Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR HIGH-THROUGHPUT SELECTION OF INTERACTING MOLECULES
The present invention relates to a method for the selection of at least one
member of a
number of specifically interacting molecules, said method being carried out in
(a)
container(s), preferably representing an arrayed form, e.g. in (a) microtiter
plate(s),
using an automated device comprising a magnetic particle processor. In another
embodiment, the present invention relates to a method for the production of a
pharmaceutical composition comprising the steps of the method of the present
invention and further the step of formulating at least one of said
specifically interacting
molecules selected and/or characterized by the above method or a functionally
and/or
structurally equivalent derivative thereof in a pharmaceutically acceptable
form.
Cellular functions are controlled by networked expression of gene catalogues.
Functional network analysis requires parallel handling of large numbers of
gene
products and selection and characterization of interacting molecules. For
studying
protein interactions, two classical library-based approaches are known, an in
vitro-
method, the so-called yeast Two-Hybrid-System (Mendelsohn & Brent, Science
284,
1999, 1948-1950) and in vitro methods, e.g. phage display. The yeast Two-
Hybrid-
System has the following disadvantages: (i) transformation efficiency in yeast
is low; (ii)
protein interactions take place in the milieu of the yeast nucleus and,
therefore,
interaction parameters can not be controlled; and (iii) only protein-protein
interactions
are possible to be investigated.
In vitro (e.g. phage surface) display enables the construction of large
recombinant
peptide and protein libraries for the selection of interacting molecules. The
basic
concept is a physical link of the phenotype, expressed as gene product (e.g.
displayed
on the phage surface) to its coding genetic information (e.g. integrated into
the phage
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genome). This allows to survey large libraries of organisms (e.g. phage,
viruses,
bacteria, eukaryotic cells) and/or organelles (e.g. ribosome) and/or soluble
molecules
(e.g. nucleic acids, protein-nucleic acid hybrids) for the presence of
specific molecules
using the discriminative power of affinity purification. The selection
procedure involves
the enrichment of a specific first molecule by binding to an immobilized
second (target)
molecule. First molecules are enriched by selection (binding and elution) on
the target
molecule. As a consequence of the physical linkage between genotype and
phenotype, sequencing the DNA of the encoding first molecule can readily
elucidate
the amino acid sequence of the selected gene product.
Peptide libraries were the first libraries to be displayed on phage (Smith,
1985). In the
meantime, a wide variety of different peptide libraries were made, with
different
degrees of randomness and special means of recombination (e.g., Fisch et al.,
1996).
Peptide libraries are especially useful for mapping interacting parts of
proteins (e.g.,
domains or epitopes). They are also a first step towards the production of
small
molecules simulating protein actions.
Recombinant immunoglobulin gene libraries cloned in phage or phagemid vectors
are
an in vitro simulation of antibody repertoires and allow the production of
antibodies
without immunisation and without the use of animals (reviewed in Winter et
al., 1994).
Human antibodies against large numbers of different antigens, including human
proteins, can be produced by phage selection of single-chain Fv (scFv; Nissim
et al.,
1994) or Fab fragments (Griffiths et al., 1994). Those antibodies should be
particularly
valuable as therapeutic agents because the patient's immune system will not
recognise them as foreign because they are completely human. Besides
antibodies,
also enzymes, enzyme inhibitors, receptors, hormones, lymphokines and DNA-
binding
molecules have been target molecules displayed on filamentous phage. The wide
range of possible applications clearly demonstrates the high potential of
linking
genotype and phenotype as a tool for the development of new molecules.
Although phage display was used extensively for the selection of peptides and
antibodies, it had its limitations when it came to the expression of unknown
sequences
from cDNA libraries. As many of these sequences contain stop codons in their
3'
untranslated regions, one cannot directly fuse these sequences to the N-
terminus of a
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phage coat protein. To overcome this problem, a specialised cloning and
expression
system has been developed that allows the display of functional cDNA
expression
products on the surface of filamentous bacteriophage (Crameri & Suter, 1993;
Crameri
& Blaser, 1995). This system exploits the high-affinity interaction of the Jun
and Fos
leucine zippers. Gene jun is expressed from a IacZ promoter as a fusion
protein with
the phage coat protein III. Using a second IacZ promoter of the phagemid
pJuFo, gene
fos is co-expressed as an N-terminal fusion peptide with the cDNA library gene
products, so that the resulting Fos-fusion proteins could become associated
with the
Jun-decorated phage particles. To avoid inter-phage exchange of fos-cDNA
fusion
products, cysteines were engineered at the N- and C-termini of each of the
leucine
zippers, providing a covalent link of the cDNA gene products to the genetic
instructions
required for their production.
The physical link between phenotype and genotype in phage display allows
selective
isolation and amplification of a particular phage encoding a desired gene
product from
pools of millions of phage (Kay et al., 1996). Selection is accomplished by
interaction
between the displayed gene product and a figand immobilized on a solid phase.
The
selected phage are amplified by infection of E. coli cells which, after helper
rescue,
produce large numbers of new phage. Successive rounds of phage selection and
amplification allow selective enrichment of phage displaying gene products
with affinity
for a desired ligand.
In the prior art, target molecules have been immobilized on plastic surtaces,
mainly
immunotubes or microtiter plate wells (Harrison et a1.,1996). Also, magnetic
particles
have been used for phage display selection (Hawkins et al., 1992, Griffiths et
al., 1994,
Low et al., 1996, Schier et al., 1996a; Schier et al., 1996b, McConnell et al.
1998,
McConnell et al., 1999, Kirkham et al., 1999). However, these methods involve
the
manual handling of the samples and are cumbersome and time-consuming.
Accordingly, these methods allow the simultaneous processing of only a very
limited
number of samples, i.e. they allow only the identification and
characterization of one or,
at most, a few pairs of interacting molecules per one selection procedure.
Moreover,
they are difficult to standardize in terms of precisely reproducible
conditions.
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Thus, the technical problem underlying the present invention was to provide a
method
that allows the reliable, simultaneous and time-saving high-throughput
selection of
various members of pairs of interacting molecules.
The solution to the above technical problem is achieved by providing the
embodiments
characterized in the claims.
Accordingly, the present invention relates to a method for the selection of at
least one
member of a number of specifically interacting molecules, said method
comprising as
the first step involving the contact of said interacting molecules:
(a) contacting a first molecule with a second molecule affixed to a magnetic
particle
under conditions that allow a specific interaction between said first and
second
molecule to occur;
and further the steps of:
(b) subjecting the product obtained in step (a) to at least one washing step;
(c) determining whether a specific interaction between said first and second
molecule
had occurred; and, if said specific interaction had occurred,
(d) providing said first and/or second molecule selected by steps (a) to (c),
wherein steps (a), (b) and (c) are carried out in (a) container(s), preferably
representing
an arrayed form, e.g. in (a) microtiter plate(s), using an automated device
comprising a
magnetic particle processor (Fig. 1 ).
The method of the present invention shows several unexpected advantages in
terms of
sensitivity, control and automation: First, the number of magnetic particles
can be
scaled down compared to the manual techniques (e.g. 10-fold to 2 NI or 1.34 x
106
Dynabeads M-280 Streptavidin, Dynal). This causes much less unspecific
background
binding resulting in a distinct reduction of false positive results.
Second, all washing and incubation conditions can be reproducibly customized.
Most
importantly, it is envisaged in accordance with the present invention that
washing
speeds are adjusted to cause different stringencies of selection. This will
enable the
predictable selection of interacting molecules with different binding
affinities. The
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washing step which may be repeated at least once in each round of selection is
designed to remove first molecules that did not specifically interact
withlbind to said
second molecules. Appropriate washing conditions can be taken from the
appended
examples or devised by the person skilled in the art without undue burden.
Third, it is envisaged in accordance with the present invention that the steps
of (i) the
provision, preferably the recombinant production of said second molecule (that
may be
a member of one library), (ii) affixing said second molecule to magnetic
particles, (iii)
optionally blocking of free binding sites on the magnetic particles (to which
no second
molecules had been affixed), (iv) contacting said first molecule (that may be
a member
of another library) with said second molecule, (v) washing steps, and (vi) the
determination whether a specific interaction between said first and second
molecule
had occurred, are carried out in (a) containers) preferably representing an
arrayed
form, e.g. in (a) microtiter plates) or containers) comprising tubes in an
arrayed form,
wherein each step is preferably performed in (a) different container(s). The
magnetic
particle processor comprised in said automated device is used to transfer the
magnetic
particles between wells of microtiter plates prefilled with the corresponding
solutions by
capturing the magnetic particles in a first well and releasing the same in a
second well
of a different microtiter plate, the position of said second well
corresponding to the
position of said first well. This allows high-throughput selection of
interacting molecules
as large numbers of, e.g., library clones can be handled in parallel, and the
selection of
interacting molecules from, e.g., two libraries can be used to create
interaction
catalogues.
In this regard it is to be understood that a "selection round" comprises steps
(a) to (c).
Accordingly, the phrase "first step involving the contact of said interacting
molecules"
denotes the contacting step of the first selection round as compared to
second, third,
etc. steps involving the contact of said interacting molecules of potential
further
selection rounds that may be performed subsequently to the first selection
round (see
below).
A preferred mode of the selection at high-throughput of the invention
comprises the
following steps: interacting molecules (e.g., anti-protein scFv antibodies)
are selected
from molecular libraries by a combination of phage display and magnetic bead
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technology. Proteins expressed from arrayed cDNA libraries are bound to
magnetic
beads via a suitable molecular tag (e.g., Hiss or Biotin). Phage displaying
specifically
interacting molecules are then fished from a library by binding to their
interaction
partners attached to the beads. This selection involves a sequence of binding
and
washing steps and was adapted to high throughput using a magnetic particle
processor (Labsystems, Helsinki, Finland). Selected molecules are then tested
for
specificity also employing the magnetic particle processor.
Said first and second molecule may be members of libraries, e.g., an antibody
and an
antigen library, respectively, i.e. two different libraries. Alternatively,
said first and
second molecule may be members of the same library. Also comprised by the
present
invention are embodiments, wherein one molecule (i.e. the first or the second
molecule) is a member of a library whereas the other molecule is a compound or
a
variety of compounds of predetermined specificity. Other options to employ
first and
second molecules from still different origins or combinations of origins are
within the
skills of the person skilled in the art.
In a preferred embodiment of the method of the present invention said first
and/or
second molecule is an organic molecule and/or a mixture of organic and/or
inorganic
molecules.
In another preferred embodiment, said first and/or second molecule is a
hapten.
In a more preferred embodiment of the method of the present invention said
first and/or
second molecule is a cDNA expression product, and/or a (poly)peptide, and/or a
nucleic acid, and/or a lipid, and/or a sugar, and/or a steroid, and/or a
hybrid of said
molecules.
In a most preferred embodiment said cDNA expression product is an antibody or
a
fragment or a derivative thereof, an enzyme or a fragment thereof, a surface
protein or
a fragment thereof, or a nucleic acid-binding protein or a fragment thereof.
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Derivatives and fragments of antibodies are well known in the art and
comprise, e.g.,
F(ab')2, Fab, Fv or single chain Fv antibody fragments (see, e.g., Harlow and
Lane,
"Antibodies, a laboratory manual", CHS Press, 1988, Cold Spring Harbor, N.Y.).
In another preferred embodiment of the method of the present invention said
first
molecule is a (poly)peptide presented on the surface of organisms (e.g. phage,
viruses, bacteria, eukaryotic cells) and/or organelles (e.g. ribosome) and/or
soluble
molecules (e.g. nucleic acids, protein-nucleic acid hybrids) and the method
further
comprises after step (b) and prior to step (c) the step of:
(b') amplifying a (poly)peptide specifically interacting with said second
molecule,
wherein step (b') is carried out in (a) containers) preferably representing an
arrayed
form, e.g. in (a) microtiter plate(s).
In one embodiment of the present invention relating to bacteriophage surface
display,
the magnetic particle processor may be used to transfer the magnetic particles
(to
which said bacteriophage is bound via said (poly)peptide specifically
interacting with
said second molecule) to bacterial culture(s). In another embodiment, the
automated
device further comprises a shaking device that may be used for shaking the
bacterial
cultures) during amplification of said bacteriophages.
It could be demonstrated in accordance with the present invention that after
binding of
the polypeptide presented on the surface of a bacteriophage to said second
(target)
molecule affixed to a magnetic particle, said bacteriophage retains its
infectivity.
Surprisingly, it is possible for the bacteriophage amplification step to
infect bacteria
directly after a specific interaction has occurred without the need of prior
detachment of
bound bacteriophage from the magnetic particle.
In a more preferred embodiment of the method of the present invention prior to
step (a)
said library of first molecules (library 1 ) is preabsorbed with unloaded
magnetic
particles and/or molecules competitive (cross-reactive) to second molecules
(target,
library 2).
For example, if a library of phages is used for performing the method of the
invention,
this step ensures that phages unspecifically interacting with the magnetic
particles are
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removed from the phage mixture and only bacteriophages displaying a
specifically
interacting molecule are selected via this specific interaction. In other
words, this step
may be performed to further reduce the number of false positive clones.
In an additional more preferred embodiment the method of the present invention
further comprises after step (c) and prior to step (d) the step of:
(c') repeating steps (a), (b) and (c) and, optionally, step (b') at least
once.
In a most preferred embodiment of the method of the present invention steps
(c) and
(c') are performed in parallel.
As mentioned above, steps (c) and (c') of the method of the present invention
are each
carried out in microtiter plates. This advantageously allows the simultaneous
performance of steps (c) and (c') in different microtiter plates which further
reduces the
time required for practicing the method of the present invention.
In a further preferred embodiment of the method of the present invention said
number
of specifically interacting molecules is a pair of interacting molecules.
In another preferred embodiment of the method of the present invention said
number
of specifically interacting molecules are three or more interacting molecules.
In yet a further preferred embodiment, the method of the present invention
further
comprises the step of characterizing said first and/or second molecule and/or
the
corresponding genetic information.
Methods for the characterization of genetic information, i.e. nucleic acids,
and
proteinaceous material are well known in the art and include, e.g., nucleic
acid
sequencing, southern-, northern-, and colony hybridization, primer extension
analysis,
RNase protection assay, gel shift analysis, western-blotting, ELISA,
immunoprecipitation assay, indirect immunofluorescence analysis, and FACS
(see,
e.g., Sambrook et al., "Molecular cloning - a laboratory manual", Cold Spring
Harbor
Laboratory (1989) N.Y., Ausubel et al., "Current protocols in molecular
biology", Green
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Publishing Associates and Wiley Interscience, N.Y. (1989), and Harlow and
Lane, loc.
cit. ).
In another preferred embodiment, said second molecule (target) is affixed to
said
magnetic particle via an affinity tag (e.g. a metal-chelating tag, an epitope
tag, an
enzyme binding domain, calmodulin, biotin, Strep-tag, protein A, protein G or
protein L)
(Fig. 2).
The use of one of the above-mentioned compounds advantageously ensures that
said
second molecule is affixed to said magnetic particle in a controlled manner
and in a
predictable orientation, thereby minimally affecting the three-dimensional
structure of
said second molecule and, consequently, the interacting capacities. Moreover,
the use
of the above-mentioned compounds allows the direct loading of magnetic
particles with
second molecules from protein mixtures like, e.g., crude extracts or cell
lysates. This is
particularly important for high-throughput selection since purification of
large numbers
of different second molecules is not necessary. However, although the use of
the
above-mentioned compounds is preferred, the present invention also encompasses
the unspecific adsorption of second molecules, e.g., to magnetic particles
coated with
a plastic surface and/or the covalent binding of second molecules, e.g., via
functional
groups such as NH2-, COOH-, SH-groups. These modes of loading may be used
especially if partial denaturation and destruction of, e.g., epitopes does not
affect the
overall efficiency of the method of the present invention.
Moreover, it has been found out in accordance with the present invention that
for
affixing said second molecule to said magnetic particle preferably saturating
concentrations of said second molecule are used so that virtually all free
binding sites
on said magnetic particle are bound by said second molecule. However, the
method of
the present invention also encompasses the work with sub-saturating
concentrations of
said second molecule.
In a most preferred embodiment, said metal-chelating tag is a His-tag, and/or
said
epitope tag is an HA-tag, a c-myc-tag, a VSV-G-tag, an a-tubulin-tag, a B-tag,
an E-
tag, FLAG, a His-tag, an HSV-tag, a Pk-tag, a protein C-tag, a T7-tag,
EpiTagT"~, a V5-
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tag or an S-tag, and/or said enzyme binding domain is cellulose binding
domain,
barnase or maltose binding protein.
In an additional preferred embodiment of the method of the present invention
step (c)
is effected by immunological means.
In a more preferred embodiment of the method of the present invention step (c)
is
effected by ELISA, RIA, western/colony blotting, FACS or immunohistochemistry.
In another more preferred embodiment of the method of the present invention
step (c)
is effected in (micro-)array format, preferably on a membrane and/or filter
and/or a glas
slide and/or in a microtiter plate.
The present invention also relates to a method for the production of a
pharmaceutical
composition comprising the steps of the method of the present invention and
further
the step of formulating said first and/or second molecule selected and/or
characterized
by the method described hereinabove or a functionally and/or structurally
equivalent
derivative thereof in a pharmaceutically acceptable form.
The pharmaceutical composition of the present invention may comprise a
pharmaceutically acceptable carrier and/or diluent. Examples of suitable
pharmaceutical carriers are well known in the art and include phosphate
buffered
saline solutions, water, emulsions, such as oil/water emulsions, various types
of
wetting agents, sterile solutions etc. Compositions comprising such carriers
can be
formulated by well known conventional methods. These pharmaceutical
compositions
can be administered to the subject at a suitable dose. Administration of the
suitable
compositions may be effected by different ways, e.g., by intravenous,
intraperitoneal,
subcutaneous, intramuscular, topical, intradermal, intranasal or
intrabronchial
administration. The dosage regimen will be determined by the attending
physician and
clinical factors. As is well known in the medical arts, dosages for any one
patient
depends upon many factors, including the patient's size, body surface area,
age, the
particular compound to be administered, sex, time and route of administration,
general
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health, and other drugs being administered concurrently. A typical dose can
be, for
example, in the range of 0.001 to 1000 pg (or of nucleic acid for expression
or for
inhibition of expression in this range); however, doses below or above this
exemplary
range are envisioned, especially considering the aforementioned factors.
Generally,
the regimen as a regular administration of the pharmaceutical composition
should be in
the range of 1 Ng to 10 mg units per day. If the regimen is a continuous
infusion, it
should also be in the range of 1 Ng to 10 mg units per kilogram of body weight
per
minute, respectively. Progress can be monitored by periodic assessment.
Dosages will
vary but a preferred dosage for intravenous administration of DNA is from
approximately 106 to 10'2 copies of the DNA molecule. The compositions of the
invention may be administered locally or systemically. Administration will
generally be
parenterally, e.g., intravenously; DNA may also be administered directly to
the target
site, e.g., by biolistic delivery to an internal or external target site or by
catheter to a
site in an artery. Preparations for parenteral administration include sterile
aqueous or
non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous
solvents are propylene glycol, polyethylene glycol, vegetable oils such as
olive oil, and
injectable organic esters such as ethyl oleate. Aqueous carriers include
water,
alcoholic/aqueous solutions, emulsions or suspensions, including saline and
buffered
media. Parenteral vehicles include sodium chloride solution, Ringer's
dextrose,
dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous
vehicles
include fluid and nutrient replenishers, electrolyte replenishers (such as
those based
on Ringer's dextrose), and the like. Preservatives and other additives may
also be
present such as, for example, antimicrobials, anti-oxidants, chelating agents,
and inert
gases and the like. Furthermore, the pharmaceutical composition of the
invention may
comprise further agents such as interleukins or interferons depending on the
intended
use of the pharmaceutical composition.
The term "functionally and/or structurally equivalent derivative" as used in
accordance
with the present invention denotes molecules modified, e.g., by deletion,
addition
and/or substitution of certain parts thereof but essentially maintaining their
capacity of
specifically interacting with said first or second molecule selected by the
method of the
present invention. Also encompassed by this term are molecules that have been
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modified in order; e.g., to increase their half-lifes in a subject to which
they have been
administered, to increase the rate of their uptake, to increase their affinity
to their
interacting counterparts or to increase the excretion rate of the
corresponding
metabolized end products. With regard to nucleic acids, such molecules may be
peptide nucleic acids or nucleic acids comprising, e.g., methylphosphonate- or
phosphorothioate-bonds instead of phosphodiester-bonds. Methods for the
synthesis
of derivatives that, e.g., show the same three-dimensional structure than the
originally
identified molecule are known in the art and include, e.g., peptidomimetics
(see, e.g.,
Hruby, V.J. et al., Biopolymers 43(3) (1997), 219-66; Bohm, H. J., J. Comput.
Aided
Mol. Des. 10(4) (1996), 265-272; Wiley, R.A. & Rich, D. H., Med. Res. Rev.
13(3)
(1993), 327-384; al-Obeidi, F. et al., Mol. Biotechnol. 9(3) (1998), 205-223;
Beeley, N.,
Trends Biotechnol. 12(6) (1994), 213-6).
Further preferred is to use the compound provided in accordance with the
present
invention as lead compound for providing downstream developments, in
accordance
with methods presently employed in the art.
In addition, the invention relates to pharmaceutical compositions comprising
at least
one of the selected interacting molecules or of derivatives as defined above,
optionally
in combination with a pharmaceutically acceptable carrier and/or diluent.
The documents cited herein are herewith incorporated by reference.
The figures show:
Figure 1: High-throughput selection of binding partners. Magnetic particles
are
transferred between wells of microtitre plates, incubated and washed
using an automated magnetic particle processor. Molecules of the
arrayed Library 2 (targets) are tag-bound to magnetic particles which are
washed, blocked and incubated with Library 1 being, e.g., a phage
display library. After washing away background phage and incubation
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with bacteria and helper phage, an enriched and amplified Library 1
enters the next round of selection against the same Library 2 molecules
for further enrichment.
Figure 2: Example for the selection of interacting molecules. Selection from
phage
display libraries of, e.g. human scFv antibody fragments recognising
targets (e.g. specific expression products of a human cDNA library) tag-
bound to magnetic particles.
Figure 3: Automated magnetic particle processor (Labsystems, Helsinki,
Finland)
in action. Left: rod-shaped magnets and plastic caps separated,
magnetic particles in solution in microtitre wells; top right: magnets in
plastic caps, collection of magnetic particles to plastic caps; bottom
right: transfer of magnetic particles to new pre-filled microtitre wells.
Figure 4: Saturation ELISA for assessment of optimal concentrations of protein
targets (e.g. bGAPDH) for loading of magnetic particles.
Figure 5: Polyclonal mixtures of phage representing the unselected (rounds)
library 1 and the results of every round of selection screened for binding
partners to the protein target used for this selection (e.g. UB18) using
magnetic particle ELISA; PTM negative control.
Figure 6: Monoclonal phage (e.g. anti-UB18) rescreened by magnetic particle
phage ELISA for binding to the same protein target (e.g. UB18); PTM
negative control.
The examples illustrate the invention.
Example 1: Automated magnetic particle-handling
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An automated device (Fig. 3, Labsystems) was used for washing and
incubation of magnetic particles. 96-well microtitre plates (e.g. CIiniPlate
200, Labsystems) were pre-filled with solutions (200 p1), and magnetic
particles were transferred between wells by capture to and release from
rod-shaped magnets covered with plastic caps. Ni-NTA Silica Beads
(Qiagen, Hilden, Germany) or Dynabeads M-280 Streptavidin (Dynal,
Oslo, Norway) were used for binding of Hiss- or biotin-tagged proteins,
respectively.
Example 2: Large-scale target production and purification
Protein targets were expressed in E. coli (strain SCS1 ) liquid cultures.
200 ml 2xTY medium (16 g/1 Bacto-tryptone, 10 g/1 yeast extract, 5 g/1
NaCI, pH 7.0) containing 100 pg/ml ampicillin were inoculated with 2 ml
of an overnight culture and shaken at 37°C until an ODsoo of 0.8 was
reached. Isopropyl-b-~-thiogalactopyranosid (IPTG) was added to a final
concentration of 1 mM. The culture was shaken for 4-6 h at 30 or 37°C.
Cells were harvested by centrifugation at 2,100 g for 10 min,
resuspended in 5 ml Lysis Buffer (50 mM NaHzP04, 0.3 M NaCI, 10 mM
Imidazole, 0.1 mM PMSF, pH 8.0) containing 0.25 mg/ml lysozyme, 10
~rg/ml DNase and 10 pg/ml RNase and incubated on ice for 30 min.
DNA was sheared with an ultrasonic homogeniser (Sonifier 250,
Branson Ultrasonics, Danbury, USA) for 3 x 1 min at 50% power on ice.
The lysate was cleared by centrifugation at 10,000 g for 30 min. Ni-NTA
agarose (Qiagen) was added and mixed by shaking at 4°C for 1 h. The
mixture was poured into a column which was subsequently washed with
ten bed volumes of Lysis Buffer containing 20 mM imidazole. Protein
was eluted in Lysis Buffer containing 250 mM imidazole and was
dialysed against Phosphate-Buffered Saline (PBS, 10 mM Phosphate
buffer, 2.7 mM KCI, 137 mM NaCI, pH 7.4) at 4°C overnight.
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Proteins were either expressed as fusion proteins with a Hisfi- and/or
biotin-tag attached (Lueking et al., 1999) or were biotinylated in vitro
using ImmunoPure NHS-SS-Biotin (Pierce, Rockford USA) and the
efficiency of biotinylation was determined by ImmunoPure HABA
(Pierce).
Example 3: High-throughput small-scale target production (native conditions)
High-throughput small-scale protein expression and purification was
modified according to (Lueking et al., 1999). Briefly, proteins were
expressed from selected clones of the arrayed human fetal brain cDNA
expression library hEx1 (Biassow et al., 1998). This library was
directionally cloned in pQE-30NST for IPTG-inducible expression of
Hiss-tagged fusion proteins. 96-well microtitre plates (e.g. CIiniPlate 200,
Labsystems) were filled with 180 p1 2xTY medium supplemented with
100 Ng/ml ampicillin. Cultures were inoculated with 20 p1 E. coli SCS1
cells from overnight cultures. After growth at 37°C with vigorous
shaking
until an ODsoo of 0.2 was reached, IPTG was added to a final
concentration of 1 mM. Cells were grown for 4-6 h at 30 or 37°C,
harvested by centrifugation at 6,000 g for 10 min, washed by
resuspension in Lysis Buffer (50 mM NaH2P04, 0.3 M NaCI, 10 mM
Imidazole, 0.1 mM PMSF, pH 8.0) containing 0.25 mg/ml lysozyme, 10
Ng/ml DNase and 10 pglml RNase and incubated on ice for 1 h.
Example 4: High-throughput small-scale target production (denaturing
conditions)
High-throughput small-scale protein expression and purification was
described (Lueking et al., 1999). Briefly, proteins were expressed from
selected clones of the arrayed human fetal brain cDNA expression
library hEx1 (Bussow et al., 1998), directionally cloned in pQE-30NST
for IPTG-inducible expression of Hiss-tagged fusion proteins. 96-well
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microtitre plates with 2 ml cavities (StoreBlock, Zinsser) were filled with
100 p1 SB medium, supplemented with 100 pg/ml ampicillin and 15 pl/ml
kanamycin. Cultures were inoculated with E. coli SCS1 cells from 384-
well library plates (Genetix, Christchurch, U.K.) that had been stored at
-80°C. For inoculation, replicating devices carrying 96 steel pins
(length
6 cm) were used. After overnight growth at 37°C with vigorous shaking,
900 p1 of prewarmed medium were added to the cultures, and
incubation was continued for 1 h. For induction of protein expression,
IPTG was added to a final concentration of 1 mM. All following steps,
including centrifugations, were also done in 96-well format. Cells were
harvested by centrifugation at 1,900 g (3,400 rpm) for 10 min, washed
by resuspension in Phosphate Buffer, centrifuged for 5 min and lysed by
resuspension in 150 NI Buffer A (6 M Guanidinium-HCI, 0.1 M NaH2P04,
0.01 M Tris-HCI, pH 8.0). Bacterial debris was pelleted by centrifugation
at 4,000 rpm for 15 min. Supernatants were filtered through a 96-well
filter plate containing a non-protein binding 0.65 pm pore size PVDF
membrane (Durapore MADV N 65, Millipore, Bedford, USA) on a
vacuum filtration manifold (Multiscreen, Millipore).
Example 5: Magnetic particle loading
Magnetic particles (10 p1 or 250 Ng Ni-NTA Silica Beads, Qiagen, or 20
p1 or 1.34 x 10' particles, Dynabeads M-280 Streptavidin, Dynal) were
washed twice in PBST (PBS, 0.1 % Tween 20) and loaded with iigands
as follows.
(a) Binding from lysates:
Magnetic particles were incubated for 1 h at RT in 200 p1 total cell
lysate. Examples were proteins like expression products of a human
cDNA library as described (Lueking et al., 1999).
(b.) Binding of purified targets:
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Magnetic particles were incubated for 1 h at RT in 200 p1 PBS
containing 1 % bovine serum albumin (BSA) and the equivalent of 5-10
NM purified targets, depending on the size of target. Examples were
proteins, like human glyceraldehyde-3-phosphate dehydrogenase
(GAPDH, Swiss-Prot P04406), a C-terminal fragment (40.3 kd) of
human heat shock protein 90 alpha (HSP90a, Swiss-Prot P07900), rat
immunoglobulin heavy chain binding protein (BIP, Swiss-Prot P06761 ),
tubulin alpha-1 chain (TUBa1, Swiss-Prot P04687), calcium-binding
protein ERC-55 precursor (ERC55, Swiss-Prot Q14257), transcription
elongation factor S-II (HS-II-T1, Swiss-Prot Q15560), transcription factor
ETR101 (ETR101, Swiss-Prot Q03827), peptidyl-prolyl cis-trans
isomerase A (EC5218, Swiss-Prot P05092) and Ubiquitin (UBIQ-
HUMAN, SWISS-Prot P02248).
After target loading, magnetic particles were washed twice in PBST.
Remaining free binding sites were blocked with PTM (PBS, 2% milk
powder, 1 % Tween) for 1 h at RT.
Example 6: Magnetic particle ELISA
Magnetic particles (10 p1 or 250 pg Ni-NTA Silica Beads, Qiagen, or 2 NI
or 1.34 x 106 particles, Dynabeads M-280 Streptavidin, Dynal) were
incubated with primary and secondary antibodies diluted in PTM for 30
min at RT each and washed twice in PBST after antibody incubations.
Secondary antibodies labelled with horseraddish peroxidase (HRP)
were detected using ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-
sulfonic acid) solution (10 ml 50 mM Na3 citrate, 10 ml 50 mM citric acid,
mg ABTS, 10 p1 30 % H202, pH 4.3) and measured as OD 405 nm
using SpectraMAX 250 (Molecular Devices, Sunnyvale, USA).
Example 7: Saturation ELISA
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Target dilutions (100 nM - 25 pM) were prepared in 200 p1 PBS
containing 1 % BSA and incubated consecutively with two aliquots of
magnetic particles (10 p1 or 250 pg Ni-NTA Silica Beads, Qiagen, or 20
p1 or 1.34 x 10' particles, Dynabeads M-280 Streptavidin, Dynal) for 1 h
at RT each. Magnetic particles were washed twice in PBST, blocked
with PTM for 1 h at RT, incubated with primary and secondary
antibodies diluted in PTM for 30 min at RT each and washed twice in
PBST after antibody incubations. Secondary antibodies labelled with
HRP were detected using ABTS solution and measured as OD 405 nm.
Example 8: Phage selection
The preparation of phage from bacterial glycerol stock of phage display
libraries was described previously (Harrison et al., 1996). Phage
suspensions (10'2 phage of unsefected libraries or the MuItiScreen flow-
through after phage amplification between selection rounds) were
equilibrated and preabsorbed by incubation with unloaded magnetic
particles (10 p1 or 250 pg Ni-NTA Silica Beads, Qiagen, or 25 p1 or 1.68
x 10' particles, Dynabeads M-280 Streptavidin, Dynal) in 200 NI PTM for
1 h at RT.
Magnetic particles (10 NI or 250 Ng Ni-NTA Silica Beads, Qiagen, or 20
NI or 1.34 x 10' particles, Dynabeads M-280 Streptavidin, Dynal) were
loaded with targets and blocked as described above, incubated with
preabsorbed phage for 1 h at RT and washed in PBST several times
according to the number of the selection round. E. coli TG1 cells were
incubated with the washed magnetic particles for 30 min at RT.
Example 9: Phage amplification between selection rounds
20 p1 2xTY containing 10 x Glu-Amp (20% glucose, 1 mg/ml ampicillin)
were added and cultures were shaken overnight at 37°C. 10 NI of these
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cultures were diluted into 200 NI 2xTY containing 2% glucose, 100 pg/ml
ampicillin and shaken at 37°C until OD600 > 0.1 (SpectraMAX 250,
Molecular Devices). 10 NI M13-K07 helper phage (10'2/m1) were added,
cultures were incubated for 30 min at RT and transferred to a Durapore
0.65 N plate (Millipore). Cultures were sucked through on a MuItiScreen
vacuum device (Millipore). TG1 cells were resuspended in 200 NI 2xTY
containing 100 Ng/ml ampiciliin and 60 pg/ml kanamycin, transferred to
a microtitre plate (e.g. CIiniPlate 200, LabSystems) and vigorously
shaken overnight at 30°C. Cultures were transferred to a Durapore 0.65
N plate (Millipore) and sucked through on a MuItiScreen vacuum device
(Millipore). The flow-through was collected in a microtitre plate (e.g.
CIiniPlate 200, LabSystems) and was used as starting material (phage
suspension) for the next round of selection.
Example 10: Magnetic particle phage ELISA
(a) Polyclonal ELISA
Phage were prepared from overnight cultures as described above.
Magnetic particles (10 p1 or 250 pg Ni-NTA Silica Beads, Qiagen, or 2 p1
or 1.34 x 106 particles, Dynabeads M-280 Streptavidin, Dynal) were
loaded with targets and blocked as described above, incubated with
phage suspensions (10'°-10" phage) for 30 min at RT and washed
twice in PBST. For detection, magnetic particles were incubated with
anti-M13 HRP (1:5,000) in PTM for 30 min at RT, washed twice in PBST
and incubated in ABTS solution which was measured at OD 405 nm.
fib) Monoclonal ELISA
The preparation of monoclonal phage was described previously
(Harrison et al., 1996). Phage suspensions were divided into two
aliquots, diluted 1:1 with PBS and incubated in parallel with magnetic
particles either loaded with targets and blocked as described above or
unloaded for 30 min at RT. Magnetic particles were washed twice in
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PBST, incubated with anti-M13 HRP (1:5,000) in PTM for 30 min at RT,
washed twice in PBST and incubated in ABTS solution which was
measured at OD 405 nm.
Example 11: PCR and DNA sequencing
PCR and DNA sequencing of antibody genes was described previously
(Walter & Tomlinson, 1996).
Example 12: BIAcore analysis
BIAcore analysis of antibody affinity was described previously (Hefta et
al., 1996).
Example 13: Specific tag-binding of targets to magnetic particles
Hiss- tagged proteins were bound to Ni-NTA Silica Beads (Qiagen), and
biotin-tagged proteins were bound to Dynabeads M-280 Streptavidin
(Dynal). This tag-binding is specific for the labelled molecules and
ensures their proper orientation on the magnetic particles. In contrast,
unspecific adsorption of proteins to plastic surfaces leads to partial
denaturation and destruction of epitopes. Tag-binding also enables
direct loading of magnetic particles with targets from protein mixtures
like crude extracts or cell lysates. This is particularly important for high-
throughput selection technology, avoiding purification of large numbers
of different proteins or other targets.
Example 14: Optimisation of target concentration by Saturation ELISA
The concentration of target molecules on solid surfaces is a critical
parameter for the selection of binding partner molecules. Optimal
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concentrations of protein targets (e.g. bGAPDH) for loading of magnetic
particles were assessed in saturation ELISA experiments (Fig. 4). For
most proteins tested, concentrations of 5-10 pM purified target (e.g.
200-400 pg/ml bGAPDH) were found to cause saturation of 10 p1 (250
Ng) Ni-NTA Silica Beads (Qiagen) or 20 p1 (1.34 x 10' particles)
Dynabeads M-280 Streptavidin (Dynal). These concentrations are in
excess of saturating concentrations of other plastic surfaces (e.g.
microtitre plates, Kala et al., 1997) and confirm the high binding capacity
of magnetic particles, reflecting their increased surface area. While it is
advisable to work with saturating concentrations if possible, some
protein targets can not be produced in sufficient amounts. In such
cases, sub-saturating concentrations of protein targets were used
successfully, due to the high sensitivity of the magnetic particle ELISA
(data not shown).
Example 15: High-throughput selection and screening of binding partners
Phage display libraries (e.g. human scFv antibody fragment libraries,
Tomlinson, unpublished) were screened for binders to various protein
targets (see above). Phage titres were recorded after each round of
selection to monitor the efficiency of phage amplification (data not
shown). Using the magnetic particle phage ELISA, polyclonal mixtures
of phage representing the unselected library and the results of every
round of selection were screened for binding partners to the protein
target used for this selection (e.g. UB18, Fig. 5). Positive mixtures were
cloned, and single colonies were rescreened by magnetic particle phage
ELISA for binding to the same protein target (e.g. UB18, Fig. 6).
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