalian cell lines include VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, W138, BHK, Cos-7 or MDCK cell lines.
  • illustrative expression control sequences are obtained from the SV-40 promoter (Science, 222:524-527, 1983), the CMV I.E. Promoter (Proc. Natl. Acad. Sci. 81:659-663, 1984) or the metallothionein promoter (Nature 296:39-42, 1982).
  • the cloning vector containing the expression control sequences is cleaved using restriction enzymes and adjusted in size as necessary or desirable and ligated with DNA coding for the arg-tag polypeptides by means well known in the art.
  • Binding of the arginine-tagged moiety to a silicate surface is accomplished simply by contacting the arginine tagged moiety with the surface. This is preferably accomplished in solution where a solution (preferably an aqueous solution) contains the arg- tag labeled moiety (e.g., arg-tag labeled polypeptide). After the arg-tag labeled moiety is bound to the surface, the solution may optionally be removed and the surface dried.
  • a solution preferably an aqueous solution
  • the arg-tag labeled moiety e.g., arg-tag labeled polypeptide
  • moieties may contain charge sites (in addition to the arginine-tag) that interact with the silicate surface.
  • This is routinely accomplished by systematically increasing the salt (e.g., NaCl) concentration of the solution until the bound moiety exhibits the desired performance characteristics.
  • salt e.g., NaCl
  • moieties bound to a silicate surface through an arginine-tag are reversibly bound; that is, they may subsequently be released with relative ease. Release can be routinely accomplished simply by competition of the surface bound arg-tag labeled moiety with another material capable of ion exchange with the silicate surface. The concentration of the competing material is simply increased until adequate release is accomplished.
  • Release can be accomplished with high enough concentrations of virtually any cation, however, as shown in Example 1, release is particularly effective using either K + or arginine.
  • the cation concentration suitable to effect release of the bound arg-tagged moiety will vary depending on the size of the arginine-tag and the nature of the bound moiety. The concentration however can be determined simply by increasing the cation concentration until adequate release is accomplished. Potassium concentrations in excess of 0.2M and arginine concentrations in excess of O.lmolar will generally effect adequate release of an arginine-tagged moiety from a layered silicate surface
  • Example 1 From the information provided in Example 1 , it will be appreciated that materials showing non-specific binding can be released from the layered silicate surface without effecting release of the arginine-tagged moiety. This is simply accomplished by contacting the surface with a cation in a concentration sufficient to release the non- specifically bound material without releasing substantial quantities of the arginine-tagged material. It was demonstrated in Example 1 , that ionic species such as Na + were highly effective in removing non-specifically bound materials, but significantly less effective in releasing the arginine-tagged moiety. Thus, in a preferred embodiment, differential release of non-specifically bound materials is accomplished by using ionic species such as Na + or Mg 2+ rather than potassium.
  • ionic species such as Na + or Mg 2+ rather than potassium.
  • the optimal ion concentration can be determined empirically by increasing the ionic concentration until acceptable removal of undesired material is accomplished without unacceptable removal of the arginine-tagged moiety.
  • suitable Na + concentrations will range from about 1 mM to about 200 or even 300 mM.
  • the materials and methods of this invention are particularly useful for immobilizing biological molecules, including, but not limited to, proteins, antibodies, polysaccharides, lipids, nucleic acids (DNA and RNA), for use in any of a number of assays.
  • assays include, but are not limited to molecular motor assays, immunoassays, nucleic acid binding assays.
  • arginine-tagged moieties on atomically smooth silicate surfaces are therefore particularly well suited for high throughput assays.
  • new chemical entities with useful properties e.g., inhibition of myosin tail interactions
  • a chemical compound called a "lead compound”
  • HTS high throughput screening
  • high throughput screening methods involve providing a library containing a large number of compounds (candidate compounds) potentially having the desired activity. Such “combinatorial chemical libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al. (1991) Nature, 354: 84-88).
  • Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT Publication No WO 91/19735, 26 Dec.
  • nucleic acid libraries see, e.g., Strategene, Corp.
  • peptide nucleic acid libraries see, e.g., U.S. Patent 5,539,083 antibody libraries (see, e.g., Vaughn et al (1996) Nature Biotechnology, 14(3): 309-314), and PCT/US96/10287)
  • carbohydrate libraries see, e.g., Liang et al. (1996) Science, 274: 1520-1522, and U.S. Patent 5,593,853
  • small organic molecule libraries see, e.g., benzodiazepines, Baum (1993) C&E ⁇ , Jan 18, page 33, isoprenoids U.S.
  • Patent 5,569,588, thiazolidinones and metathiazanones U.S. Patent 5,549,974, pyrrolidines
  • U.S. Patents 5,525,735 and 5,519,134, morpholino compounds U.S. Patent 5,506,337, benzodiazepines 5,288,514, and the like.
  • Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA).
  • a number of well known robotic systems have also been developed for solution phase chemistries.
  • any of the assays for compounds inhibiting the virulence described herein are amenable to high throughput screening.
  • likely drug candidates either inhibit expression of the gene product, or inhibit the activity of the expressed protein.
  • Preferred assays thus detect inhibition of transcription (i.e., inhibition of mRNA production) by the test compound(s), inhibition of protein expression by the test compound(s), or binding to the gene (e.g., gDNA, or cDNA) or gene product (e.g., mRNA or expressed protein) by the test compound(s).
  • the assay can detect inhibition of the characteristic activity of the gene product or inhibition of or binding to a receptor or other transduction molecule that interacts with the gene product.
  • High throughput assays for the presence, absence, or quantification of particular nucleic acids or protein products are well known to those of skill in the art.
  • binding assays are similarly well known.
  • U.S. Patent 5,559,410 discloses high throughput screening methods for proteins
  • U.S. Patent 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays)
  • U.S. Patents 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding.
  • high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configuarable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols the various high throughput. Thus, for example, Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like. B Assay Types.
  • the materials and methods of this invention are particularly well suited for binding polypeptides to layered silicate solid supports for use in assays related to protein- protein interactions and/or protein-nucleic acid interactions.
  • the methods of this invention are well suited for investigation of protein-protein interactions that underlie the structure and function of molecular motors.
  • the methods of this invention facilitate the creation and execution of assays that screen for compounds that inhibit or enhance interactions (e.g., binding) between cytoskeletal protein components.
  • the methods involve immobilizing a first cytoskeletal component on a surface (preferably a layered silicate surface) and detecting the presence, absence, affinity and/or specificity of binding of a second component to the first component in the presence and/or absence of a test compound.
  • the assay can involve immobilizing, according to the methods of this invention, either the molecular motor or the "track” upon which the motor runs and detecting the respective movement of the non-immobilized component (motor or track).
  • Molecular motor activity assays are well known to those of skill in the art (see, e.g., Gittes et al. (1996) Biophys. J. 1: 418-429 and Shirakawa et al. 1995) J. Exp. Biol, 198: 1809- 1815). The number and identify of cytoskeletal components that have been identified thus far are legion, and far too numerous to be completely listed here.
  • the assays of this invention typically involve the interaction of two or more components. Assays involving interactions between two components can be viewed as assays for enhancers or inhibitors of binding between members of "binding pairs". Preferred binding parirs include ⁇ -actinen/actin, tropomyosin/actin, vinculin/actin, villin/actin, kinesin/microtubule, dynein/microtubule, myosin actin, myosin tail/myosin tail, and the like. It will be appreciated that either member of the binding pair can be immobilized (attached to the surface) while the other member is in a solution contacted to the surface. Alternatively, both members can be attached surfaces which are then juxtaposed to perform the assay.
  • the art has long employed the technique of utilizing indicator or carrier particles or surfaces upon which is carried the appropriate immunological material.
  • the types of particles used are extremely varied, ranging from biological materials such as red blood cells and tissue culture cells to immunologically inert polymeric particles.
  • the specific nature of the arginine-tag site permits the attachment of molecules in a uniform orientation and allows reactive (e.g., binding) sites to be positioned in a manner that optimizes their interaction with applied reagents (e.g., the reactive site can be positioned away from the substrate). Consequently immunoassays performed with the arginine-tagged moieties on a layered silicate surface are expected to provide higher sensitivity and specificity and therefore a higher signal to noise ratio. It will be appreciated that whether an antigen or an antibody is attached to the surface through an arginine-tag depends on the assay format.
  • Formats for immunoassays include, but are not limited to competitive (e.g., ELISA, hapten inhibition, etc.) and non-competitive assays.
  • Various immunoassay formats are well known to those of skill in the art (see, e.g., U.S. Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168; Asai (1993) Methods in Cell Biology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. New York; Stites & Terr, eds. (1991) Basic and Clinical Immunology 7th Edition, etc.) and the references cited therein).
  • the materials and methods of this invention are used to immobilize components in nucleic acid binding protein assays.
  • such assays detect binding of a nucleic acid by one or more binding proteins.
  • the assays are generally used to screen for agents that improve (specificity or affinity) or inhibit nucleic acid binding by one or more particular nucleic acid binding proteins.
  • Nucleic acid binding proteins include, but are not limited to endonucleases, polymerases, nuclear transcription factor receptors, API proteins (e.g., fos and jun).
  • the assays are facilitated by attachment to a surface of either the nucleic acid containing the protein binding site or the binding protein itself.
  • attachment of either of these moieties to an atomically flat (e.g. layered silicate) surface through an arginine tag of this invention is expected to improve sensitivity, selectivity and hence the signal to noise ratio of such an assay.
  • Formats for nucleic acid/binding protein assays are well known to those of skill in the art.
  • assays require particular conditions for the subject assay components (e.g., enzymes) to maintain their desired activity.
  • such assays are optimized to maintain physiologically compatible conditions for elements (e.g., pH, ion composition) critical to component activity.
  • elements e.g., pH, ion composition
  • the arginine tag attachments of this invention are stable to physiological concentrations of most ionic species (e.g., Na + , Ca 2+ , Mg 2+ , etc.)
  • the assays can be run in standard physiologically compatible compositions (e.g., phosphate buffered saline (PBS) standard ringers solutions, and the like.).
  • PBS phosphate buffered saline
  • the arginine tags of this invention can also be used to purify the moiety (e.g., polypeptide(s)) to which they are linked.
  • the arginine tag can be used in conjunction with virtually any anion or cation exchange resin. (It will be appreciated that an anion resin will be used to capture other species and exclude the arg-tagged moieties.) Because the arginine tags are more charged than other tags in current use, the argimne tags are expected to provide greater affinity to cation resins resulting in more effective purification. Suitable anion and cation exchange resins are well known to those of skill in the art and are commercially available.
  • Cation exchange resins for example include, but are not limited to, carboxymethylcellulose, while anion exchange resins include, but are not limited to, DEAE cellulose, DEAE sepharose, heparin, and the like.
  • anion exchange resins include, but are not limited to, DEAE cellulose, DEAE sepharose, heparin, and the like.
  • the interaction of an arginine tag with a layered silicate can be exploited for purification purposes. This accomplished in a manner analogous to a cation exchange resin or in a manner analogous to the polyhistidine/nickel separation systems substituting mica for the cation exchange resin or the nickel resin, respectively.
  • the mica is provided as a bed (either of mica flakes or powder) through which the sample is flowed.
  • the mica bed is housed in any of a variety of suitable vessels, including cartridges that are attachable to syringes, chromatography columns, and the like.
  • the arginine tag labeled moiety e.g. recombinantly expressed arginine tag labeled fusion protein
  • Non-specifically binding components can be eluted away using salt solutions at concentrations sufficient to release undesired components while retaining the arginine-tagged moiety.
  • the arginine-tagged moiety can then be released by application of sufficient salt concentrations and/or arginine as described above.
  • the layered silicate can be added to a vessel containing a sample (e.g. cell lysate) from which the arginine-tagged moiety it to be isolated under conditions in which the arginine tag binds to the silicate.
  • the silicate can then be separated from the sample (e.g., by centrifugation) and the arginine-tagged moiety is then optionally separated from the silicate.
  • the arginine tag can be attached to the moiety via a cleavable linker as described above.
  • a cleavage site can be provided between the arginine tag and the polypeptide.
  • Such cleavable linkages are well known to those of skill in the art (see, e.g., U.S. Patent No: 5,532,142). It is noted that Example 1 describes a system in which proteins contain either a hexa-histidine or both a hexa-histidine and an arginine tag.
  • the hexa-histidine tagged protein was eluted from an Ni /NT A matrix using 500 mM inidazole, while 500 mM NaCl was required for the dual-tagged proteins due to the strong arginine-tag intreaction with the Ni 2+ /NTA matrix. This indicates that the arginine tag can be used to separate proteins.
  • biomolecules e.g., protein
  • Both techniques are improved by smooth support surfaces, however smooth surfaces are particularly beneficial to atomic force microscopy measurements so that the molecule being measured and the measurement itself display minimal surface variation-induced artifacts.
  • protein structure can be investigated by making 2-dimensional crystals and then using electron microscopes or atomic force microscopes to probe the crystal structure at atomic resolution. Again this requires the use of atomically smooth surfaces to minimize surface induced artifacts.
  • a regular repeating "crystal" pattern is required which can be provided by the regular attachment of the molecule via an arginine tag.
  • Control of the orientation of individual molecules attached to a surface via the arginine tags of this invention permits the creation of lithography masks having resolution at an atomic scale. Precise attachment of masking proteins allows the precision etching of microcircuits, nanomachines, and the like.
  • the attachment methods can be used to make arrays of moieties (e.g., biomolecules).
  • the arrays can include one or more different biomolecules (either the same or different type, e.g., all protein arrays, or mixed protein/nucleic acid arrays) where the spatial location of each species of array element is known.
  • the moieties can be arranged in extremely precise patterns (e.g., rows of dots, rows of squares, lines, etc.) on a surface.
  • kits for the Attachment of Moieties to Arginine Tags This invention also provides kits for practice of the methods of this invention.
  • the kits include materials for chemical conjugation of an arginine tag to a moiety.
  • the kits include materials for the recombinant expression of a polypeptide fused to an arginine tag.
  • the kits can also include materials for the immobilization, isolation and/or purification of an arginine-tagged moiety.
  • A.) Kits for chemical conjugation of arg-tags A.
  • kits include materials for chemical conjugation of an arginine tag to a moiety.
  • the kits include one or more containers containing linkers and/or reagents for the chemical conjugation of an arginine tag to an appropriately functionalized moiety (e.g., a polypeptide, an antibody, etc.) as described above.
  • the kit can optionally include an arginine tag either bound to a linker or functionalized for binding to a linker or direct conjugation to a suitable moiety.
  • the kits may optionally contain any of the buffers, reagents, and or media that are useful for the practice of the methods of this invention.
  • the kits may include instructional materials containing directions
  • instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.
  • kits include materials for the recombinant expression of a polypeptide fused to an arginine tag.
  • the kit can include one or more vectors designed for the production of polypeptide fused to a polyarginine tag.
  • the vectors will contain restriction sites that facilitate the insertion of a nucleic acid encoding a polypeptide.
  • the vectors will also contain sequences for the expression of an arginine tag of this invention.
  • the vectors may also encode a cleavage site between the polypeptide and the arginine tag.
  • Such vectors are well known for the expression of histidine tagged fusion proteins (see, e.g., the pRSETA His-tag expression plasmid (Invitrogen)).
  • kits of this invention are preferably analogous substituting the arginine tag coding sequences for the polyhistidine coding sequences.
  • the kits can optional include any of the instructional materials, buffers, reagents, and/or media that are useful for the practice of the methods of this invention.
  • Preferred instructional materials include protocols for the expression and/or purification of the arginine-tagged fusion polypeptides of this invention.
  • the kits of this invention can also include layered silicate materials or ion exchange resins for the attachment of arginine-tagged moieties.
  • the layered silicate materials are preferably powdered, flaked, or atomically smooth sheets.
  • This example describes the specific binding of polyarginine tagged proteins to atomically flat negatively charged mica surfaces.
  • the polyarginine tags were expressed as fusion proteins. It is shown herein that the arginine (e.g., hexaarginine) tagged proteins bind to mica via the Arg-tag based on ion exchange of naturally occurring potassium cations. Only nonspecific binding was observed with the control protein that is free of the Arg-tag. This novel technology facilitates the uniform and specific orientation of immobilized proteins on a standard substrate used for many surface-related applications.
  • Muscovite mica was obtained from Provac (Liechtenstein).
  • the plasmid pGFPuv was from Clontech (Palo Alto, CA) and the vector pET28a(+) was from Novagen (Madison, WI). All other reagents were from Sigma Chemical (St. Louis, MO) and of highest available grade. Ultrapure water with a resistance of 18 MOhm was used for all aqueous buffers (purified by passage through a Milli-Q purification system).
  • oligodeoxyribonucleotide primers were designed: one corresponding to the N-terminal part of the GFP gene (5'-GGAATTCCATATGAGTAAAGGAGAAGAACTTTTC-3 ⁇ designated primer #1, SEQ ID No: 1) and a second corresponding to the C-terminal part (5'- GACCGGCGCTCAGTTGGAATTC-3', designated primer #2, SEQ ID No: 2). These oligodeoxyribonucleotides were used for PCR with 20 ng of linearized pGFPuv as template.
  • the resulting plasmid pGFPH ⁇ was used for transformation of E. coli BL21(DE3). Standard protocols were followed for DNA handling and bacterial transformation (Sambrook et al. (1989) Molecular cloning: A laboratory manual, Cold Spring Harbor: Cold Spring Harbor Laboratory Press).
  • All of the expressed proteins carry a vector-encoded tag of a hexa-histidine sequence for purification by metal chelate affinity chromatography on a Ni + /NTA matrix (Qiagen, Santa Clarita, CA).
  • the cells were grown at 37°C by shaking in LB-medium containing 25 mg/ml Kanamycin. At an OD 60 o of 0.8 the cells were induced with 1 mM IPTG, and 5h later, they were harvested by centrifugation at 6000xg for 10 min.
  • the cells were lysed by addition of lysozyme at a concentration of 100 mg/ml and 10 % (v/v) of 1% Triton X-100 in 50 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM EDTA. After incubation for 30 min on ice, MgC12 was added to a final concentration of 40 mM. The liberated DNA was digested by adding 0.2 mg DNasel per ml lysate. The lysate was incubated for 15 min on ice and then centrifuged at 30,000xg for 40 min.
  • the clear supernatant was dialysed against buffer containing 10 mM Hepes/NaOH pH 7.4, 50 mM NaCl, and then applied to a Ni /NTA column.
  • Weakly bound proteins were eluted with 10 mM imidazole pH 8.0.
  • the His-tagged proteins were eluted with 500 mM imidazole in the case of the GFPH6 and with 500 mM imidazole, 500 mM NaCl for all the other variants (the Arg-tag caused a strong ionic interaction with the Ni 2+ /NTA matrix).
  • the eluted proteins were dialyzed against buffer containing 10 mM Hepes/NaOH pH 7.4, 50 mM NaCl, 50 % glycerol and stored at - 20°C.
  • the purity of the recombinant proteins was estimated by SDS-polyacrylamide gel electrophoresis and found to be greater than 95%.
  • mice were cut into pieces of 5x5 cm 2 and freshly cleaved immediately before use. Droplets of protein solutions (GFPH6, GFPR6, GFPH6R6) at a concentration of 10 mg/ml were applied onto the previously unexposed, hydrophilic surfaces resulting in aqueous films of approximately 4 cm 2 in size. After incubation for 5 min, the mica sheets were washed with 10 ml of water. The central parts, 1 cm in size, were then cut out to ensure that no contaminants from the edges could falsify the subsequent analyses. For each data point four surfaces were analyzed and the readings were averaged.
  • GFPH6, GFPR6, GFPH6R6 protein solutions
  • XPS X-ray photoelectron spectroscopy
  • the strategy presented here for the site-specific immobilization of proteins is based on the ion exchange capacity of naturally occurring cations on the negatively charged cleavage plane of atomically flat mica.
  • Positively charged polypeptide tags with high affinity to mica were genetically fused to either the N- or C-terminus of GFP.
  • this tag the interaction of positively charged amino acids to the mica surface was investigated in a preliminary experiment by XPS measurement.
  • Table 1 shows the extent of release of arginine, lysine and histidine from the mica surface and its dependence on the salt concentration in the wash buffer.
  • the emitted photo electrons of the nitrogen atoms of the adsorbed molecules were used to monitor the residual amount of the amino acids on the mica after consecutive washing with increasing concentrations of salt.
  • Table 1 Extent of release of arginine, lysine, and histidine from a mica surface and its dependence on the salt concentration in the wash buffer.
  • FIG. 2 gives an overview of the three different GFP constructs that were designed for this experiment.
  • GFPH6 carries an N-terminal His-tag alone in order to facilitate the purification of the protein by Ni 2+ /NTA affinity chromatography.
  • GFPR6H6 carries in addition a stretch of six arginine residues at the N-terminal region, whereas GFPR6 has the same Arg-tag at the C-terminal region and the His-tag at the N-terminus.
  • GFPR6 came off with NaCl.
  • the complete release could be achieved by elution with arginine-containing wash buffer (final column in Fig. 3). It is rather likely that this arginine-releasable protein was exclusively bound via its Arg-tag, whereas that released in the NaCl washing steps stemmed primarily from protein electrostatically bound to the surface via other charged groups in the protein.
  • the monovalent cation K + is similar to arginine in its ability to release the GFPH6R6 and the GFPR6 from the mica substrate, although higher concentrations are necessary.
  • Potassium is the naturally occurring cation in muscovite mica and has a lower enthalpy of hydration than sodium, explaining its "power" for inducing Arg-tagged GFP desorption from the mica.
  • mica is atomically flat could help to investigate the structure of uniformly oriented biomolecules by electron and scanning probe microscopy and other surface-related biophysical assays. It should be noted, however, that the charge distribution on the surface of a protein of interest could influence its adsorption properties. Patches of arg-rich areas could act as additional adsorption sites and jeopardize any attempts to achieve uniform orientation.
  • the stability of immobilized Arg-tagged proteins allows functional studies under physiological conditions and even at high ionic strength. In many cases, proteins lacking the polyarginine sequence should not bind at such high salt concentrations, which could also facilitate in situ purification directly on the mica substrate.
  • This concept should be widely applicable to a large number of proteins and represents a powerful strategy to design anisotropic protein surfaces for applications in structural biology, biosensing and biophysics.

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Abstract

Cette invention concerne des matériels et des méthodes de fixation spécifique à un site, de pratiquement n'importe quelle fraction à une surface de silicate en couches. Les méthodes consistent à fixer la fraction par covalence à un marqueur d'arginine; et à mettre le marqueur d'arginine en contact avec la surface de silicate en couches (par exemple, du mica).
PCT/US1998/018531 1997-09-04 1998-09-03 Immobilisation reversible de fractions marquees a l'arginine sur une surface de silicate WO1999012036A1 (fr)

Priority Applications (3)

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AU92225/98A AU9222598A (en) 1997-09-04 1998-09-03 Reversible immobilization of arginine-tagged moieties on a silicate surface
US09/486,480 US6960457B1 (en) 1997-09-04 1998-09-03 Reversible immobilization of arginine-tagged moieties on a silicate surface
US10/850,207 US20050283003A1 (en) 1997-09-04 2004-05-19 Reversible immobilization of arginine-tagged moieties on a silicate surface

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US5792997P 1997-09-04 1997-09-04
US60/057,929 1997-09-04

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DE19938369B4 (de) * 1999-08-09 2004-08-19 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren und Vorrichtung zur Erfassung von Molekülwechselwirkungen über molekulare Motoren
WO2014090905A1 (fr) * 2012-12-11 2014-06-19 Imaxio Protéines modifiées à superhélice et à propriétés améliorées
CN105143251A (zh) * 2013-03-18 2015-12-09 艾马克西欧 流感核蛋白疫苗

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19938369B4 (de) * 1999-08-09 2004-08-19 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Verfahren und Vorrichtung zur Erfassung von Molekülwechselwirkungen über molekulare Motoren
WO2014090905A1 (fr) * 2012-12-11 2014-06-19 Imaxio Protéines modifiées à superhélice et à propriétés améliorées
CN104981541A (zh) * 2012-12-11 2015-10-14 艾马克西欧 具有改进特性的经修饰的卷曲螺旋型蛋白
CN105143251A (zh) * 2013-03-18 2015-12-09 艾马克西欧 流感核蛋白疫苗
CN113307881A (zh) * 2013-03-18 2021-08-27 奥西瓦科斯公司 流感核蛋白疫苗

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