US20040096895A1 - Biosensor with covalently attached membrane-spanning proteins - Google Patents

Biosensor with covalently attached membrane-spanning proteins Download PDF

Info

Publication number
US20040096895A1
US20040096895A1 US10/250,682 US25068203A US2004096895A1 US 20040096895 A1 US20040096895 A1 US 20040096895A1 US 25068203 A US25068203 A US 25068203A US 2004096895 A1 US2004096895 A1 US 2004096895A1
Authority
US
United States
Prior art keywords
protein
membrane
product according
substrate
product
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/250,682
Other languages
English (en)
Inventor
Jeremy Lakey
Horst Vogel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Newcastle University Ventures Ltd
Original Assignee
Newcastle University Ventures Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0101279A external-priority patent/GB0101279D0/en
Priority claimed from GB0108947A external-priority patent/GB0108947D0/en
Application filed by Newcastle University Ventures Ltd filed Critical Newcastle University Ventures Ltd
Assigned to NEWCASTLE UNIVERSITY VENTURES LIMITED reassignment NEWCASTLE UNIVERSITY VENTURES LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LAKEY, JEREMY HUGH, VOGEL, HORST
Publication of US20040096895A1 publication Critical patent/US20040096895A1/en
Priority to US12/232,393 priority Critical patent/US8242077B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12Q1/001—Enzyme electrodes
    • C12Q1/002—Electrode membranes
    • C12Q1/003—Functionalisation
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G01N33/5438—Electrodes

Definitions

  • the invention relates to a product, typically referred to as a biosensor, comprising a membrane-spanning protein linked to a substrate, including methods to manufacture the product and uses thereof.
  • Amphiphilic molecules for example lipids
  • membrane structures which may be monolayers, micelles or liposomes. These structures have been shown to have semi-permeable properties which, in some examples, show selective passage of molecules (eg ions, ligands, antagonists, agonists).
  • the selective permeability relates to the chemistry of the lipids used in the construction of the membrane structures.
  • these synthetic membranes may incorporate larger molecules such as polypeptides or proteins which function to, for example, facilitate the transport of molecules (for example, ion-channels which facilitate the transport of ions across membranes are referred to as ionophores), act as receptors for ligands, form pores through which polypeptides may be translocated (eg nuclear pore forming structures, mitochondrial protein import structures).
  • polypeptides or proteins which function to, for example, facilitate the transport of molecules
  • ionophores which facilitate the transport of molecules
  • ligands act as receptors for ligands
  • pores through which polypeptides may be translocated eg nuclear pore forming structures, mitochondrial protein import structures.
  • membrane proteins have the intrinsic ability to assemble at interfaces in combination with other amphiphiles, they are suited to the construction of biomimetic surfaces for use in biocompatible devices and biosensors.
  • Biosensors incorporating membrane polypeptides have many potential applications including, by example and not by way of limitation, ligand based biosensors for clinical diagnostics; the detection of contaminants in water and environment; memory devices; screening devices for pharmaceutical applications; the provision of biologically functionalised surfaces; binding sites for, and thus sensors for, small molecules such as drugs, pesticides, molecules required to be analysed during process control (i.e.
  • a biosensor may contain membrane polypeptides combined with other components independently attached to the surface of the biosensor which themselves act as specific binding sites and for which the membrane polypeptide provides a stable non-denaturing surface and/or a ion-channel dependent sensor function. Biosensors can be employed in high throughput screening for pharmaceutical applications using ion channel modulation or the other methods describe above.
  • Biosensors can provide an inert, stable, biologically compatible assembly of biologically functionalised surfaces including peptides, nucleic acid proteins and other large molecules. These structures can be used in screening and biosensor systems. They may also be used to create surfaces compatible with cell culture or implantation in living tissue.
  • enzymes may be engineered into the membrane polypeptides or co-assembled with the membrane polypeptides such that they can be functionally and precisely assembled at surfaces. This could have applications in bioreactor systems where catalytic surfaces are required or sensor systems where the enzymatic product is more easily detected than the substrate.
  • the enzyme plus membrane polypeptide could be specifically printed or applied such that 1, 2 or even 3 dimensional spatial arrangements can be defined. If combined with flow systems this may allow for sequential enzymatic synthesis/degradation along a small scale bioreactor.
  • the modifiable permeability of the biosensor could be used to allow the controlled release of molecules across the surface from a reservoir below the layer.
  • a layer composed of thiolipids and membrane polypeptides could also be used to trap molecules such as drugs in a reservoir.
  • a biological signal such as pH, protease activity or ligand binding could trigger the release of the drug.
  • One example could be microbeads targeted to specific tissues which release drugs through the membrane polypeptide channel when they bind to or enter the target cell.
  • the membrane polypeptide could also be engineered to carry peptide sequences which by binding to specific cellular receptors would target the microbead to specific tissues. This could be in addition to or separate from the drug release function.
  • Ionophores are polypeptides or protein structures with a tertiary and in many cases quarternary structure forming pores embedded in cell membranes. Ionophores function to control the flow of ionic currents in response to either electrical excitation (referred to as voltage gating) or the presence of stimulatory ligands, for example neurotransmitters (referred to as ligand gating).
  • voltage gating electrical excitation
  • stimulatory ligands for example neurotransmitters
  • porins which is a sub-group of the ionophores.
  • Other groups include GPCRs, pentameric ligand gated channels, ABC transporters etc.
  • Prior art biosensors are based on the tethering of a biolayer to the surface of a substrate in such a way that a layer is left attached to the substrate (eg by the use of thio-lipids). These biolayers serve as a substrate to which ion-channel forming polypeptides can be inserted during or after formation to provide a functional biological transducer.
  • a problem associated with the synthetic membranes of this type is that when used with large integral membrane proteins of biological origin they lack durability, are expensive to manufacture and protein density and orientation is poorly controlled.
  • the device of Cornell uses a fluid lipid bilayer composed of half membranes spanning lipids and membrane spanning lipids which is tethered to the surface of thiolipids.
  • the membrane allows for ease of diffusion in the plane of the bilayer and this is exploited by the use of gramicidin peptides.
  • These synthetic half membrane spanning ionophore peptides only allow ion flow when a dimer is formed spanning the whole membrane.
  • one monomer is tethered to the substrate and the upper monomer is connected to a receptor molecule. Binding to the receptor alters the amount of conducting dimers present and results in a conductance change. This allows for biosensors of high sensitivity to be manufactured.
  • the present invention directly immobilises membrane-spanning proteins, for example, ion-channel forming membrane proteins, at the surface of a substrate to form a high density layer of coupled proteins to which is added a lipid layer to form a membrane structure.
  • membrane-spanning proteins for example, ion-channel forming membrane proteins
  • amphiphile lipid layer provides a stable layer in which the normal protein structure and function are conserved. This is contrary to the teaching in the art, which taught that the direct tethering of membrane proteins to a substrate will result in the denaturation of the polypeptide thereby providing a non-functional membrane protein.
  • Biosensors constructed according to the invention have ease of manufacture and are more robust and are more widely applicable than prior art sensors since in addition to membrane based biosensor applications the method allows for the controlled assembly of dense, mimics of native protein layers with controlled orientation on solid substrates.
  • the proteins can be engineered to incorporate specific functions in their exposed surfaces.
  • an ion-channel forming protein such that it can be directly coupled to a metal surface, in this example gold, to which is added thio-lipids which complete the membrane structure and provide a functional biosensor.
  • a metal surface in this example gold
  • thio-lipids which complete the membrane structure and provide a functional biosensor.
  • One example uses the bacterial OmpF polypeptide which is modified to incorporate a cysteine amino acid which facilitates the linkage of the polypeptide to a treated substrate.
  • this technology can be applied to other ion-channel forming polypeptides and other membrane polypeptides using thiol containing amino acids.
  • any protein which presents a flat surface close to the membrane interface such as the outer membrane proteins of bacteria or bacteriorhodopsin
  • the polypeptides may naturally include a thiol containing amino acid or be genetically engineered to include a thiol containing amino acid.
  • the amino acids may be naturally occurring or modified amino acids.
  • the present invention relies upon large integral membrane proteins directly fixed to the substrate surface and lateral diffusion in the bilayer does not occur.
  • the role of the bilayer is to stabilise the assembled protein layer, reduce non-specific binding to the substrate surface and provide electrical insulation.
  • the tethered lipids may comprise 100% of the lipids in the half bilayer next to the ice.
  • the upper half bilayer is then completed with membrane phospholipids such as diphytanoyl-phosphatidylcholine.
  • the proteins employed all span the complete lipid bilayer, rather the being half-membrane spanning peptides (as in the prior art of Cornell), the option is available to form the entire membrane from tethered lipids capable of spanning the entire thickness of the membrane.
  • the conductance changes observed by impedance spectroscopy when using porins as the membrane-spanning protein in the present invention are due to changes in the pore of each protein and rely upon the intrinsic gating of individual channels rather than the formation and disruption of dimers.
  • purified recombinant membrane proteins can be employed, the method offers a broad technique to create immobilised engineered protein surfaces that are not achievable with synthetic peptides of Georgia.
  • the ⁇ -barrel membrane protein family are good targets for protein engineering and thus a wide variety of protein interfaces may be constructed in this manner.
  • the polypeptides which are directly coupled to the substrate are referred to as “membrane-spanning proteins”.
  • the polypeptides are generally not simple linear short synthetic amino acid sequences with little or no secondary/tertiary structure (eg protein folding).
  • the protein is of a size and shape which means that it would reside in a membrane and would extend at least mostly across the width of the membrane bilayer. It is generally expected that polypeptides which reside essentially only at or in one or other periphery of a membrane bilayer will not be advantageously useful in this invention.
  • membrane-spanning should not be interpreted strictly so as to exclude from this invention proteins extending partially beyond the membrane boundaries or extending only across the majority of the region between the membrane boundaries.
  • a product comprising a membrane-spanning protein; a lipid membrane formed from amphiphilic molecules and membrane-spanning protein molecules; and a substrate: characterised in that the membrane protein is directly coupled to the substrate.
  • the product is a biosensor or protein array.
  • the membrane-spanning protein comprises an ⁇ -helix structure or a ⁇ -barrel structure.
  • Porins are outer membrane proteins which, in monomeric, dimeric or trimeric form, constitute a water-filled transmembrane channel (“pore”). This pore allows the passage of ions and numerous other, non-specific, molecules through a membrane. Porins are found in the outer membrane of mitochondria and in many Gram-negative bacteria. Porins include the OmpA family of polypeptides having an eight stranded ⁇ -barrel, and OmpF which is a homotrimer of 16 stranded ⁇ -barrels.
  • TonB dependent and related outer membrane transporters are also included in the scope of this invention. These proteins are usually monomeric polypeptides, are not channel forming and are highly specific for particular nutrients.
  • Membrane-spanning proteins can be modified to facilitate the coupling of the polypeptide to the substrate.
  • Gene names are: OMPF or TOLF or CMLB or COA or CRY or B0929
  • the membrane-spanning protein may be alpha helix rich and has no large extra membrane projections on at least one of the two membrane surfaces.
  • Examples whose high resolution structures are known include bacteriorhodpsin and the bacterial potassium channel KcsA. Many members of the GPCR family may be applicable to this form of immobilisation.
  • the membrane-spanning protein may be selected from those presented in Table 1 or Table 2.
  • the linking of a membrane-spanning protein directly to a substrate may be achieved by the reaction of a sulphur atom (found, for example, in cysteine) with a substrate, for example gold, by a direct sulphur-gold linkage. This results in a protein retaining functional activity.
  • a sulphur atom found, for example, in cysteine
  • a substrate for example gold
  • the substrate can be rendered hydrophobio by preincubation with a small hydrophilic thiol, for example ⁇ -mercaptoethanol or thio glycerol.
  • the porins are stable proteins and can be added to the surface in detergent solutions (such as SDS or Dodecyl-glucoside) which do not denature the protein but ensure non specific hydrophobic interactions with the surface are reduced or do not occur.
  • detergent solutions such as SDS or Dodecyl-glucoside
  • the cooperative nature of the cysteine reaction means that the OmpF protein readily reacts with the pre-treated surface and is not inhibited by the presence of thiols.
  • the cooperativity may arise from having three cysteines per trimer, or more than one cysteine per monomer.
  • the self assembly properties of the proteins in creating a monolayer of protein with non-polar contacts between neighbouring proteins is likely to be important. This may explain the high density achievable.
  • the contact of the hydrophilic protein loops with the hydrophilic surface is non-denaturing and separates the core of the protein from the surface.
  • the protein layer is then further stabilised by adding amphiphiles to the surface, preferably thio-lipids, which will bond via a gold sulphur bond and fill the gaps between the proteins.
  • amphiphiles to the surface, preferably thio-lipids, which will bond via a gold sulphur bond and fill the gaps between the proteins.
  • the lipid is a thiolipid.
  • the thiolipids are a very variable group.
  • ZD16 is based on dipalmitoylphosphatidic acid, which is extended at the lipid phosphate by a hydrophilic spacer chain of ethoxy groups of variable length, with a terminal disulfide group at the end of hydrophilic spacer.
  • These anchor-bearing “thiolipids” can attach to substrates by forming stable substrate-sulfur bond. In this way we can couple lipid bilayers to substrates with the possibility of preserving a water layer between the substrate and the first monolayer.
  • the lipid portion may be any fatty acid including branched chain (e.g. phytanoyl groups) or unsaturated (e.g. oleoyl) or a sterol such as cholesterol or the lipid may be based on a ceramide.
  • the lipid portion may also consist of lipids such as those from archaea in which the membrane contains lipids which span its entire width from one aqueous phase to another.
  • Thiolipids based upon this design will covalently link the entire membrane to the surface.
  • Examples of this design are the Di-Bi-Phytanyl glycerol tetraethers which are found in species such as Methanobacterium, Methanobrevibacter, Sulfolobus, Thermoplasma, Thermoproteus.
  • the hydrophilic groups may be any kind of chemical unit which links the thiol group to the hydrophobic groups.
  • the carbon backbone is hydrophilic to stabilise the hydrophilic protein loops adjacent to it.
  • the thiolipid is selected from 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol; 1,2-doleoyl-sn-gly 3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate]; 1,2-Dipalmitoyl-sn-glycero-3-Phosphoethanolamine-N-[3-(2-pyridyldithio)propionate]; N-14′-mercapto-1′,11′-dioxo-3′,6,9′-trioxa-12′-azatetradecyl)-2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine; (8′-mercapto-3′,6′-dioxa-octyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid.
  • the invention also includes the use of amphiphilic molecules other than lipids.
  • Amphiphilic molecules have one or two hydrophilic head portions and at least one hydrophobic region and are typically referred to as surfactants. Examples are cationic surfactants (eg quaternary ammonium salts); aniomic surfactants (eg organosulphate salts) and zwitterionic surfactants (eg phosphatidyl cholines).
  • Other amphiphilic molecules include detergents, fatty acids. Less water soluble molecules as a alkane thiols may be suitable. These may terminate with a thiol at one end and a water soluble group such as a hydroxy group or a sugar at the other. These may, for example, have carbon chain lengths between 10 and 18.
  • the substrate is a metal.
  • the metal may be selected from gold; chromium platinum; silver.
  • the substrate is selected from: glass, silicon, hydrogen terminated silicon or plastic polymers.
  • Modification of a membrane-spanning protein means deletion, addition or substitution of at least one amino acid residue which the modification provides a protein capable of binding directly to a substrate to provide a product according to the invention.
  • OmpF is modified at position. 183 of the amino acid sequence by mutation of glutamate to cysteine. This corresponds to position 205 of the unprocessed OmpF polypeptide and position 183 of the mature polypeptide.
  • the vector may encode, and thus said modified recombinant protein is provided with a secretion signal to facilitate purification of the protein.
  • a product comprising a biosensor.
  • the product may be a single element biosensor using eletrochemical or optical methods to detect the specific interaction of a defined analyte, protein, nucleic acid, sugar, steroid etc with the engineered protein layer. It might consist of a protein/lipid layer on a gold surface combined with electronic circuitry or as part of a surface plasmon resonance device.
  • the protein layer would, for example, contain binding sites for a specific antibody which is diagnostic of an infection. A sample of blood or other biological fluid could be applied to the surface via a wick which will filter out large particles, cells etc. On reaching the protein layer the binding would cause a change in the electrical or optical properties which could be read via an electronic output.
  • Another related product might be a sensor implanted in the body to monitor in real time the levels of specific biomolecules in blood or tissue.
  • the device might consist of an electrical component containing a small (eg. ⁇ 1 mm diameter) gold electrode covered with the membrane protein layer. Binding of specific molecules would be read off as a change in the impedance properties.
  • the product may also be a device for making and analysing two dimensional arrays of proteins for analytical screening purposes.
  • the device would include apparatus for the printing of modified proteins onto surfaces for the creation of the arrays.
  • the arrays would then be used to screen samples containing proteins, small molecules, cells, enzyme inhibitors, nucleic acids which will bind specific sites on the array.
  • the sites where protein has bound would be detected by surface plasmon resonance, fluoresence, mass spectrometry, electrochemical methods etc.
  • the product could allow for rapid screening of samples for a range of proteins and thus increase productivity in clinical or research labs.
  • Another product would be a bioreactor which used catalytic surfaces created by inserting catalytic domains into the membrane protein. Samples would be pumped by microfluidics along channels lined by immobilised catalytic proteins. In this way sequential modification of samples could be achieved and precise synthesis or modification of biological molecules achieved. Such a bioreactor could be formed on micromachined silicon structure to modify small amounts of sample which would then be used for subsequent analysis. Fusions of enzymes and the membrane protein will also allow arrays of bioreactor spots to be used in high throughput screening.
  • a further product could be the creation of biocompatible surfaces for in vitro implantation or cell cube technology.
  • a coronary stent could have improved biocompatibility by being coated in a monolayer of engineered proteins which improve the interaction with epithelial cells.
  • Other examples are artificial joints or continuous in vivo monitoring devices for controlled drug delivery.
  • a further example would be cell culture plates which are modified with a protein monolayer to encourage defined patterns of cellular growth. These may be from Petri dish size down to micro machined silicon devices. Applications of the product may involve the inducement of morphological changes in cells especially nerve cells or the patterned growth of different cells lines adjacent to one another to study and exploit cell-cell communication in research and analog. The product would consist of pained monolayers of engineered proteins which present surfaces recognised by each cell type. The patterned monolayers could be incorporated into a range of sizes of cell culture vessels.
  • the product of the invention could be produced from a ready made solution (not necessarily aqueous) of membrane proteins, also optionally with all amphiphile (detergent/lipid), which can be used to create controlled mono-molecular protein surfaces.
  • the solution could consist of a defined mixture of proteins which would provide a similarly defined mixture of modified proteins on the surface. This would enable the manufacture of substrates with a controlled density of binding sites to be applied by self assembly with non-binding proteins acting as spacers.
  • a product according to the invention would be ready made gold surfaces bearing defined protein/lipid layers which could be utilised in a surface plasmon resonance biosensor.
  • the product is a biosensor.
  • the membrane protein is an outer membrane protein, such as a porin.
  • the hydrophilic coating agent may be selected from: 2-mercaptoethanol; mercaptopropionic acid; 1-mercapto-2-propanol; 2,3 dimercapto-1-propanol; 2-mercapto-3-butanol; dithioeythritol (erythro-1,4-dimercapto-2,3-butanediol, DTE); Diothiothreitol (Cleland's Reagent threo-dimercapto-2,3-butanediol, DTT); mixtures of DTE and DTT; thiol glycerol.
  • the coating agent is 2-mercaptoethanol.
  • Conditions for coating substrates may be varied.
  • the concentration of hydrophilic coating agent is between about 1 mM and 1M, preferably between about 50 MM and 500 mM, more preferably between about 100 mM and 200 mM.
  • Incubation time may also vary from hours to many days without detrimental effects to the prepared surface.
  • the substrate is gold and the outer membrane polypeptide is a porin. More preferably still the porin is OmpF or modified OmpF.
  • the amphiphilic molecule is a lipid, ideally thio-lipid.
  • biosensor obtainable by the method according to the invention
  • biosensor to detect at least one of following molecules: colicin N R-domain; polypeptides; antigenic polypeptides; antibodies or fragments of antibodies; receptors; ligands, antibiotics; drugs; pesticides; sugars; amino acids; fatty acids; peptides; hormones; steroids; nucleic acids (DNA, RNA, cDNA); peptide nucleic acids; metals; inorganic ions.
  • Table 1 represents porin genes of Gram-ve bacteria
  • Table 2 represents Ton B dependent receptors from various bacterial species
  • FIG. 1 is the nucleic acid sequence encoding OmpF (wild type) and the encoded protein sequence of OmpF; [SEQ ID No. 1] and [SEQ ID No. 2];
  • FIG. 2 represents binding of anti OmpF polyclonal antibodies to tethered OmpF-Cys
  • Wild type OmpF (OmpF-WT) from E coli BE3000 was produced as previously described using the plasmid pGBF96 (Bainbridge, G., Mobashberi, H., Armstrong, G. A, Lea, E. J. A. and Lakey, J. H. (1998)) and purified in SDS detergent or subsequently resuspended in Octyl-POE.
  • the mutant of OmpF with a single cystein in turn 1 (OmpF-CYS) was created using the QuikChangeTM method (Stratagene).
  • the mutagenesis used two complementary 37 mer oligonucleotides which coded for a TGT cysteine codon instead of a GAA glutamate codon.
  • Forward [SEQ ID No. 3] 5′-GGTTCTATCAGCTAC Reverse [SEQ ID No. 4] 3′-CCAAGATAGTAGATG ATGCTTCCGAAACCATAGT-5′
  • the mutation in the OmpF BE3000 gene caused by our introduction of an XbaI restriction site is Lysine279 to Arginine (K279R).
  • the DNA sequence is different at “wobble” positions from the database E. Coli OmpF DNA sequence
  • the protein used in the example differs from this database example only by the mutations EI 83C and k279R.
  • the phospholipids used were di-oleoyl phosphatidyl choline (from the Sigma Chemical Company, Fancy Road Poole Dorset UK) or Di-phytanoyl phosphatidyl choline (from Avanti Polar Lipids Birmingham Ala. USA).
  • the thiolipid (laboratory designation ZD16) was of the formula below and synthesised using the methods previously descried Lang (1994).
  • Gold chips for the Biacore were either plain gold Biacore J1 type (Biacore AB, St Albans UK) or recycled Biacore chips cleaned with piranha solution.
  • Glass slides of refractive index 1.7 were cleaned by sonication in a water bath sonicator in 2% Helmanex cleaning solution (Helma GmbH Germany) followed by extensive rinsing in Nanopure water and stored in ethanol with or without 2-mercaptoethanol until required. Residual ethanol was evaporated under nitrogen and the slide was placed in an Edwards High Vacuum Evaporator. When the vacuum reached 5 ⁇ 10 ⁇ 6 millibar the slide was coated on one side by a 3 nm chromium layer by evaporation of chromium.
  • the system was allowed to cool for 15 minutes before a 100 nm layer of gold was evaporated on top of the chromium substrate. Following a further 15 minutes cooling the vacuum was relieved by argon and the slide was assembled into the SPR device. This consisted of placing the slide with the gold face forming the remaining side of a Teflon cuvette which was open towards the top for exchange of fluids. A Leica 60° prism was placed on the opposite side of the slide with the junction completed by refractive index matching fluid. The assembly was clamped together and placed in the SPR device. Solutions were added and removed from the cuvette by Pasteur pipette or graduated syringe.
  • the minimum of reflected laser light was detected by scanning the reflected signal with a photodiode connected to a personal computer. The exact minimum was calculated using curve fitting. To follow time dependent changes in SPR signal the diode angle was set to a value approximately 2 degrees below the initial minimum where the intensity change per unit angle is large. Increases in the angle of the minimum were thus represented as increases in signal strength.
  • the Biacore experiments were carried out with the machine in the standard format.
  • the mercaptoethanol treated chips were placed in the machine and the required solutions pumped over the chip at a defined flow rate.
  • the flow rates are 1 ⁇ l per minute for the proteins thiolipid assembly steps and 30 ⁇ l per minute for the binding assays with colicin N R-domain. Two lanes in series were used. Time dependent changes in SPR signal were measured as changes in resonance units as defined in the Biacore Users Manual.
  • Impedance spectroscopy was carried out in an electrochemical cell comprising a membrane covered gold electrode and a reference Ag/AgCl electrode in 0.1M KCl 5 mM sodium phosphate buffer pH 7.4.
  • the surface area of the gold disk electrode was 3.34 ⁇ 10 ⁇ 2 cm 2 .
  • No DC voltage was applied
  • a sinusoidal voltage of 10 mV (RMS) was applied to the cell at 199 successive frequencies equally spaced on a logarithmic scale from 1 Hz to 20 kHz.
  • the resulting current was recorded via a phase sensitive lock-in amplifier to calculate the complex impedance and admittance.
  • FTIR Fourier Transform Infra-Red Spectroscopy
  • Samples for FTIR analysis were assembled on Helmanex cleaned glass microscope slides onto which was evaporated 3 nm of chromium followed by 100 nm of gold as for the SPR experiment apart from the gold thickness. Pretreatment with mercaptoethanol was as for the SPR glass slides. Assembly of protein, thiolipid or phospholipid was performed by laying the solution onto the slide which was placed in a plastic Petri dish and covered to prevent evaporation. Successive layers were assembled by replacement of the initial solution by the subsequent solution without drying or rinsing. Before spectra were collected the slides were washed under running Nanopure water, dried under argon and immediately placed in the spectrometer.
  • Infra red spectra were recorded using a Bruker IFS 28 spectrometer equipped with an HgCdTe detector. One thousand scans were recorded at 1 cm ⁇ 1 resolution and apodised with a boxcar function. Background spectra were recorded from the respective bare supports. The corrected spectra were Fourier smoothed to 1 cm ⁇ 1 resolution with triangular apodisation. The reflectance absorbance spectra a molecular layers on gold was recorded at an angle of incidence of 85° using only parallel-polarised light. All peak positions were derived from second derisive spectra. For further details of the technique, see Boucheva (1997).
  • FIG. 2 shows the binding of anti-OmpF loop 6 rabbit polyclonal antibody (at concentrations) to OmpF-Cys tethered to gold and embedded in the fluid hybrid bilayer of DOPC (top layer, towards the solution) and thiolipids (bottom layer, tethered to gold).
  • Sensorgrams were recorded in PBS running buffer at flow rate of 30 ⁇ l/min on a Biacore X (Biacore AB B, Uppsala, Sweden). Fitted black curves for a Langmuir 1:1 binding model are superimposed.
  • association rate constant K a was evaluated at 4.4 ⁇ 10 4 M ⁇ 1 s ⁇ 1
  • dissociation rate constant K d was 6.0 ⁇ 10 ⁇ 5 s ⁇ 1 for a calculated affinity constant K D of 1.4 nM.
  • the binding interaction indicates that the protein is immobilized with its extracellular loops exposed.
  • a plain gold surface is first treated with ⁇ -mercaptoethanol (1 mM in buffer A (100 mM NaCl 100 mM Na-phosphate pH 7.0.)), followed by a wash with buffer A.
  • OmpF-Cys in Octyl-POE 0.3 mg/ml was then applied to the surface and rapidly bound to the surface achieving an equilibrium. Washing with Buffer A removes the previous buffer effect and leaves an increased signal of 1000 resonance units (this can be increased with longer incubation).
  • the surface is washed with 0.05% SDS until no more protein is removed OmpF is stable in SDS and this treatment removes non-specifically bound OmpF and leaves covalently bound trimers of OmpF on the gold surface.
  • any wild type protein which binds non-specifically to gold surfaces is washed off by this treatment.
  • Thiolipid (ZD16) added from 1% Octyl-glucoside solution binds via its thiol group to the remaining gold surface and completes a monolayer of covalently bound lipid and protein.
  • Addition of thiolipid to a control gold layer shows a much higher level of lipid binding confirming that OmpF-Cys and ZD16 compete for the surface layer.
  • Addition of phosphatidylcholine vesicles completes the bilayer since they attach and become disrupted on the exposed hydrophobic surface. We have since found that incubation ex situ overnight followed by washing in the Biacore device provides the most complete layer. Further treatment with ⁇ -mercaptoethanol does not dislodge the layers showing that the self assembled proteins and lipids are effectively irreversibly bound to the surface.
  • the protein immobilisation gave an angle shift of 031°+/ ⁇ 0.17 whether the protein was dissolved in SDS or Octyl-POE (0.3 mg/ml). This was a very reproducible result which confirms the efficiency of the self-assembly process.
  • the angle erected from a two-dimensional crystal of OmpF is 0.75° and thus the protein is immobilised at 40% of the maximum coverage. This is a very high level considering that no special precautions are taken to maximise self assembly, such as preformation of 2D crystals.
  • FTIR spectra was collected of porin trimers directly self assembled (1% Octyle-POE0.3 mg/ml OmpF-Cys) for one hour at room temperature and after a second stage of self-assembly using 1 mg/ml ZD-16 thiolipid in 9 omM Octyl-glucoside. Neither sample showed spectra with beta structure remaining.
  • the coding sequence of mature OmpA (1-147) was amplified from Escherichia coli XL1-Blue (Stratagene) genomic DNA using colony PCP and the following primers Forward 5′-TTTTCTGAGCTGTCCTCCGAAAGATAACACC-3′ [SEQ ID No. 5] Reverse 5′-TTTTGCGCAAAGTGCCACGGCCTCGACCTCC-3′ [SEQ ID No. 6]
  • the primers include MluI and XhoI restriction sites respectively, which allowed cloning into the pET8c vector based upon the pET3c vector from Novagen.
  • the expressed protein thus contains an N-terminal insertion of an MHHHHHHS sequence [SEQ ID No.
  • the plasmid was named pET8c-(CysOmpA1-147).
  • the absence of the natural signal peptide means that the protein is expressed in the cytoplasm and forms inclusion bodies (Arora et al., 2001; Pautsch & Schulz, 2000).
  • the protein was expressed in BL21 DE3 pLysE (Novagen) using Luria broth and 100 ⁇ g/ml Ampicillin and 30 ⁇ g/ml Chloramphenicol. 6 liters of culture were incubated in Erlenmeyer flasks until the OD reached 0.6 when OmpA expression was induced by IPTG. After a further three hours growth the cells were harvested by centrifugation, broken by French Press and unbroken cells removed by a low speed centrifugation (3000 rpm 10 mins). The supernatant was then centrifuged at 10,000 rpm in Beckman 55.2 rotor for 1 hour at 4° C.
  • This crude inclusion body sample was washed three times by homogenising in 20 mM Tris pH 8.0 1% Triton X-100 and centrifuging at 8000 rpm for 15 hours (twice) and for 30 min (fix) to yield a pure inclusion body preparation.
  • This pellet was resuspended in 20 ml buffer (20 mM Tris pH 8.0, 8.0 M urea) and 20 ml isopropanol homogenised at 55° C. for 30 min.
  • the homogenate was centrifuged at 39,000 rpm for 1.5 h at 4° C.
  • the supernatant contained the solubilised His-Cys-OmpA1-147.
  • the protein was further purified using Ni-agarose column and elution using imidazole.
  • the protein was a single band on SDS-PAGE and its identity was confirmed by Western Blot using an anti-His tag antibody (SIGMA). Isopropanol was removed by dialysing the protein sample into a solution of 8M urea 20 mM Tris, pH 8.0 and 1% v/v Genapol (Fluka) using Spectra-Por 6-8,000 MW dialysis tubing.
  • SIGMA anti-His tag antibody
  • the protein concentration at this stage was 0.34 mM.
  • the protein was refolded by dilution into an 200 fold molar excess of DG (n-docecyl- ⁇ -D-glucopyranoside Calbiochem) at pH 8.1 following the procedures of Kleinschmidt et al., (1999).
  • the refolded protein was then examined by circular dichroism spectroscopy which showed it to contain largely ⁇ -structure as expected from a fully refolded OmpA transmembrane domain.
  • the protein was also resistant to proteolysis which is another property of a folded OmpA monomer.
  • a gold J1 chip was cleaned with Piranha solution (9% v/v H 2 O 2 and 70% conc H 2 SO 4 ), rinsed with ethanol and water and dried with nitrogen.
  • Piranha solution 9% v/v H 2 O 2 and 70% conc H 2 SO 4
  • the chip was purified with 0.05% SDS solution, then 0.2%, ⁇ -mercaptoethanol to provide the hydrophilic layer.
  • SDS wash the folded protein His Cys-OmpA (1-147) at 0.2 mg/ml in n-docecyl- ⁇ -D-glucopyranoside showed strong binding to the surface.
  • a subsequent SDS wash removed non-specifically bound protein and this was followed by a second assembly step of thio-lipid ZD16 (0.5 mg/ml in 1% Octyl-polyoxyethylene (Bachem)) to complete the protein-lipid layer.
  • Porin proteins suitable for use in the invention There are more than 340 identified genes which code for Gram-ve porins, of these many are variants of Neisseria meningitidis outer membrane porins which show great variability in their outer loops. The following list is by species Additional porins, which have little sequence homology, are found at the end of the list. Figures in brackets are the total number of porin-like genes identified in that species. Some proteins may not be true porins.
  • TonB dependent receptors which may be immobilised by the method have been identifed in the following species of bacteria Species (total number of proteins of this class so far identified in this species) Helicobacter pylori (6) Campylobacter coli (1) Haemophilus influenzae (17) Pasteurella haemolytica (1) Haemophilus ducreyi (3) Actinobacillus pleuropneumoniae (5) Vibrio vulnificus (1) Vibrio anguillarum (1) Vibrio orientalis (1) Vibrio cholerae (3) Aeromonas salmonicida (1) Pseudomonas stutzeri (1) Moraxella catarrhalis (4) Acinetobacter sp(1) Pseudomonas aeruginosa (11) Pseudomonas putida (2) Shewanella sp(1) Stenotrophomonas maltophilia (1) Salmonella typhimurium (2) Shigella dysenteriae (1) Pe

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Urology & Nephrology (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
US10/250,682 2001-01-18 2002-01-18 Biosensor with covalently attached membrane-spanning proteins Abandoned US20040096895A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US12/232,393 US8242077B2 (en) 2001-01-18 2008-09-16 Biosensor

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
GB0101279A GB0101279D0 (en) 2001-01-18 2001-01-18 Biosensor
GB0101279.8 2001-01-18
GB0108947.3 2001-04-10
GB0108947A GB0108947D0 (en) 2001-04-10 2001-04-10 Biosensor
PCT/GB2002/000222 WO2002057780A1 (en) 2001-01-18 2002-01-18 Biosensor with covalently attached membrane-spanning proteins

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/232,393 Continuation US8242077B2 (en) 2001-01-18 2008-09-16 Biosensor

Publications (1)

Publication Number Publication Date
US20040096895A1 true US20040096895A1 (en) 2004-05-20

Family

ID=26245580

Family Applications (2)

Application Number Title Priority Date Filing Date
US10/250,682 Abandoned US20040096895A1 (en) 2001-01-18 2002-01-18 Biosensor with covalently attached membrane-spanning proteins
US12/232,393 Expired - Fee Related US8242077B2 (en) 2001-01-18 2008-09-16 Biosensor

Family Applications After (1)

Application Number Title Priority Date Filing Date
US12/232,393 Expired - Fee Related US8242077B2 (en) 2001-01-18 2008-09-16 Biosensor

Country Status (13)

Country Link
US (2) US20040096895A1 (de)
EP (1) EP1352245B1 (de)
JP (1) JP4313039B2 (de)
AT (1) ATE336723T1 (de)
AU (1) AU2002219413B2 (de)
CA (1) CA2435123A1 (de)
DE (1) DE60213948T2 (de)
DK (1) DK1352245T3 (de)
ES (1) ES2271253T3 (de)
HK (1) HK1059469A1 (de)
NZ (1) NZ527036A (de)
PT (1) PT1352245E (de)
WO (1) WO2002057780A1 (de)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004092394A2 (en) * 2003-04-08 2004-10-28 The Children's Hospital Of Philadelphia Fluid planar lipid layer-based membrane-anchored ligand system with specific binding pair members and methods of use thereof
US20060068503A1 (en) * 2004-09-10 2006-03-30 John Cuppoletti Solid support membranes for ion channel arrays and sensors: application ro rapid screening of pharmacological compounds
DE102006025344A1 (de) * 2006-05-31 2007-12-06 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Anordnung für eine biologisch funktionelle Membran, Sensoranordnung, Filteranordnung sowie deren Verwendungen
US20090275066A1 (en) * 2006-11-13 2009-11-05 Universite Paris 7 - Denis Diderot Immobilization of membrane porteins onto supports via an amphiphile

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4484553B2 (ja) * 2004-03-22 2010-06-16 幸宏 杉山 膜たんぱく質固定化基板および固定化方法
GB0505971D0 (en) * 2005-03-23 2005-04-27 Isis Innovation Delivery of molecules to a lipid bilayer
US8038885B2 (en) * 2005-10-14 2011-10-18 The Regents Of The University Of California Formation and encapsulation of molecular bilayer and monolayer membranes
US9233846B2 (en) 2005-10-14 2016-01-12 The Regents Of The University Of California Formation and encapsulation of molecular bilayer and monolayer membranes
PL380105A1 (pl) * 2006-07-04 2008-01-07 Instytut Immunologii I Terapii Doświadczalnej Im. L. Hirszfelda Pan Białko, jego fragment oraz ich zastosowania
US8728025B2 (en) 2008-03-10 2014-05-20 S.E.A. Medical Systems, Inc. Intravenous fluid monitoring
EP2293921A4 (de) * 2008-05-22 2013-05-22 Univ California Membranvorläufer und daraus hergestellte membrane
US9052276B2 (en) * 2009-06-08 2015-06-09 S.E.A. Medical Systems, Inc. Systems and methods for the identification of compounds using admittance spectroscopy
CA2817671A1 (en) * 2009-11-17 2011-05-26 Universite De Montreal Heteropeptides useful for reducing nonspecific adsorption
WO2011085047A1 (en) 2010-01-05 2011-07-14 The Regents Of The University Of California Droplet bilayer formation using throughput liquid handling techniques
US9150598B2 (en) 2011-10-05 2015-10-06 The Regents Of The University Of California Masking apertures enabling automation and solution exchange in sessile bilayers
US9744193B2 (en) 2012-09-06 2017-08-29 Orbis Health Solutions Llc Tumor lysate loaded particles
GB201400562D0 (en) 2014-01-14 2014-03-05 Orla Protein Technologies Ltd Protein coated polymeric substrate
EP3113759B1 (de) 2014-03-05 2021-05-12 Orbis Health Solutions LLC Impfstoffabgabesysteme mit hefezellwandpartikeln
CA2979712C (en) 2015-03-25 2024-01-23 The Regents Of The University Of Michigan Nanoparticle compositions for delivery of biomacromolecules

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5234566A (en) * 1988-08-18 1993-08-10 Australian Membrane And Biotechnology Research Institute Ltd. Sensitivity and selectivity of ion channel biosensor membranes
US5516890A (en) * 1989-11-02 1996-05-14 Synporin Technologies Biologically mimetic synthetic ion channel transducers and methods of making the same
US5736342A (en) * 1993-09-21 1998-04-07 Washington State University Research Foundation Biosensor for detecting the presence of chosen analytes

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU1360297A (en) 1996-01-11 1997-08-01 Australian Membrane And Biotechnology Research Institute Ion channel sensor typing
GB9806449D0 (en) * 1998-03-25 1998-05-27 Peptide Therapeutics Ltd Attenuated bacteria useful in vaccines
JP3657790B2 (ja) 1998-11-09 2005-06-08 独立行政法人科学技術振興機構 Ｓｐｒセンサー用金属薄膜、その製法およびそれを用いた測定方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5234566A (en) * 1988-08-18 1993-08-10 Australian Membrane And Biotechnology Research Institute Ltd. Sensitivity and selectivity of ion channel biosensor membranes
US5516890A (en) * 1989-11-02 1996-05-14 Synporin Technologies Biologically mimetic synthetic ion channel transducers and methods of making the same
US5736342A (en) * 1993-09-21 1998-04-07 Washington State University Research Foundation Biosensor for detecting the presence of chosen analytes

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004092394A2 (en) * 2003-04-08 2004-10-28 The Children's Hospital Of Philadelphia Fluid planar lipid layer-based membrane-anchored ligand system with specific binding pair members and methods of use thereof
WO2004092394A3 (en) * 2003-04-08 2005-03-31 Philadelphia Children Hospital Fluid planar lipid layer-based membrane-anchored ligand system with specific binding pair members and methods of use thereof
US20060068503A1 (en) * 2004-09-10 2006-03-30 John Cuppoletti Solid support membranes for ion channel arrays and sensors: application ro rapid screening of pharmacological compounds
DE102006025344A1 (de) * 2006-05-31 2007-12-06 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Anordnung für eine biologisch funktionelle Membran, Sensoranordnung, Filteranordnung sowie deren Verwendungen
US20090275066A1 (en) * 2006-11-13 2009-11-05 Universite Paris 7 - Denis Diderot Immobilization of membrane porteins onto supports via an amphiphile
US8207263B2 (en) * 2006-11-13 2012-06-26 Centre National De La Recherche Scientifique (Cnrs) Immobilization of membrane proteins onto supports via an amphiphile
US8674044B2 (en) 2006-11-13 2014-03-18 Centre National De La Recherche Scientifique (Cnrs) Immobilization of membrane proteins onto supports via an amphiphile

Also Published As

Publication number Publication date
US8242077B2 (en) 2012-08-14
ES2271253T3 (es) 2007-04-16
AU2002219413B2 (en) 2007-03-15
EP1352245A1 (de) 2003-10-15
CA2435123A1 (en) 2002-07-25
DE60213948D1 (de) 2006-09-28
DE60213948T2 (de) 2007-03-08
JP4313039B2 (ja) 2009-08-12
ATE336723T1 (de) 2006-09-15
JP2004533601A (ja) 2004-11-04
NZ527036A (en) 2005-03-24
PT1352245E (pt) 2007-01-31
US20110111985A1 (en) 2011-05-12
EP1352245B1 (de) 2006-08-16
DK1352245T3 (da) 2006-12-18
HK1059469A1 (en) 2004-07-02
WO2002057780A1 (en) 2002-07-25

Similar Documents

Publication Publication Date Title
US8242077B2 (en) Biosensor
AU2002219413A1 (en) Biosensor with covalently attached membrane-spanning proteins
Heyse et al. Emerging techniques for investigating molecular interactions at lipid membranes
Richards et al. Membrane protein mobility and orientation preserved in supported bilayers created directly from cell plasma membrane blebs
Beseničar et al. Surface plasmon resonance in protein–membrane interactions
Chapman Biomembranes and new hemocompatible materials
Dumas et al. Consequences of hydrophobic mismatch between lipids and melibiose permease on melibiose transport
Terrettaz et al. Stable self‐assembly of a protein engineering scaffold on gold surfaces
WO1996010178A1 (en) Method of producing bilayer lipid membranes
Chadli et al. New tethered phospholipid bilayers integrating functional G-protein-coupled receptor membrane proteins
Bolduc et al. Peptide self-assembled monolayers for label-free and unamplified surface plasmon resonance biosensing in crude cell lysate
Kang et al. Specific adsorption of histidine-tagged proteins on silica surfaces modified with Ni2+/NTA-derivatized poly (ethylene glycol)
Lee et al. The lateral diffusion of selectively aggregated peptides in giant unilamellar vesicles
EP1410028A2 (de) Immobilisierung von proteinen und elektroaktiven verbindungen auf einer sensoroberfläche mittels einer hydrophobin-beschichtung
Merzlyakov et al. Directed assembly of surface-supported bilayers with transmembrane helices
Pollheimer et al. Reversible biofunctionalization of surfaces with a switchable mutant of avidin
Arfin et al. Proteins and their novel applications
Bompard et al. BALM: Watching the Formation of Tethered Bilayer Lipid Membranes with submicron lateral resolution
Lakey et al. Biosensor
Ottova et al. The lipid bilayer principle: A historic perspective and some highlights
KR100718207B1 (ko) 금속 결합 단백질을 이용한 바이오-금속 칩 및 그 제조방법
Novikova et al. Nanostructured Biosensors Based on Components of Bacterial Membranes
JP4598469B2 (ja) 検査用チップの製造方法
JP3823178B2 (ja) 認識多様性を有するセンサーチップ
Naser The Development of Complex Biomembrane Platforms to Investigate the Impact of Membrane Disrupting Agents on Membrane Biophysical Properties

Legal Events

Date Code Title Description
AS Assignment

Owner name: NEWCASTLE UNIVERSITY VENTURES LIMITED, UNITED KING

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LAKEY, JEREMY HUGH;VOGEL, HORST;REEL/FRAME:014817/0673;SIGNING DATES FROM 20031118 TO 20031127

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION