U.S. patent application number 10/250682 was filed with the patent office on 2004-05-20 for biosensor with covalently attached membrane-spanning proteins.
Invention is credited to Lakey, Jeremy Hugh, Vogel, Horst.
Application Number | 20040096895 10/250682 |
Document ID | / |
Family ID | 26245580 |
Filed Date | 2004-05-20 |
United States Patent
Application |
20040096895 |
Kind Code |
A1 |
Lakey, Jeremy Hugh ; et
al. |
May 20, 2004 |
Biosensor with covalently attached membrane-spanning proteins
Abstract
The invention provides 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 invention also provides a method for producing such a product
which i) comprises treating a substrate with a hydrophilic coating
agent; ii) providing at least one membrane-spanning protein; iii)
bringing the protein into contact with the treated substrate under
conditions for the coupling of the protein directly to the treated
substrate; iv) adding amphiphilic molecules to the protein-coupled
substrate to form a lipid membrane. The product is useful for
biosensors, protein arrays and the like.
Inventors: |
Lakey, Jeremy Hugh;
(Northumberland, GB) ; Vogel, Horst; (Preverenges,
CH) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W.
SUITE 600
WASHINGTON
DC
20004
US
|
Family ID: |
26245580 |
Appl. No.: |
10/250682 |
Filed: |
October 17, 2003 |
PCT Filed: |
January 18, 2002 |
PCT NO: |
PCT/GB02/00222 |
Current U.S.
Class: |
435/7.1 ;
435/287.2 |
Current CPC
Class: |
C12Q 1/003 20130101;
G01N 33/5438 20130101 |
Class at
Publication: |
435/007.1 ;
435/287.2 |
International
Class: |
G01N 033/53; C12M
001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 18, 2001 |
GB |
0101279.8 |
Apr 10, 2001 |
GB |
0108947.3 |
Claims
1. 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 coupled to the substrate by a direct covalent
coupling.
2. A product according to claim 1 wherein at least some of the
amphiphilic molecules are coupled to the substrate by a direct
covalent coupling.
3. A product according to claim 1 or 2 wherein the protein is
coupled to the substrate via at least one exposed cysteine residue
located in the periplasmic side of the protein.
4. A product according to any preceding claim wherein the product
is a biosensor.
5. A product according to any preceding claim wherein the product
is a protein array.
6. A product according to any preceding claim wherein the protein
comprises a polypeptide of more than 20 L-amino acid residues
purified from a cell extract.
7. A product according to any preceding claim wherein the protein
comprises a D-barrel structure.
8. A product according to any of claims 1-6 wherein the protein
comprises a .alpha.-helix structure.
9. A product according to any preceding claim wherein the protein
is an integral membrane protein in which secondary structural units
span the membrane at least once and join regions exposed to an
aqueous phase.
10. A product according to any preceding claim wherein the protein
is an ion channel forming protein (ionophore).
11. A product according to any of claims 1-9 wherein the protein is
a receptor for a ligand or family of ligands.
12. A product according to any of claims 1-9 wherein the protein is
an enzyme.
13. A product according to any of claims 1-12 wherein the protein
is an engineered (recombinant) protein comprising a
membrane-spanning protein and a synthetic polypeptide.
14. A product according to any of claims 1-12 wherein the protein
is an engineered (recombinant) protein comprising a
membrane-spanning protein and all or part of a soluble protein of
more than 20 L-amino acid residues purified from a cell
extract.
15. A product according to any of claims 1-12 wherein the protein
is an engineered (recombinant) protein into which has been
introduced, by insertion or mutation, at least one exposed cysteine
residue located in the periplasmic side of the protein.
16. A product according to claim 10 wherein the protein is a
porin.
17. A product according to claim 16 wherein the porin is selected
from Table 1.
18. A product according to any preceding claim wherein the
amphiphilic molecules comprise or consist of thiolipid.
19. A product according to claim 18 wherein the thiolipid is
selected from the group consisting of:
1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol;
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propi-
onate];
1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[3-(2-pyridyldi-
thio)propionate];
N-(14'-mercapto-1',11'-dioxo-3',6',9'-trioxa-12'-azatetr-
adecyl)-2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidylethanolamine;
(8'mercapto-3',6'-dioxa-octyl)-1,2-dipalymitoyl-sn-glycero-3-phosphatidic
acid.
20. A product according to any of claims 1-19 wherein the substrate
is a metal.
21. A product according to claim 20 wherein the metal is selected
from: gold; chromium; platinum; silver.
22. A product according to any of claims 1-21 wherein the substrate
has a surface modified to react with thiol groups.
23. A product according to any of claims 1-19 wherein the surface
is selected from: glass; plastic; silicon; hydrogen terminated
silicon.
24. A method for the preparation of a product according to any of
claims 1-23 comprising: i. treating a substrate with a hydrophilic
coating agent; ii. providing at least one membrane-spanning
protein; iii. bringing the protein into contact with the treated
substrate under conditions for the covalent coupling of the protein
directly to the treated substrate; iv. adding amphiphilic molecules
to the protein coupled substrate to form a lipid membrane.
25. A method according to claim 24 wherein the hydrophilic agent is
a thiol.
26. A method according to claim 25 wherein the thiol is of general
formula OH--(CH.sub.2).sub.n--SH, wherein n is an integer and the
alkyl chain is straight or branched.
27. A method according to claim 24 wherein the substrate is treated
with a hydrophilic agent selected from: 2-mercaptoethanol;
mercaptopropionic acid; 1-mercapto-2-propanol; 2,3
dimercapto-1-propanol; 2-mercapto-3-butanol;
erythro-1,4-dimercapto-2,3-butanediol, (DTE);
threo-1,4-dimercapto-2,3-butanediol, (DTT); mixtures of DTE and
DTT; thiol glycerol.
28. A method according to claim 27 wherein the hydrophilic agent is
2-mercaptoethanol.
29. A method according to any of claims 24-28 wherein the substrate
is gold.
30. A method according to any of claims 24-29 wherein the protein
is an ionophore.
31. A method according to claim 24 wherein the amphiphilic
molecules are covalently coupled to the substrate.
32. A method according to claim 31 wherein the protein is a
porin.
33. A method according to any of claims 24-32 wherein at least one
exposed cysteine residue is provided in the periplasmic side of the
protein, whereby coupling of the protein to the substrate is via
coupling of at least one cysteine to at least one thiol group on
the substrate.
34. A method according to claim 33 wherein the porin is modified
OmpA, formed by N-terminal addition of MHHHHHHSS-Cys- to the mature
OmpA protein.
35. A method according to claim 33 wherein the porin is modified
OmpF, formed by mutation of glutamate 183 to cysteine.
36. A product obtainable by the method according to any of claims
24-35.
37. Use of the product according to any of claims 1-23 to interact
with at least one of following molecules: 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.
Description
TECHNICAL FIELD
[0001] 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.
BACKGROUND
[0002] Amphiphilic molecules, for example lipids, are known to
aggregate in solution to form 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. It
is also known that 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). These membrane polypeptides are typically referred to
as membrane proteins.
[0003] Since 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. food stuffs, fermenter
products, chemicals); larger molecules such as proteins for
research screening (eg array technology) or diagnostics (cancer
markers, infectious disease markers, hormones); nucleic acids;
carbohydrate polymers; cells such as pathogenic bacteria;
eukaryotic cells such a cancer cells and small single or
multicellular organisms especially parasites. Moreover, 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.
[0004] 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.
[0005] Additionally, 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.
[0006] It will be apparent to the skilled artisan that this
technology also has broad applicability to the provision of
surfaces which allow the linking of membrane polypeptides to a
substrate in a particular orientation and density which allow these
surfaces to function as binding surfaces for a range of molecules
the interaction of which can be monitored by established methods.
For example, optical methods such as surface plasmon resonance;
fluorescence; ellipsometry; or electrical methods such as
spectroscopy impedance, cyclic volametry, conductance measurement.
Membrane polypeptides could be used in biosensors in devices to
sense physical signals such as voltage, pressure or
temperature.
[0007] 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.
[0008] 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).
[0009] An example of a group of functionally related membrane
proteins is the porins which is a sub-group of the ionophores.
Other groups include GPCRs, pentameric ligand gated channels, ABC
transporters etc.
[0010] The signal transducing properties of ion-channels, combined
with their high sensitivity and their minimal size, has led to the
development of biosensors based on synthetic membrane structures
incorporating ion-channel proteins. Although there is no difficulty
in manufacturing these membranes incorporating ion-channels they
are often expensive to obtain or are fragile and unstable.
[0011] Prior art biosensors (see U.S. Pat. No. 5,234,566; U.S. Pat.
No. 5,736,342; U.S. Pat. No. 5,516,890; WO9725616) 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. In a particular case
the device of Cornell (Cornell et al., 1997) 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. Generally
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.
BRIEF DISCLOSURE OF THE INVENTION
[0012] 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. The combination of chemisorbed protein and 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.
[0013] 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.
[0014] For example, according to the invention, we have engineered
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. 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. It will be apparent that this technology can be applied
to other ion-channel forming polypeptides and other membrane
polypeptides using thiol containing amino acids. In particular any
protein which presents a flat surface close to the membrane
interface (such as the outer membrane proteins of bacteria or
bacteriorhodopsin) may be considered suitable for this method. 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.
[0015] In comparison to the prior art of Cornell, the present
invention relies upon large integral membrane proteins directly
fixed to the substrate surface and lateral diffusion in the bilayer
does not occur. Hence the role of the bilayer is to stabilise the
assembled protein layer, reduce non-specific binding to the
substrate surface and provide electrical insulation. Since the
bilayer need not be fluid, 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. However, as 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. Furthermore since 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 Cornell. The
.beta.-barrel membrane protein family are good targets for protein
engineering and thus a wide variety of protein interfaces may be
constructed in this manner.
[0016] In the context of the present invention the polypeptides
which are directly coupled to the substrate are referred to as
"membrane-spanning proteins". The implication of this is that the
polypeptides are generally not simple linear short synthetic amino
acid sequences with little or no secondary/tertiary structure (eg
protein folding). The implication is also that 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.
Similarly, very large/complex proteins which mainly reside outside
a membrane bilayer but which may extend partly into the bilayer are
unlikely to be advantageously useful in this invention. Those
skilled in the art will appreciate that the term
"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.
STATEMENTS OF INVENTION
[0017] According to a first aspect of the invention there is
provided 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.
[0018] In a preferred embodiment of the invention the product is a
biosensor or protein array.
[0019] In a further preferred embodiment of the invention the
membrane-spanning protein comprises an .alpha.-helix structure or a
.beta.-barrel structure.
[0020] 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 .beta.-barrel, and OmpF which is a homotrimer of 16
stranded .beta.-barrels.
[0021] 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.
[0022] Membrane-spanning proteins can be modified to facilitate the
coupling of the polypeptide to the substrate.
[0023] We have genetically engineered OmpF by the mutation
glutamate 183 to cysteine E183C). The insertion of a cysteine, or
other thiol containing amino acid, into any of the periplasmic
turns of the OmpF polypeptide will give similar results to those
disclosed. More than one cysteine residue could be added to each
protein to increase the interaction with the surface, hence this
could be tailored to individual requirements. Insertion of cysteine
into the extremities of the extracellular loops will also work but
will result in a protein layer of the opposite orientation.
[0024] Synonyms of OmpF are: Porin ompF
[0025] Outer membrane protein 1A
[0026] Outer membrane protein 1A
[0027] Outer membrane protein B
[0028] Gene names are: OMPF or TOLF or CMLB or COA or CRY or
B0929
[0029] We have also genetically engineered OmpA to insert a Lys
residue.
[0030] In an embodiment of the invention 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.
[0031] The membrane-spanning protein may be selected from those
presented in Table 1 or Table 2.
[0032] 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. The substrate can be rendered hydrophobio by
preincubation with a small hydrophilic thiol, for example
.beta.-mercaptoethanol or thio glycerol.
[0033] 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.
[0034] 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. In general 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. Additionally, the
contact of the hydrophilic protein loops with the hydrophilic
surface is non-denaturing and separates the core of the protein
from the surface.
[0035] 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.
This provides a membrane-like environment which satisfies the
polypeptide's needs for hydrophilic and hydrophobic surfaces and
thus ensures the stability of the protein.
[0036] In a further embodiment of the invention the lipid is a
thiolipid.
[0037] The thiolipids are a very variable group. For example, 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.
[0038] Thiolipids without spacers such as
1,2-dipalmitoyl-sn-glycero-3-pho- sphothioethanol also have utility
with respect to the biosensor of the invention. 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. 1
[0039] 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.
Preferably the carbon backbone is hydrophilic to stabilise the
hydrophilic protein loops adjacent to it.
[0040] Preferably the thiolipid is selected from
1,2-dipalmitoyl-sn-glycer- o-3-phosphothioethanol;
1,2-doleoyl-sn-gly 3-phosphoethanolamine-N-[3-(2-p-
yridyldithio)propionate];
1,2-Dipalmitoyl-sn-glycero-3-Phosphoethanolamine-
-N-[3-(2-pyridyldithio)propionate];
N-14'-mercapto-1',11'-dioxo-3',6,9'-tr-
ioxa-12'-azatetradecyl)-2-oleoyl-1-palmitoyl-sn-glycero-3-phosphatidyletha-
nolamine;
(8'-mercapto-3',6'-dioxa-octyl)-1,2-dipalmitoyl-sn-glycero-3-pho-
sphatidic acid.
[0041] 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.
[0042] In a further embodiment of the invention the substrate is a
metal. The metal may be selected from gold; chromium platinum;
silver.
[0043] In a further embodiment of the invention the substrate is
selected from: glass, silicon, hydrogen terminated silicon or
plastic polymers.
[0044] It will be apparent to one skilled in the art that methods
to generate mutations which delete or change codon usage are
available. Modification of a membrane-spanning protein (such as an
ion-channel forming polypeptide) 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.
[0045] In one embodiment of 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.
[0046] There is provided a method for the production of the
modified protein used in the invention, comprising:
[0047] i) providing a cell transformed/transfected with a nucleic
acid molecule which is part of a vector adapted to facilitate
recombinant expression of the protein;
[0048] ii) growing said cell in conditions conducive to the
expression of said protein; and
[0049] iii) purifying said protein from said cell, or its growth
environment.
[0050] The vector may encode, and thus said modified recombinant
protein is provided with a secretion signal to facilitate
purification of the protein.
[0051] According to a further aspect of the invention there is
provided a product comprising a biosensor.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] The product could allow for rapid screening of samples for a
range of proteins and thus increase productivity in clinical or
research labs.
[0056] 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.
[0057] A further product could be the creation of biocompatible
surfaces for in vitro implantation or cell cube technology. For
example, 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] According to a yet another aspect of the invention there is
provided a method for the preparation of a product comprising)
[0062] i) treating a substrate with a hydrophilic coating
agent;
[0063] ii) providing at least one membrane-spanning protein;
[0064] iii) bringing the protein into contact with the treated
substrate under conditions for the coupling of the protein directly
to the treated substrate;
[0065] iv) adding amphiphilic molecules to the protein-coupled
substrate to form a lipid membrane.
[0066] In one method of the invention the product is a biosensor.
In a further method of the invention the membrane protein is an
outer membrane protein, such as a porin.
[0067] In the method of the invention 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.
[0068] Preferably the coating agent is 2-mercaptoethanol.
[0069] Conditions for coating substrates may be varied. For
example, 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.
[0070] Preferably the substrate is gold and the outer membrane
polypeptide is a porin. More preferably still the porin is OmpF or
modified OmpF. Preferably the amphiphilic molecule is a lipid,
ideally thio-lipid.
[0071] According to an aspect of the invention there is provided a
biosensor obtainable by the method according to the invention
[0072] According to a further aspect of the invention there is
provided the use of the biosensor according to the invention 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.
[0073] An embodiment of the invention will now be provided by way
of example only and with reference to the following Figures and
Tables wherein:
[0074] Table 1 represents porin genes of Gram-ve bacteria;
[0075] Table 2 represents Ton B dependent receptors from various
bacterial species;
[0076] 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];
[0077] FIG. 2 represents binding of anti OmpF polyclonal antibodies
to tethered OmpF-Cys;
MATERIALS AND METHODS
[0078] Protein Purification
[0079] 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 QuikChange.TM. method
(Stratagene).
[0080] Mutagenesis of OmpF
[0081] The mutagenesis used two complementary 37 mer
oligonucleotides which coded for a TGT cysteine codon instead of a
GAA glutamate codon.
1 Forward [SEQ ID No. 3] 5'-GGTTCTATCAGCTAC Reverse [SEQ ID No. 4]
3'-CCAAGATAGTAGATGATGCTTCCGAAACCATAGT-5'
[0082] These mutagenic primers were used with the plasmid pGBF96 in
the Quikchange protocol described in the QuikChange kit supplied by
Stratagene. The transformants in XL1-blue E coli cells were used to
produce pure plasmid DNA by use of Promega Wizars SV miniprep kits.
The OmpF gene was sequenced by an ABI-Prism 377 device in the
University of Newcastle Facility for Molecular Biology.
[0083] The mutation in the OmpF BE3000 gene caused by our
introduction of an XbaI restriction site is Lysine279 to Arginine
(K279R). Thus although 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.
[0084] Amphiphillic Agents
[0085] 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). 2
[0086] Surface Plasmon Resonance (SPR)
[0087] 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.times.10.sup.-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.degree. 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.
[0088] 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 .mu.l per minute for the
proteins thiolipid assembly steps and 30 .mu.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.
[0089] Impedance Spectroscopy (LS.)
[0090] 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.times.10.sup.-2 cm.sup.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.
[0091] Fourier Transform Infra-Red Spectroscopy (FTIR).
[0092] 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.sup.-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.sup.-1 resolution with triangular apodisation. The reflectance
absorbance spectra a molecular layers on gold was recorded at an
angle of incidence of 85.degree. using only parallel-polarised
light. All peak positions were derived from second derisive
spectra. For further details of the technique, see Boucheva
(1997).
[0093] Binding of Anti-OmpF Polyclonal Antibodies to OmPF-Cys
[0094] 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 .mu.l/min on a Biacore X (Biacore AB B, Uppsala, Sweden).
Fitted black curves for a Langmuir 1:1 binding model are
superimposed. The association rate constant K.sub.a was evaluated
at 4.4.times.10.sup.4M.sup.-1s.sup.-1, and the dissociation rate
constant K.sub.d was 6.0.times.10.sup.-5s.sup.-1 for a calculated
affinity constant K.sub.D of 1.4 nM. The binding interaction
indicates that the protein is immobilized with its extracellular
loops exposed.
EXAMPLE 1
[0095] Immobilisation Experiment Using OmpF-Cys.
[0096] A plain gold surface is first treated with
.beta.-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). Next 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
.beta.-mercaptoethanol does not dislodge the layers showing that
the self assembled proteins and lipids are effectively irreversibly
bound to the surface.
EXAMPLE 2
[0097] Data from the SPR device is provided directly in angle shift
of the SPR signal on a time axis. This showed similar results to
the Biacore data but can be related directly to surface densities
which can then be used to calculate the mean thickness of the
layer.
[0098] The protein immobilisation gave an angle shift of
031.degree.+/-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.degree. 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.
[0099] Thiolipid self assembly results in another 0.42+/-0.06
increase and PC addition is about 0.30.degree.+1-0.17.
[0100] The values are very constant and show that a very
reproducible system of self assembly has been created
EXAMPLE 3
[0101] 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.
[0102] Pre-treating the gold with .beta.-mercaptoethanol (10 mM in
ethanol) (at least 1 hour but now these surfaces are stored in 10
mM .beta.-mercaptoethanol until use) causes an improvement in the
retention of OMPF secondary structure. This was followed by
OmpF-Cys self assembly as above followed by rapid replacement of
this solution by the ZD16 solution. This self assembles the
thiolipid layer without a drying and also solubilises and removes
non-specifically bound protein. The result of this is a spectrum
which closely resembles the published spectrum (Nabedryk et al.,
1988) and confirms that the secondary structure of OmpF is retained
by this process. Incubation with 1 mg/ml phosphatidylcholine
vesicles in buffer A for 1 hour generates an extra intensity in the
peak at a wavenumber of 1740 cm-1. This is from the lipid and
exactly doubles the existing thiolipid peak showing that the
surface layer of the bilayer has been completed by this process.
The protein spectrum showed a slight increase in beta secondary
structure.
[0103] Even without subsequent thiolipid assembly the pre treatment
with .beta.-mercaptoethanol causes a significant improvement in
retention of secondary structure. A similar benefit could be
achieved by using another small hydrophilic Thiol molecule.
EXAMPLE 4
[0104] The maximum density of self assembled OmpF-Cys followed by
thiolipid provides a layer which is very conducting and this cannot
be analysed by IS. The impedance spectroscopy can be measured at
lower protein densities and clear results were obtained using a
layer made using a 1 hour incubation with a 0.003 mg/ml OmpFC-Cys
solution, this is 100 fold less than the concentration used above.
At 100 Hz the resistance of the surface layer dominates the real
part of the signal. Addition of 10.sup.-6 M colicin R domain caused
the resistance to increase showing that the layer is being blocked
by the protein interaction. This effect is concentration dependent
and resembles the measured affinity of the previous paper.
[0105] Binding of colicin N R domain is being studied by SPR in
Newcastle and confirms the impedance data shown here.
EXAMPLE 5
[0106] Self-Assembly of Refolded OmpA Monomeric Porin
Transmembrane-Domain Layers on Gold.
[0107] In order to use outer membrane receptor proteins in the
development of membrane protein monolayer for bio-nanotechnology,
we need to refold the proteins in vitro prior to assembling them on
surfaces. Here we show that the outer membrane protein OmpA
refolded by published methods can be assembled in the
protein-thiolipid layers already demonstrated for OmpF. The result
also shows that monomeric OMP's such as OmpA can be immobilised
using the same methods as for trimeric porins.
[0108] Cloning
[0109] 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. 7] coded by the
plasmid and an additional Cys residue coded by the primer, and
amino acids 1-147 of the mature OmpA protein. 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).
[0110] The protein was expressed in BL21 DE3 pLysE (Novagen) using
Luria broth and 100 .mu.g/ml Ampicillin and 30 .mu.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.degree. 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.degree. C. for 30
min. The homogenate was centrifuged at 39,000 rpm for 1.5 h at
4.degree. 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.
[0111] 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-.beta.-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 .beta.-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.
[0112] Finally its was shown by SPR that the protein assembles on
gold electrodes, and that thiolipids can be added to complete the
monolayer as shown for OmpF layers. The results demonstrate that
refolded monomeric porins such as OmpA can be used in the same
manner as previously described for OmpF.
[0113] A gold J1 chip was cleaned with Piranha solution (9% v/v
H.sub.2O.sub.2 and 70% conc H.sub.2SO.sub.4), rinsed with ethanol
and water and dried with nitrogen. In the Biacore-X surface plasmon
resonance spectrometer the chip was purified with 0.05% SDS
solution, then 0.2%, .beta.-mercaptoethanol to provide the
hydrophilic layer. After a second SDS wash the folded protein His
Cys-OmpA (1-147) at 0.2 mg/ml in n-docecyl-.beta.-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.
REFERENCES
[0114] 1. Stora, T., Lakey, J. H. and Vogel, H. (1999). Ion-channel
Gating in Transmembrane Receptor Proteins: Functional Activity in
Tethered Lipid Membranes. Angew. Chem. Int. Ed. 38(3), 389-392.
[0115] 2. Cornell B. A., Braach-Maksvytis V. L. B., King, L. G.,
Osman P., Raguse, B., Wieczorek, L. and Pace, R. J. (1997). A
biosensor that uses ion-channel switches Nature 387(6633),
580-583.
[0116] 3. Bainbridge, G., Mobasheri, H., Armstrong, G. A., Lea, E.
J. A. and Lakey, J. H. (1998). Voltage gating of Escherichia coli
porin a cystine scanning mutagenesis study of loop 3. Journal Of
Molecular Biology 275, 171-176.
[0117] 4. Evans, L. J. A., Cooper, A, and Lakey, J. H. (1996).
Direct measurement of the association of a protein with a family of
membrane receptors. Journal Of Molecular Biology 255(4),
559-563
[0118] 5. Nabedryk, E., Garavito, R. M. and Breton, J. (1988). The
orientation of beta-sheets in porin--a polarized fourier-transform
infrared spectroscopic investigation. Biophysical Journal 53(5),
671-676.
[0119] 6. Evans, L. J. A., Labeit, S., Cooper, A., Bond, L. H. and,
Lakey, J. H. (1996). The central domain of colicin N possesses the
receptor recognition site but not the binding affinity of the whole
toxin. Biochemistry 35(48), 15143-15148.
[0120] Stora, T., Lakey, J. H. and Vogel, H. (1999). Ion-Channel
gating in Transmembrane Receptor Proteins: Functional Activity in
Tethered Lipid Membranes. Angew. Chem. Int. Ed. 38(3), 389-392.
[0121] 7. Lang, H., Duschl, C., and Vogel, H. (1994). "A new class
of thiolipids for the attachment of lipid bilayers on gold
surfaces." Langmuir, 10(1), 197-210.
[0122] 8. Boncheva, M., and Vogel, H. (1997). "Formation of stable
polypeptide monolayers at interfaces: Controlling molecular
conformation and orientation" Biophysical Journal, 73(2),
1056-1072.
[0123] 9. Arora, A., Abildgaard, F., Bushweller, J. H. & Tamm,
L. K. (2001). Structure of outer membrane protein A transmembrane
domain by NMR spectroscopy. Nature Structural Biology 8(4),
334-338.
[0124] 10. Kleinschmidt, J. R., Wiener, M. C. & Tamm, L. K.
(1999). Outer membrane protein A of E-coli folds into detergent
micelles, but not in the presence of monomeric detergent, Protein
Science 8(10), 2065-2071.
[0125] 11. Pautsch, A. & Schulz, G. E. (2000). High-resolution
structure of the OmpA membrane domain. Journal of Molecular Biology
298(2), 273-282.
2TABLE 1 Examples of 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. Species (total number of proteins of this class so far
identified in this species) siphoviridae pa(1) lambda phage group
p21(1) Ectothiorhodospira vacuolata(1) Vibrio cholerae(1)
Photobacterium profundum(1) Haemophilus influenzae(30) Pasteurella
multocida(7) Rahnella aquatilis(2) Salmonella typhimurium(4)
Enterobacter cloacae(1) Salmonella typhi(5) Citrobacter freundii(1)
Calymmatobacterium granulomatis(1) Klebsiella pneumoniae(7)
Yersinia pestis(2) Xenorhabdus nematophilus(1) Klebsiella
oxytoca(1) Escherichia coli(9) Serratia marcescens(2) Burkholderia
cepacia(1) Eikenella corrodens(1) Neisseria polysaccharea(1)
Neisseria lactamica(2) Neisseria gonorrhoeae(91) Neisseria
flavescens(2) Neisseria sicca(2) Neisseria meningitidis(160)
Bordetella pertussis(1) Rhodobacter blastica(1) Rhodobacter
capsulatus Brucella sp.; Chlamydia sp Shigella Comamonas
acidovorans Aeromonas Thermotoga maritima OmpA 8 stranded beta
barrel proteins and hmologous membrane proteins Chlamydophila
pneumoniae(1) Chlamydia trachomatis(1) Helicobacter pyori(5)
Haemophilus influenzae(4) Haemophilus sp(1) Pasteurella
haemolytica(1) Haemophilus parainfluenzae(1) Pasteurella
multocida(1) Haemophilus somnus(1) Haemophilus ducreyi(3)
Actinobacillus pleuropneumoniae(1) Actinubacillus
actinomycetemcomitans(1) Vibrio alginolyticus(2) Vibrio chlerae(1)
Vibrio parahaemolyticus(3) Methylococcus capsulatus(1) Aeromonas
salmonicida(2) Pseudomonas syringae(1) Moraxella catarrhalis(1)
Pseudomonas aeruginosa(3) Pseudomonas putida(2) Pseudomonas
fluorescens(7) Escherichia vulneris(3) Escherichia hermanii(1)
Escherichia fergusonii(1) Escherichia blattae(1) Salmonella
typhimurium(2) Shigella dysenteriae(1) Serratia odorifera(1)
Enterobacter aerogenes(2) Citrobacter freundii(1) Klebsiella
pneumoniae(1) Serratia marcescens(1) Escherichia coli(6) Legionella
pneumophila(1) Neisseria gonorrhoeae(1) Neisseria meningitidis(1)
Leptothrix discophora(1) Bordetella avium(1) Brucella abortus(1)
Bartonella bacilliformis(1) Sinorhizobium meliloti(1) Rickettsia
prowazekii(1) Aquifex aeolicus(3) Treponema phagedenis(1) Borrelia
burgdorfei(2) Treponema pallidum(3) Sugar selective porins Vibrio
parahaemolyticus(1) Vibrio cholerae(1) Aeromonas salmonicida(1)
Yersinia enterocolitica(1) Klebsiella pneumoniae(2) Salmonella
typhimurium(2) Escherichia coli(2) Nucleoside specific channel
forming protein TSX KLEBSIELLA PNEUMONIA ENTEROBACTER AEROGENENES
ESCHERICHIA COLI SALMONELLA TYPHIMURIUM
[0126]
3TABLE 2 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)
Pectobacterium chrysanthemi(1) Yersinia pseudotuberculosis(1)
Citrobacter freundii(1) Yersinia enterocolitic(4) Erwinia
amylovora(1) Salmonella paratyphi(1) Salmonella cholerae(1)
Yezsinia pestis(2) Serratia marcescens(1) Escherichia coli(17)
Neisseria gonorrhoeae(3) Neisseria meningitidis(12) Bordetella
pertussis(1) Bordetella bronchiseptica(3) Paracoccus
denitrificans(1) Sphingomonas aromaticivorans(3) Bradyrhizobium
japonicum(1) Rhizobiaceae sp(1) Synechocystis sp(3) Proteobacteria
sp(1)
[0127]
Sequence CWU 1
1
7 1 1089 DNA Escherichia coli CDS (1)..(1089) 1 atg atg aag cgc aat
att ctg gca gtg atc gtc cct gct ctg tta gta 48 Met Met Lys Arg Asn
Ile Leu Ala Val Ile Val Pro Ala Leu Leu Val 1 5 10 15 gca ggt act
gca aac gct gca gaa atc tat aac aaa gat ggc aac aaa 96 Ala Gly Thr
Ala Asn Ala Ala Glu Ile Tyr Asn Lys Asp Gly Asn Lys 20 25 30 gta
gat ctg tac ggt aaa gct gtt ggt ctg cat tat ttt tcc aag ggt 144 Val
Asp Leu Tyr Gly Lys Ala Val Gly Leu His Tyr Phe Ser Lys Gly 35 40
45 aac ggt gaa aac agt tac ggt ggc aat ggc gac atg acc tat gcc cgt
192 Asn Gly Glu Asn Ser Tyr Gly Gly Asn Gly Asp Met Thr Tyr Ala Arg
50 55 60 ctt ggt ttt aaa ggg gaa act caa atc aat tcc gat ctg acc
ggt tat 240 Leu Gly Phe Lys Gly Glu Thr Gln Ile Asn Ser Asp Leu Thr
Gly Tyr 65 70 75 80 ggt cag tgg gaa tat aac ttc cag ggt aac aac tct
gaa ggc gct gac 288 Gly Gln Trp Glu Tyr Asn Phe Gln Gly Asn Asn Ser
Glu Gly Ala Asp 85 90 95 gct caa act ggt aac aaa acg cgt ctg gca
ttc gcg ggt ctt aaa tac 336 Ala Gln Thr Gly Asn Lys Thr Arg Leu Ala
Phe Ala Gly Leu Lys Tyr 100 105 110 gct gac gtt ggt tct ttc gat tac
ggc cgt aac tac ggt gtg gtt tat 384 Ala Asp Val Gly Ser Phe Asp Tyr
Gly Arg Asn Tyr Gly Val Val Tyr 115 120 125 gat gca ctg ggt tac acc
gat atg ctg cca gaa ttt ggt ggt gat act 432 Asp Ala Leu Gly Tyr Thr
Asp Met Leu Pro Glu Phe Gly Gly Asp Thr 130 135 140 gca tac agc gat
gac ttc ttc gtt ggt cgt gtt ggc ggc gtt gct acc 480 Ala Tyr Ser Asp
Asp Phe Phe Val Gly Arg Val Gly Gly Val Ala Thr 145 150 155 160 tat
cgt aac tcc aac ttc ttt ggt ctg gtt gat ggc ctg aac ttc gct 528 Tyr
Arg Asn Ser Asn Phe Phe Gly Leu Val Asp Gly Leu Asn Phe Ala 165 170
175 gtt cag tac ctg ggt aaa aac gag cgt gac act gca cgc cgt tct aac
576 Val Gln Tyr Leu Gly Lys Asn Glu Arg Asp Thr Ala Arg Arg Ser Asn
180 185 190 ggc gac ggt gtt ggc ggt tct atc agc tac gaa tac gaa ggc
ttt ggt 624 Gly Asp Gly Val Gly Gly Ser Ile Ser Tyr Glu Tyr Glu Gly
Phe Gly 195 200 205 atc gtt ggt gct tat ggt gca gct gac cgt acc aac
ctg caa gaa gct 672 Ile Val Gly Ala Tyr Gly Ala Ala Asp Arg Thr Asn
Leu Gln Glu Ala 210 215 220 caa cct ctt ggc aac ggt aaa aaa gct gaa
cag tgg gct act ggt ctg 720 Gln Pro Leu Gly Asn Gly Lys Lys Ala Glu
Gln Trp Ala Thr Gly Leu 225 230 235 240 aag tac gac gcg aac aac atc
tac ctg gca gcg aac tac ggt gaa acc 768 Lys Tyr Asp Ala Asn Asn Ile
Tyr Leu Ala Ala Asn Tyr Gly Glu Thr 245 250 255 cgt aac gct acg ccg
atc act aat aaa ttt aca aac acc agc ggc ttc 816 Arg Asn Ala Thr Pro
Ile Thr Asn Lys Phe Thr Asn Thr Ser Gly Phe 260 265 270 gcc aac aaa
acg caa gac gtt ctg tta gtt gcg caa tac cag ttc gat 864 Ala Asn Lys
Thr Gln Asp Val Leu Leu Val Ala Gln Tyr Gln Phe Asp 275 280 285 ttc
ggt ctg cgt ccg tcc atc gct tac acc aaa tct aaa gcg aaa gac 912 Phe
Gly Leu Arg Pro Ser Ile Ala Tyr Thr Lys Ser Lys Ala Lys Asp 290 295
300 gta gaa ggt atc ggt gat gtt gat ctg gtg aac tac ttt gaa gtg ggc
960 Val Glu Gly Ile Gly Asp Val Asp Leu Val Asn Tyr Phe Glu Val Gly
305 310 315 320 gca acc tac tac ttc aac aaa aac atg tcc acc tat gtt
gac tac atc 1008 Ala Thr Tyr Tyr Phe Asn Lys Asn Met Ser Thr Tyr
Val Asp Tyr Ile 325 330 335 atc aac cag atc gat tct gac aac aaa ctg
ggc gta ggt tca gac gac 1056 Ile Asn Gln Ile Asp Ser Asp Asn Lys
Leu Gly Val Gly Ser Asp Asp 340 345 350 acc gtt gct gtg ggt atc gtt
tac cag ttc taa 1089 Thr Val Ala Val Gly Ile Val Tyr Gln Phe 355
360 2 362 PRT Escherichia coli 2 Met Met Lys Arg Asn Ile Leu Ala
Val Ile Val Pro Ala Leu Leu Val 1 5 10 15 Ala Gly Thr Ala Asn Ala
Ala Glu Ile Tyr Asn Lys Asp Gly Asn Lys 20 25 30 Val Asp Leu Tyr
Gly Lys Ala Val Gly Leu His Tyr Phe Ser Lys Gly 35 40 45 Asn Gly
Glu Asn Ser Tyr Gly Gly Asn Gly Asp Met Thr Tyr Ala Arg 50 55 60
Leu Gly Phe Lys Gly Glu Thr Gln Ile Asn Ser Asp Leu Thr Gly Tyr 65
70 75 80 Gly Gln Trp Glu Tyr Asn Phe Gln Gly Asn Asn Ser Glu Gly
Ala Asp 85 90 95 Ala Gln Thr Gly Asn Lys Thr Arg Leu Ala Phe Ala
Gly Leu Lys Tyr 100 105 110 Ala Asp Val Gly Ser Phe Asp Tyr Gly Arg
Asn Tyr Gly Val Val Tyr 115 120 125 Asp Ala Leu Gly Tyr Thr Asp Met
Leu Pro Glu Phe Gly Gly Asp Thr 130 135 140 Ala Tyr Ser Asp Asp Phe
Phe Val Gly Arg Val Gly Gly Val Ala Thr 145 150 155 160 Tyr Arg Asn
Ser Asn Phe Phe Gly Leu Val Asp Gly Leu Asn Phe Ala 165 170 175 Val
Gln Tyr Leu Gly Lys Asn Glu Arg Asp Thr Ala Arg Arg Ser Asn 180 185
190 Gly Asp Gly Val Gly Gly Ser Ile Ser Tyr Glu Tyr Glu Gly Phe Gly
195 200 205 Ile Val Gly Ala Tyr Gly Ala Ala Asp Arg Thr Asn Leu Gln
Glu Ala 210 215 220 Gln Pro Leu Gly Asn Gly Lys Lys Ala Glu Gln Trp
Ala Thr Gly Leu 225 230 235 240 Lys Tyr Asp Ala Asn Asn Ile Tyr Leu
Ala Ala Asn Tyr Gly Glu Thr 245 250 255 Arg Asn Ala Thr Pro Ile Thr
Asn Lys Phe Thr Asn Thr Ser Gly Phe 260 265 270 Ala Asn Lys Thr Gln
Asp Val Leu Leu Val Ala Gln Tyr Gln Phe Asp 275 280 285 Phe Gly Leu
Arg Pro Ser Ile Ala Tyr Thr Lys Ser Lys Ala Lys Asp 290 295 300 Val
Glu Gly Ile Gly Asp Val Asp Leu Val Asn Tyr Phe Glu Val Gly 305 310
315 320 Ala Thr Tyr Tyr Phe Asn Lys Asn Met Ser Thr Tyr Val Asp Tyr
Ile 325 330 335 Ile Asn Gln Ile Asp Ser Asp Asn Lys Leu Gly Val Gly
Ser Asp Asp 340 345 350 Thr Val Ala Val Gly Ile Val Tyr Gln Phe 355
360 3 37 DNA Artificial Sequence Synthetic oligonucleotide 3
ggttctatca gctactgtta cgaaggcttt ggtatcg 37 4 37 DNA Artificial
Sequence Synthetic oligonucleotide 4 ccaagatagt agatgacaat
gcttccgaaa ccatagc 37 5 32 DNA Artificial Sequence Synthetic
oligonucleotide 5 ttttctcgag ctgtgctccg aaagataaca cc 32 6 31 DNA
Artificial Sequence Synthetic oligonucleotide 6 ttttgcgcaa
agtgccacgg cctcgacctc g 31 7 9 PRT Artificial Sequence N-terminal
tag sequence encoded by pET8c vector 7 Met His His His His His His
Ser Ser 1 5
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