Note: Descriptions are shown in the official language in which they were submitted.
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Protein Binding Polypeptides
Field of Invention
This invention relates to protein binding polypeptides and applications for
such
polypeptides.
Background
Hitherto the only small molecules known to bind to ligand binding and receptor
proteins
were those natural molecules of biological origin which are known to bind to
the usually
highly specific functional sites of such proteins, or synthetic analogues of
those natural
molecules. Common examples of these small molecules are the haptens,
polypeptides and
epitopes binding to the variable or complimentarity determining region (CDR)
of
antibodies and other immunoglobulins, biotin binding to avidin or
streptavidin, glucose
binding to concanavalin A and the tachykinins binding to tachykinin receptors.
Whilst
other protein molecules are known to bind to other sites on such ligand
binding and
receptor proteins, such as Protein A or G binding to immunoglobulins, or
antibodies raised
to bind to epitopes on the surface of many proteins, these binding proteins
are of a
particularly large size which limits their usefulness in separation, detection
and treatment.
The use of antibodies, another major tool in the life sciences, relies heavily
on affinity
chromatography as a purification tool. In particular, antibody purification by
immobilised
Protein A is a frequently-used separation technique both in the laboratory and
for pilot
scale manufacture.
Protein A, which is isolated from the cell walls of the pathogenic bacterium
Staphylococcus aureus, and Protein G, which is isolated from the cell wall of
a ~i
haemolytic Streptococcus G strain, are extensively used as ligands for the
affinity
purification of polyclonal and monoclonal antibodies. A number of affinity
supports to
which these proteins are immobilised are available. Protein A and Protein G
offer some
differences in selectivity for the source and subtype of the antibody to be
purified. The use
of proteins such as Protein A and Protein G in affinity chromatography is
summarised in an
article by S R Narayaman (Journal of Chromatography 658, 1994, pages 237-258).
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Large proteins such as Protein A and Protein G are fragile molecules and their
biological
activity is prone to changes in their protein structure. Thus the storage and
use of such
protein matrixes requires expert handling and careful attention. Retaining the
biological
activity after each purification cycle is essential for the re-use of affinity
chromatography
columns. However, each purification cycle involves the use of harsh conditions
(such as
low pH) which can denature protein structure. Consequently, affinity
chromatography
columns ng Protein A or Protein G have a limited life, which is progressively
reduced in
normal use and accelerated by improper maintenance or use in difficult
conditions.
Commercially available Protein A and (in particular) Protein G are relatively
expensive. W
R Trumble et al (Protein Engineering, 7, No. 5 1994, pages 705-713) have
investigated
whether shortening the protein used on an affinity column would reduce the
likelihood of
the protein structure of the protein being changed in use or storage, and
report an attempt to
idcntify the minimum portion of a monodomain IgG F~ binding protein that
retained F
binding ability. This paper indicates that the smallest F~ binding protein
which could be
produced would be of the order of 45-55 amino acids long. Another paper has
recently
reduced this sequence to 33 residues (Proc.Natl.AcadSci. 1996, 93,5688-5692;
Biophysical Journal 1992, 62, 87-91.). Frick et al (Proc. Natl. Acad. Sci. USA
1992, 89,
8532-8536 derived a 11 mer peptide from Protein G and found it to bind
specifically to IgG
Fc site.
A further paper (J.Neuroimmunol. 1993; 48:2,199-203) has shown that the
covalently
membrane bound 12-28 amino acid domain, derived from the natural amyloid
polypeptide, binds Immunoglobulin G at its hinge region. This 17 residue
polypeptide
however binds with very high affinity resisting dissociation by denaturants.
An analogue of
the 17 mer polypeptide with almost identical hydropathic profile also showed
binding to
Ig G while a control polypeptide having a scrambled amino acid sequence and
thus
different hydropathic profile, shows minimal binding.
G Fassina et al (J. Molecular Recognition 1996, 9, 561-569) have identified a
synthetic
tetrameric tripeptide which mimics Protein A in its ability to recognise the
Fc portion of
immunoglobulin G.
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However, this tetrameric polypeptide is not a linear ammo acid sequence. It
comprised 4
copies of a 3-mer peptide (Y'TIt) on a common glycine core which branches with
lysyl
residues and in addition the latter tetrameric polypeptide is shown to bind
specificaily to
the F~ fragment of IgG. In this context it is also noteworthy that Sloostra et
al (Molecular
Diversity, 1 ( 1995) 87-96) made all possible trimers (8000 peptides) and
carried out
screens against three different antibodies . These studies reveal that the
only linear
sequence trimer peptides which bound the antibodies were those which
corresponded to
mimic the linear or non linear part of the native epitopes. The YTR linear
peptide was not
identified as an antibody binding sequence.
Other groups have produced paralogs, which are short polypeptides that
simulate the
binding site for the antigen on a molecule antibody. Such polypeptides have
high
specificity for the antigen, and are reviewed in the article by Narayaman
(supra), but
require knowledge of an antibody amino acid sequence and are therefore not
appropriate
for general use. Peptides which bind specifically to a protein have been
identified using
Phage or chemical libraries screened against known specificities or binding
receptors (Eur.
J. Biochem. 1974,43 71-375. Anal Biochem. 1979, 97, 302-308. Biotech. Bioeng
1995, 47,
288-297.). Again such peptides are specific ligands of those proteins.
The inventors have surprisingly found short polypeptide sequences which have
the ability
to bind both antibodies and several other proteins, typically those with
ligand binding and
receptor functions, but not to the majority of enzymes tested.
The protein binding polypeptides can be distinguished according to this
invention in three
important and previously unknown and unexpected ways: their particularly small
size
(between 2 and 50 and typically 4 to 30 amino acids); their binding at a
different site to
those sites known to define the function of the proteins bound; and their
binding
substantially to two or more unrelated proteins which may or may not have
ligand binding
functions. Typical proteins bound by the polypeptides are immunoglobulins (eg
antibodies)
and related receptors (eg antibody receptors on B cells, and T cell receptors
on T cells of
the cellular immune system); immunoglobulin binding proteins (eg Protein A,
Protein G),
lectins (eg concanavalin A), vitamin binding proteins (eg avidin,
streptavidin). The
polypeptides of the invention are advantageous in the fields of separation,
detection and
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treatment, particularly in applications involving the binding of the
polypeptides to proteins
with protein binding and receptor functions.
Disclosure of the Invention
A first aspect of the invention provides a protein binding polypeptide not
directly derived
from a na~~ral protein-binding protein, the protein binding polypeptide
comprising 2-50
amino acids preferably 4 to 30. The polypeptide prefcrably comprises less than
17 amino
acids. In a preferred embodiment, the polypeptide comprises 13 amino acids.
The precise nature of the polypeptide protein interaction in the present
invention may be
elucidated by detailed structural analysis. It is possible that the
polypeptides of the present
invention may interact with themselves via aggregation or with other proteins.
The
binding and dissociation may in many, but not all cases, be governed by
aggregation
phenomena. Indeed the polypeptides in the invention may influence aggregation
properties
of other proteins including ~ amyloid polypeptides.
The term "not directly derived" from a protein binding protein means that the
sequence
identity between polypeptide and any stretch, especially a contagious stretch,
of sequence
taken from said protein is under 90%. The term "polypeptide" is used herein in
a broad
sense to indicate that a particular molecule comprises a plurality of amino
acids joined
together by peptide bonds. It therefore includes within its scope substances,
which may
sometimes be referred to in the literature as peptides, polypeptides or
proteins.
The polypeptide may be further truncated below 13 amino acids.
Preferably, the amino acid sequence of the protein binding polypeptide
includes at least
two of non polar amino acids which are separated by n polar amino acids where
n is 0 or 1
and the said sequence has the ability to bind to at least two or more
unrelated other proteins
and where the full length of the sequence bears less than 90% identity with
any stretch of
sequence present in the said protein
Preferably, the protein binding polypeptides of the present invention include
at least one
Gln residue adjacent to at least one non-polar residue.
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The protein binding polypeptides of the present invention may be non-branching
and are
capable of binding to a greater range of polypeptides Such as Iectins, vitamin
binding
proteins and immunoglobulin binding proteins (including Protein A itself).
The polypeptide may bind to sites on a protein which do not compete with
natural protein
active sites. For example, the polypeptide preferably binds an antibody at a
site or sites not
competing-with the antigen binding site of the antibody.
The polypeptide of the invention has several advantages over the prior art
protein-binding
protein-molecules such as Proteins A and G.
The polypeptide of the present invention is distinguished from the
polypeptides described
in J. Neuroimmunol (1993) supra. Unlike the peptides of the present invention,
these
peptides were membrane bound polypeptides binding specifically to IgG at hinge
region
only and failed to show binding to Fc or Fab fragments. There are further
distinctions that
the amyloid peptides required specific hydropathic profile, were longer in
nature and
showed reluctance to reversing the binding in the presence of strong
denaturing conditions.
Due to its smaller size, the polypeptide of the invention has a higher
capacity than
conventional protein binding proteins that is to say it binds more protein per
unit weight of
polypeptide. The polypeptide of the present invention has been found to bind
strongly to
enzyme labelled rabbit and goat IgG antibody, and native antibodies from a
range of animal
species (goat, human, dog, cat, horse), but not to the common enzymes used in
labels in
such immunological procedures.
The use of immobilised proteins to purify other proteins by affinity
chromatography is well
known in the art. Affinity chromatography, which is based upon the ability of
molecules in
solution to bind specifically to immobilised Iigands or receptors on solid
phase is simple in
concept. It is performed in a column containing a ligand derivatised matrix,
molecules to
be separated from crude preparations binding specifically and tightly to the
matrix, whereas
most of the contaminants, which lack specific binding sites, are washed away.
The
specifically absorbed molecules are then eluted with desorbing agents and
collected.
Affinity chromatography has become an important method for the purification of
molecules
for use as research probes, diagnostic tools and therapeutic agents.
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In the case of relatively non-specific Iigands (eg dyes and other short
polypeptides), the
binding of proteins is typically not very efficient, serving only to resolve
some of the
desired proteins into "bands" or separated fractions eluting from separation
media. In
contrast, the polypeptide ligands according to this invention typically allow
the
contaminants to be washed away whilst retaining the desired proteins on or
within the
separation media.
The short length of the polypeptide sequence means that the polypeptide is
more stable in
the harsh conditions used in affinity chromatography columns and also has
improved
storage properties. It is an important aspect of this invention that such
harsh conditions can
be avoided which is of particular advantage to the proteins being separated.
Typically
bound proteins can be eluted from the polypeptides of the invention using
reagents such as
dilute acid solutions or by using buffers or electrolytes of higher ionic
strength than used
to bind the polypeptides. In one example, antibodies bound onto one
polypeptide in
accordance with the invention in weak buffer (eg 10 mM Tris HCl pH 7) can be
eluted in a
stronger buffer. It will be apparent to those skilled in the art that loading
and elution buffers
of this type can be designed for particular proteins and polypcptide
combinations according
to this invention.
The polypeptides of this invention may be made by recombinant DNA methods.
Alternatively, the polypeptides of the invention may be made synthetically.
This reduces
the risk of pyrogenic substances, typically from the cell envelope of
bacteria, contaminating
a particular product when the polypeptides of the invention are used with
affinity columns.
Conventional protein binding proteins Protein A and Protein G are derived from
the cell
envelope of pathogenic bacteria, and may comprise such pyrogenic substances as
contaminants. The pyrogenic substances produce fever in animals, which often
makes
traditional protein preparations unsuitable for medical use or requires
careful and extensive
production and quality analysis procedures.
The production of the short polypeptides of the invention by synthetic means
has the
additional advantage that the cost of producing them is significantly reduced
in comparison
to the conventional isolation of known protein binding polypeptides. This
results in a
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significant reduction in the cost of affinity chromatography columns including
the
polypeptides of the invention compared with conventional columns.
Production of the polypeptides of the invention synthetically has the further
advantage that
the structure of the polypeptide can be altered by known techniques to improve
its stability.
For example, some or all labile bonds of the polypeptide can be chemically
modified to
prevent at~k by proteolytic enzymes, thus making the polypeptide non-
biodegradable and
resistant to microbiological attack. This is very difficult to achieve with
native Protein A
or Protein G even through recombinant DNA technology.
The structure of the polypeptide may be modified to enhance its binding
properties. For
example, residues such as cysteine may be introduced into the sequence via
which residues
the polypeptide can be attached to an affinity chromatography matrix.
In a preferred embodiment, the polypeptide is capable of binding an antibody
or a protein
enzyme conjugate. Preferred protein-enzyme conjugates includes: Protein A -
horseradish
peroxidase (HRP), concanavalin A - HRP, and avidin - HRP. Such conjugates find
application in immunoassays and related immunological procedures.
A second aspect of the invention provides a polypeptide including the
following amino
acid sequence:
TRNGQVLQGAIKG
and functional equivalents thereof.
A third aspect of this invention provides a polypeptide including the
following amino acid
sequence:
GQVLQGAIKG
and functional equivalents thereof.
A fourth aspect of this invention provides a polypeptide including the
following amino acid
sequence:
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DMHDFFVGLM
and functional equivalents thereof.
A fifth aspect of this invention provides a polypeptide including the
following amino acid
sequence:
APVGTDKELSDLLDF
and functional equivalents thereof.
A sixth aspect of the invention provides a polypeptide including the following
amino acid
sequence:
SRAQILQAAG
and functional equivalents thereof.
A seventh aspect of the invention provides a polypeptide including the
following amino
acid sequence:
KIGQFLIQFAGAFLSILQGLT'L,RAAEKQAG
and functions equivalents thereof.
Functional equivalents include polypeptides comprising additions, deletions,
and/or
substitutions to the above sequence having the same or similar protein binding
abilities as
the above polypeptide. The determination of functional equivalents of the
above sequence
is within the scope of the skilled worker. For example a polypeptide having
the sequence
AIKG derived from the polypeptides of the second and third aspects of the
invention binds
protein.
Functional equivalents of the polypeptide of the invention may have improved
protein
binding abilities. One or more amino acids of the polypeptide may be replaced
by an
amino acid having similar properties.
Amino acids having similar properties include
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Amino acids having aliphatic side chains: gly, ala, val, leu, ile, pro.
Aliphatic hydroxyl amino acids: ser, thr.
Aromatic amino acids: phe, tyr, trp
Basic amino acids: lys, arg, his, asn, gln.
Acidic amino acids: asp, glu.
Sulphur containing amino acids: cys, met.
Non-polar amino acids are amino acids not including acidic and basic amino
acids.
The polypeptide may also contain synthetic amino acids such as aminoadipic
acid,
aminobutyric acid, desmosine, sarcosine, norvaline, norleucine and ornithine,
~
-alanine,homocysteine,citrulline,cyclohexylalanine,chlorophenylalanine,cystine,
dehydrproli
ne,homocitrulline,homoserine,hydroxyproline, ~ hydroxyvaline, penicillamine,
statine.
Preferably the polypepdde binds to a site or sites on a protein which do not
compete with
the natural protein active sites, that is to say the normal function of the
active site is not
substantially affected. Where the protein is an antibody, the polypeptide may
bind to the
antibody at a site or sites which do not compete with a normal antigen binding
site of the
antibody.
In a preferred embodiment, polymers of a polypeptide according to the
invention may be
prepared. These have the advantage that several protein binding sites may be
provided on
the same molecule at the same time. The polymer may be a homopolymer or a
copolymer
of a polypeptide in accordance with the invention together with another
suitable
polypeptide.
In a preferred embodiment, a polypeptide of the invention is used to modify
another
protein, by incorporation of the sequence of the polypeptide into the amino
acid sequence
of the protein, so that that protein can bind to other proteins. Proteins
which may have the
polypeptide of the invention added or substituted to their amino acid sequence
include
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Protein A and Protein G. This improves the protein's ability to bind
antibodies and other
proteins, and increases its binding affinity to other molecules.
Alternatively, the
polypeptide sequence of a polypepdde in accordance with the invention may be
added to
other proteins to enable novel protein conjugates to be made.
The polypeptides of the invention may be used as affinity chromatography
agents.
According the polypeptides of the invention may be conjugated to one or more
solid
materials suitable for use in affinity chromatography such as; acrylic
polymers cross-linked
dextran, silica, glass, agarose, methacrylamide-methylbisacrylamide,
cellulose, vinyl
polymers, polyacrylamide or combinations thereof. The polypeptides may be
covalently or
non-covalently attached to such substrates by any means known in the art.
For instance the sequence GQVLQGAIKG can be assembled on solid phase removing
a
portion for testing after each amino acid. In this example a Lys residue is
incorporated with
temporary side chain protection such as Fmoc which can be removed (prior to
testing) with
20% piperidine in DMF without peptide cleavage from the resin In this way the
inventors
were able to scan the whole of GQVLQGA1KG polypeptide of the invention and
found
that relative to control experiment even the short sequcnces for instance AIKG
are able to
bind proteins.
Preferably the polypeptides of the invention are used to purify antibodies, or
similar
immunoglobulins, such as T cell receptor, lectins, streptavidin, avidin, or
fragments or
derivatives thereof, their ligands, ligands, substrates, antigens or other
analytes.
Antibody binding proteins, such as protein A and G, are also widely used in
the
implementation of diagnostics tests or assays, including those assays, tests
and monitoring
methods using biosensor devices (eg surface plasmon resonance, surface
acoustic wave, or
other such microelectronic, optoelectronic devices). It will be apparent to
those skilled in
the art that the polypeptides of the invention may similarly be used, where
their unique
ability to bind a broad range of commonly used diagnostic molecules very close
to the
active surface of such sensor devices is particularly advantageous. The small
size of the
polypeptide of the invention enables further improvements to be made in such
assays, tests
and monitoring methods, particularly those where the distance between reacting
components needs to be short, which methods are typically called proximity
assays.
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Another typical example would be the attachment of a fluorescent dye (e.g.
dansyl) to the
polypeptide such that the binding of the peptide-dye complex to the protein
changes the
fluorescence measured. Alternatively, the natural (e.g. tryptophan)
fluorescence of the
protein may be used or fluorescence may be introduced into the polypeptide. In
both cases,
it will be apparent to those skilled in the art that the fluorescence of the
polypeptide or
protein may be coupled in such a way as to change the nature of the
fluorescence (its
intensity, wavelength of emission or excitation, or the time scale or
polarisation of the
fluorescence). In the particular case of coupling, the fluorescence between
two or more
molecules, it is of particular advantage that the polypeptide enables the
distance between
the two molecules to be much shorter than would be possible using a protein
such as
Protein A or G. While the polypeptide may not bind to the functional site of
proteins, its
binding to the protein can present a molecule, such as a fluorophore,
sufficiently close to
the functional site that the fluorophore responds to binding processes
occurring at the
functional site. For example, the well known processes of fluorescence
quenching or
resonance energy transfer may be used. It will be similarly apparent to those
skilled in the
art that molecules or other materials may be attached to the polypeptide so as
to interfere
with the normal operation of the functional site of the protein.
In some cases, it can be advantageous to bind the polypeptide covalently to
its site of
binding on the protein molecule by procedures well established in the art,
which typically
involve the use of heterobifunctional cross-linking agents, whose reaction to
couple the
polypeptide to the protein may include photochemical methods.
Similar methods used in separation and diagnostic tests and assays may be used
in the
context of microbiological, animal cell or viral diagnostics tests, assay and
monitoring
procedures. Some of these cells can possess the proteins to which the
polypepddes of the
invention bind, or the proteins can be introduced to bind to the micro-
organisms, cells or
viruses, by methods well known to those skilled in the field.
In a similar fashion, the polypeptides of the invention can be used to treat
micro-organisms,
cells or viruses by attaching a bioactive agent, drug or their carriers to the
polypeptide by
well established methods.
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The polypeptides of the invention can also be introduced onto the surface of
larger drug
molecules, such as those produced by biotechnology processes, commonly known
as
biopharmaceuticals, cells, micro-organisms, viruses, macromolecules, polymers
or other
particles or materials, such as medical implants, that are introduced into
biological samples
or the body of an animal. A particular problem in these procedures is that the
molecule,
cell, particle or material so introduced is often treated as foreign by the
animal body, such
that various processes (e.g. immune responses) result in unfavourable
reactions in the body.
One of the early phases of these unfavourable responses is the attachment of
proteins, such
as antibodies, which label or opsonise the foreign matter introduced, which
provokes the
unfavourable response. The binding of proteins, such as immunoglobulins, by
the
polypeptides of the invention attached to such foreign matter by the processes
described in
this invention, notably not involving the functional site of the oposonising
protein (e.g.
antibody), may prevent or minimise the unfavourable biological response. The
foreign
matter bearing the polypeptides bind proteins present in the host which are
not recognised
as foreign by the host, and furthermore may bind them in such a way that they
do not
present their normal labelling or opsonisation function.
As the polypeptide of the invention is able to bind to more than one protein
at once it is
possible to target one protein to another via interaction with the
polypeptide. Similarly an
antibody bound to the polypeptide of the invention may be targeted to specific
site and
another protein could then be targeted to the same site , and vice versa, by
interaction with
the polypeptide.
The binding of the polypeptide to proteins may influence the specific
functional properties
of those proteins and this can be exploited to control the function of
protein. In one
embodiment the aggregation of Alzheimer polypeptides may be controlled by
binding to
polypeptides of the present invention leading to treatment by minimising
fibril formation.
It is known that some small synthetic molecules (e.g. dyes) do bind to protein
molecules.
These are distinguished from the polypeptides of the invention by their
binding to a much
wider or different range of proteins (e.g. including enzymes) and their
binding at both
functional and other sites on the protein. In principle, short polypeptides
may also be
designed to bind to proteins in the manner exemplified by the above dyes, for
example by
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presenting the basic, acidic or hydrophobic properties of the amino acid side
chains, which
would be expected to bind to many such sites on many protein molecules.
Similarly
polypeptides can be designed to bind to particular motifs on a protein.
However, it will be
clear to those skilled in the art that the polypeptides according to this
invention
demonstrate markedly different and unexpected properties. Indeed, the
polypeptides
according to this invention may indicate the presence on ligand binding and
receptor
proteins of a previously unknown common or similar site or structural motif,
which is
substantially absent at least from one other major class of proteins with
catalytic functions -
the enzymes.
Brief Description of the Drawings
The polypeptides in accordance with the invention and their production will
now be
described by way of example only, with reference to the accompanying drawings
Figures 1
to 11 in which:
Fig. 1 is an HPLC trace of purified peptide TRNGQVLQGAIKG;
Fig. 2 shows antibody binding to lOmer peptide GQVLQGAIKG and its Ala scan
derivatives;
Fig. 3 is binding and elution profile from peptide affl-prep-10 column.
Fig. 4 is a fluorescence spectrum recorded by excitation at 490nm showing
quenching of
FITC fluorescence by anti-FITC in the presence and absence of the 10 mer
peptide;
Fig. S shows binding of HRP,GARP,Fc fragment and Fab fragment to lOmer peptide
GQVLQGAIKG. Control experiments in which no peptide was present are marked by
m
Fig.6 Shows binding of goat anti rabbit peroxidase to 11 mer peptide
(NDNGVDGETW~
derived from natural antibody binding protein (Proc. Natl. Acad. Sci.USA 1992,
89,
8532-8536) compared to peptide GQVL.QGAIKG. Control experiments in which no
peptide was present are marked by m.
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Fig. 7 shows binding of peroxidase conjugated antibody and fragments Fab and
Fc to
peptide GQVLQGAIKG which has been immobilised on affinity matrix affi-prepl0.
Control experiments in which no peptide was present are marked by 4.
Fig 8. Shows association and dissociation of different concentrations of Goat
IgG with
BSA-peptide conjugate immobilised on CM 5 chip. The curves from top to bottom
are for
IgG concen'~'ations 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05 and 0.025 p.M
respectively.
Fig 9. Shows association and dissociation of different concentrations of Goat
IgG with
multimeric 10 mer peptide immobilised on CM 5 chip. The curves from top to
bottom are
for IgG concentrations 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125 and
0.00625 ~t.M
respectively.
Fig. 10. Shows binding of goat antirabbit peroxidase (GARP) to a 30 mer
peptide
(KIGQFLIQFAGAFLSILQGLTLRAAEKQAG) and also a conjugate of 10 mer peptide
(GWVLQGAIKG) with BSA measured by FLISA.
Fig. 11. Shows binding of GARP to a peptide (SRAQILQQAG) sequence taken from
Flagella protein (J. Mol. Biol 1991 219: 471-480) and the same figure also
shows that the
protein itself does not bind.
Fig. 12 is a chromatographic profile indicating binding and elution of Goat
IgG from
multimeric peptide (GWVLQGAIKG) column.
The preparation of polypeptides in accordance with this invention is now
described by way
of example only.
Example 1: Preparation, purification and characterisation of polypeptides.
Many methods are known for synthesising polypeptides by solution phase and
solid phase
chemistries. The polypeptide can be readily prepared by solid-phase synthesis
as follows
using well established protocols. For instance we used Boc chemistry developed
by
Merrifield to synthesise a polypeptide having the sequence TRNGQVLQGAIKG.
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MBHA resin (O.SmMoles) was used. The side chain protecting group for Lys was 2-
C1Z.
Each synthetic cycle consisted of (i) a 2min and 25min deprotection with 50%
TFA/DCM
(ii) neutralisation with 5% DIPEA/DCM and (iii) coupling with I.SmMoles amino
acid,
I.SmMoles BOP and 4.Smmoles DIPEA in DMF for 40 rains. A second coupling was
used when necessary to drive the reaction to almost completion (>99.8% yield).
At the end
of synthesis the polypeptide was cleaved with HF by known procedure. Typically
the
polypeptide resin was treated with 20m1 HF, O.Sg thiocresol and 0.75g p-cresol
and after
evaporation of HF, extraction was carried out with 50% acetic acid/water. The
polypeptide
was purified on C-8 reverse phase Vydac semi-prep column using linear gradient
of 20%
acetonitrile/0.1 % TFA to 80% acetonitrile/0.136 TFA over 45 rains. The
product peak was
lyophilised and analysed by HPLC.
Other sequences accrding to the invention could be similarly produced. The
side chain
protected amino acids used in other sequences were Boc-Arg(Tos)-OH,
Boc-Asp(OcHx)-OH, Boc-Glu(OBzI)-OH, Boc-Lys(2-CL-Z)-OH, Boc-Lys(Fmoc)-OH,
Boc-Ser(BzL)-OH and Boc-Thr(Bzl)-OH.
Biotin could be coupled in identical manner to amino acids using BOP
activation as
described above. For multimeric peptide the first residue to couple was Fmoc-
Lysine
(Fmoc)-OH. The Fmoc groups were removed using 20% Piperidine in DMF. Repeating
this procedure again yielded the lysine core for extending four peptide chains
in the usual
manner.
Figure 1 shows an HPLC trace of the purified polypeptide : TRNGQVLQGAIKG 25
~cg
polypeptide applied to a C-18 Vydac column running gradient of 0.1 % TFA to
80%
acetonitrile/0.1 %TFA in 30 rains. Detection wavelength was 218nm.
Techniques for synthesising polypeptides with different sequences and similar
properties
are well known. For instance new sequences may be discovered by the common
method of
constructing polypeptide libraries. Similarly existing sequences can be
chemically modified
by removing, adding or replacing or substituting amino acids or analogues
which are not
required for activity. Sections of sequences may be combined from different
polypeptides
to make a new polypeptide. In one typical example of the method, the sequence
TRNGQVLQGAIKG was reduced to 10 residues to give the sequence GQVLQGAIKG and
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this was further modified by substituting each amino acid at a time, with
another residue
(Ala) generating several sequences (termed "Ala scan" polypeptides) as below:
( 1 ) AQVLQGAIKG
(2) GAVLQGAIKG
(3) GQ~QGAIKG
(4) GQVAQGAIKG
(5) GQVLAGAIKG
(6) GQVLQAAIKG
(7) GQVLQGAIKG
(8) GQVLQGAAKG
(9) GQVLQGAIAG
( 10) GQVLQGAIKA
In Fig. 2, polypeptides (1) - (10) with Ala residue at positions 1-10 were
screened for their
binding to GAItP (Goat anti-rabbit peroxidase) as described in Example 3. The
absorbance
reflects the binding of each analogue. The polypeptides produced by this
substitution
technique of Fig. 2 show that the Ala scan polypeptide sequences 1 to 8 each
bound the
protein to a different level.
Using amino acids other than Ala, thousands of analogues can be made. In this
way
information can be gained regarding the significance of each residues leading
to discovery
of new polypeptides.
Example 2. Screening of proteins binding to a polypeptide
There are many techniques known for measuring the binding of proteins to
molecules
including polypeptides either in solution or by attaching to solid surfaces.
We typically
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employed a 96 microwell ELISA plate which was coated, in replicates of eight,
overnight
with 100 ~Cl of 20 ~tg/ml of polypeptide dissolved in buffer such as SOmM
sodium
carbonate pH 9.6. The coated plats then went through series of steps:
( 1 ) Wash- times) with Phosphate buffered saline pH 7.4 containing ~.1 %
Tween
(PBS-T),
(2) Block the plate by incubating for 90 rains with 100 ~Cl per well of PBS-T
and wash
three times with the same.
(3) Incubation for 60 rains with 100 ~1 of binding protein solution. The
protein may be a
labelled protein such as antibody or avidin or any other labelled with HRP or
other
enzymes or reported groups. The concentration of protein solution will depend
on the
amount of label.
(4) Wash three times with PBS-T and monitor response by a technique depending
on the
label attached. For instance when Horse Radish Peroxides (HRP) is the label
the wells
could be incubated with a substrate such as 5-amino salicylic acid dissolved
in SOmM
Sodium phosphate pH 6 buffer containing 0.01% (W) of fresh hydrogen peroxide.
The
response may then be measured colourimetrically after short incubation (e.g.
30 mina)
by a reader or visually. Fig 5 shows binding of immunoglobulins and two
fragments
where the enzyme peroxidase is used as label.
In an alternative format, the label could be attached or bound secondary to
the protein (step
2 above). For instance when rabbit IgG is used, the procedure following
coating of
polypeptide would then comprise:
(1) Wash (3 times) with Phosphate buffered saline pH 7.4 containing 0.1% Tween
(PBS-T)
(2) Block the plats by incubating for 90 rains with 100 ,ul per well of PBS-T
and wash
three times with the same.
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(3) Incubation for 60 mins with 1001 of binding protein solution prepared in
PBS-T.
(4) Wash three times with PBS-Tween and incubate with GAItP
(5) Wash three times with PBS-T and monitor response with a substrate such as
5-aminosalicylic acid dissolved in SOmM Sodium phosphate pH 6 buffer
containing
0.01°Jb-('VlV) of fresh hydrogen peroxide.
There are numerous variations which are well known in the art of solid phase
assays and
which can easily be made to these protocols.
For instance for indirect Peptide-Antibody Binding Assay the following
procedure was
adopted. Plates were coated by incubation for 1 hr at 37°C with 100 pU
well of IgG
solution at 20pg/ ml in carbonate buffer, pH 9.6 (15 mM Na2C03; 35 mM NaHC03);
plates were washed three times with tris-buffered saline (25 mM, pH 7.4)
containing 0.1 %
Tween-20 (TBS-T) ; 100 ItU well of TBS-T was used to block the uncoated well
surface by
incubation for 3 hr at 37°C; plates were washed as before; 100 pl of
biotinylated peptide at
20 pg/ml in TBS-T containing 0.2 % DMSO was placed into each well followed by
incubation at 37°C for 1 hr; excess and unbound biotinylated peptide
was washed;
IgG-bound biotinylated-peptide in each well was detected by incubation with
100 pl of
ExtrAvidin-peroxidase conjugate in TBS-T (1:1000 dilution as supplied and
recommended
by manufacturer) for 1 hr at 37°C; plates were washed as before to
remove excess and
unbound conjugate; bound conjugate was then detected by incubation with 5-
amino
salicylic acid and optical readings at 450 nm were determined as described
previously.
Biotinylated GQVLQGAIKG showed significant binding to polyclonal IgG from
various
sources and some proteins relative to the appropriate controls (Table 1 and 2
). In this
assay avidin exhibited some non-specific binding to the proteins.
Nevertheless, signal
from peptide-protein interaction was apparent.
Accordingly biotinylated GQVLQGAIKG is an example of a polypeptide in
accordance
with the invention which binds more than one protein.
Example 3. Screening of polypeptides binding to a typical prntein
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In this example Goat antirabbit peroxidase is used as typical protein.
Different poiypeptides were coated in microwells as above and following steps
performed.
( 1 ) Wash (3 times) with Phosphate buffered saline pH 7.4 containing 0.1 %
Tween
(PBS-T)
(2) Block the plate by incubating for 90 rains with 100 ,ul per well of PBS-T
and wash
three times with the same.
(3) Incubation for 60 rains with 100 ~cl of CARP.
(4) Wash three times with PBS-T and monitor response by incubating with a
substrate
such as S-aminosalicylic acid dissolved in SOmM Sodium phosphate pH 6 buffer
containing 0.01 % (V/V) of fresh hydrogen peroxide. The response may then be
measured colourimetrically after short incubation {e.g 30 rains) by a reader
or visually.
In one typical example the sequence GQVLQGAIKG was compared to 11 mer sequence
derived from Protein G {Proc. Natl. Acad. Sci.USA 1992, 89, 8532-8536) .
Figure 6 shows
that the peptide derived from natural protein is unable to bind IgG in the
same manner as
our sequence.
Fig. 10 shows the binding of GARP to a 30mer peptide of sequence
KIGQFLIQFAGAFLSIC,QGLTLRAAEKQAG. In these cases an improved response is
apparent due to the longer peptide than a shorter peptide as adsorption and or
binding may
be improved. A 10 mer peptide of sequence GQVLQGAIKG immobilised on BSA
protein
as in Example 9 also shows comparable binding to the 30 mer peptide (Fig. 10).
Using a similar screening method we coated the plates with Flagellin protein
(sequence
published J.MoI..Biol 1991,219,471-480) and found that it was unable to bind
to GARP.
Based on the possible binding motifs in our sequence we were able to
synthesise the
peptide SRAQIL,QQAG and show that this binds to GARP. It is thus possible to
derive
useful protein binding polypeptide sequences which show different binding
properties to
their natural full protein sequence (Fig 11).
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Example 4: Screening of proteins binding to polypeptides directly on resin
Short polypeptides do not usually adsorb efficiently to microwells. There are
several ways
such polypeptides can be screened for their binding ability. One of the ways
is to
immobilise the polypeptide covalently to solid surface. In this regard, ELISA
plates with
derivatised-surfaces are commercially available for linking molecules to
surfaces. Such
methods can easily be applied to bind shorter sequences and then screen in the
usual
manner described above. In an alternative way, the sequences can be
synthesised on the
solid phase by well known techniques and portions of the resin removed at
various stages
of the synthesis . The resin can then be used instead of the microwell as a
support for the
protein binding polypeptide of the invention. The washing steps analogous to
the ELISA
method reported above can be carried out by mixing the resin with desired
solution and
separation effected by bench top microcentrifuge. In this way the inventors
were able to
scan the whole of TRNGQVL.QGAIKG polypeptide of the invention. and found that
even
short sequences, for instance AIKG, are able to bind proteins. In a typical
example the
polypeptide (GQVLQGAIKG) was assembled on acid resistant resin but using Fmoc
amino acids instead of the Boc used in example 1. A small amount of resin
(lOmg) was
removed after assembly of each amino acid and treated with 95% TFA/ 59'o water
mixture
to cleave side chains. The resin was washed with dichloromethane and methanol
and dried.
Next the resin was incubated in lml solution of PBS-T to block non specific
sites. The
ELISA steps as described in examples 2,3 and 4 could be performed on the resin
using lml
solution volumes followed by centrifugation to recover the resin after each
wash step. At
the last stage after adding the substrate and allowing reaction to take place
the response
was measured by recording absorbance reading of the supernatant.
Using this method the binding of sequences as short as 4 residues, for
instance AIKG
linked to resin, to proteins (e.g. GAItP) can be shown.
Example 5: Preparation of polypeptide affinity column
There are several ways and chemistries available to prepare affinity columns
with a wide
range of matrices. (c.f Immobilised affinity ligand techniques (Academic Press
1992) or
Bioaffinity Chromatography (Elsevier Science Publications 1993)). In a typical
example
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the inventors used a commercially available preactivated Affi-prep column and
immobilised the polypeptide of the invention in a high performance stainless
steel column.
A l amer polypeptide was synthesised with a 6 carbon spacer, aminohexanoic
acid, and was
immobilised in dilute buffer at pH7.8 at polypeptide concentration of lOmglml.
The
coupling was allowed to proceed overnight by recirculating the polypeptide
solution
through the column. The next day any remaining activated groups were treated
with O.1M
ethanolam~e solution.
In one example Proteins-HRP conjugates binding to Affiprep-10 immobilised
peptide
were measured by incubation of small amount of matrix (Preblocked to minimise
non
specific binding) in PBS-T buffer and washing off unbound material by
centrifugation.
The bound conjugates were then estimated by incubation with 5- aminosalicylic
acid as
described for ELISA measurement in example 2 except that the reaction mixture
was
centrifuged at low speed and supernatant used for recording the optical
density. Affi-prep
matrix blocked with ethanolamine was used as a control matrix. Figure 7 shows
that the
immobilised sequence is able to bind proteins.
immobilisation of multimeric GQVLQGAIKG peptide on amino sepharose matrix
(AH-Sepharose 4B Pharmacia) was done as follows. lg of AH-Sepharose in 4ml of
PBS
was treated with O.SmI of 8°~o Glutaraldehyde solution. for 30 rains.
Excess reagent was
removed by washing the resin with distilled water on a sintered funnel. A 2
fold molar
excess of multimeric peptide in SOmM bicarobnate buffer pH 9.6 containing 10%
DMSO
was coupled to this activated matrix for 3hrs. Unbound peptide was washed by
filtration
with buffer followed by 10°lo acetic acid followed by water and
ethanol. The matrix was
resuspended in 25m1 buffer and few crystals of sodium borohydride added. The
washings
used to remove unbound peptides were repeated. The coupling of peptide was
qualitatively
measured using a ninhydrin test. The matrix was packed into a short column
(0.8cm X
lOcm) and equilibrated with binding buffer.
Example b: Separation and/or screening of proteins using polypeptide affinity
column
The column produced in Example 6 was attached to the HPIrC system and proteins
were
detected by using UV detector fixed at 280nm wavelength. Typically protein
(0.2 to 0.5 to
mg) was loaded onto the polypeptide column, and equilibrated with suitable
buffer such as
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IOmM Tris-HCL pH 8, by rheodyne injector at a flow rate of 0.2m1/min. The
effluent was
continuously monitored at wavelength of 280nm and an elution profile obtained.
The
bound protein was then eluted by applying elution buffer such as 3M Guanidine
hydrochloride or 0.1% TFA. The binding and elution profile can be seen in Fig.
3 which
represents the binding and elution profile from affinity column when HRP
enzyme (trace
A) and goat anti-rabbit IgG antibody (trace B) were applied. The binding was
achieved in
IOmM tris-HCL pH7.4 buffer also used to equilibrate the column while the
elution was
made possible with the 0.1% TFA~ solution. In this example, the HItP is not
appreciably
bound and is thus eluted almost in the void. In contrast, the IgG is bound and
eluted when
0.1 % TFA is applied
The multimeric peptide column prepared in example 5 was equilibrated with 6m1
of
Phosphate buffered saline (PBS) at pH 7.4. A 0.2mg amount of Goat IgG(Sigma)
in O.Sm1
PBS was applied to the column at a flow rate of 0.4mUmin. After loading the
sample the
binding buffer was applied to wash off unbound protein and absorbance measured
continually at 280nm. We then applied 3m1 solution of 3M Guanidine
hydrochloride to
elute off the bound protein. Figure 12 shows the chromatographic profile
indicating
binding and elution of typical protein from this column.
Example 7 : Screening different compounds for binding to polypeptides.
Using the methodologies of F..LISA and affinity chromatography the inventors
screened
several antibodies from different sources and the data is tabulated below to
indicate binding
to typical polypeptide sequence. The proteins marked showed either no binding
or
insignificant levels of binding.
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Table 1.
Antibody Binding Determination by
Goat IgG Yes ELISA & Column
Human IgG Yes ELISA dt Column
Rat IgG Yes ELISA 8c Column
Mouse IgG weak ELISA
Human IgA Yes
Donkey IgG Yes
Guinea pig weak ELISA
~g Yes Column
Sheep Yes Column
Horse Yes Column
Pig Yes Column
Cw Yes Column
The results depicted in Table I indicate that almost all sources of antibody
showed binding
and elution to varying degrees.
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Similarly other compounds can be screened and some of the ones screened are
tabulated
below.
Table 2.
Protein Binding Determination
by
Alkaline phosphateNo ELISA & Column
HLtP No F.LISA & Column
BSA No Column
Galactosidase No Column
Human transferrinYes F.LISA
Strepatvidin/avidinYes ELISA
Protein A Yes ~A
Protein G Yes Rl~l~p
Concanavalin Yes Rl~ltp
Chymotrypsin Weak Column
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Example 8. Binding site on IgG.
In an experiment to demonstrate that the polypeptide of the invention does not
bind directly
at the normal binding site of the protein we used anti-F1TC antibody. This
antibody is
known to quench >95% fluorescence of fluorescein upon specific binding at the
antigen
binding site. This quenching assay was performed in 2tn1 of IOmM Tris-HCL pH
?.4 buffer
containing 3:1 of 10 ~tg/ml fluorescein solution in the presence and absence
of antibody. As
shown in Fig. 4 5:1 of antibody was able to quench most of the fluorescence
upon binding.
The same level of binding was observed in the presence of 10:g of poIypeptide
(GQVLQGAIKG). The data indicates that the binding of the polypeptide does not
influence the normal functioning of protein to appreciable extent. However it
can not be
ruled out whether the polypeptide of the invention binds close to the
functional site or
whether the non immobilised polypeptide behaves differently than that free in
solution.
Example 9 Measurement of binding by optical biosensor
The peptide sequence GQVL.QGAIKG was immobilised on BSA using the
glutaraldehyde
method as follows. Bovine Serum albumin (4mg) was dissolved in 0.75m1 of IOmM
Sodium Phosphate pH 7.4 buffer. Glutaraldehyde(0.25m1 of 8% solution) was
added and
mixture stirred for 30mins at room temperature. The Excess Glutaradehyde was
removed
by Gel filtration on PD-10 column. The peptide (lOmg dissolved in minimum
volume of
DMSO) was added to the activated BSA and conjugation allowed to proceed for 3
hrs.
Unconjugated Peptide was removed by dialysis, centrifugation and further Gel
filtration.
The conjugate was immobilised on CMS chip ,using EDC coupling, by flowing
across the
sensor chip according to the manufacturers description (BIAcore). BSA was used
in the
control flow cell. The peptide protein interaction was studies using different
concentrations of proteins in order to obtain optimum conditions for measuring
binding
constants. In a typical example the antibody was bound in Phosphate buffered
saline and
regeneration effected with 3M Guanidine hydrochloride solution. The binding
affinities
(kD) were determined. Figure 8 and 9 shows the association and dissociation
progress
curves. The multimeric peptide could be immobilised in identical manner and
binding
evaluated. The kD values obtained were estimated to be 4 x 10''M and 1 x 10-'
M
respectively for the multimeric and BSA conjugated 10 mer peptide.
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