From the Department of Pathology and Laboratory Medicine, Texas A&M
University Health Science Center, College Station, Texas 77843
Received for publication, December 18, 2000, and in revised form, January 19, 2001
Previous work from our laboratory demonstrates
that the
4
1 integrin is an adhesion
receptor for OPN and that
4
1 binding site(s) are present in the N-terminal thrombin fragment of osteopontin (OPN) (Bayless, K. J., Meininger, G. A., Scholtz, J. M., and Davis, G. E. (1998) J. Cell Sci. 111, 1165-1174). The work presented here identifies two
4
1 binding sites within a recombinantly produced N-terminal thrombin fragment of human OPN. Initial
experiments, using wild-type OPN containing an RGD sequence or an
OPN-RGE mutant, showed identical
4
1-dependent cell adhesive
activity. A strategy to localize
4
1
binding sites within the thrombin fragment of osteopontin involved
performing a series of truncation analyses. Removal of the last 39 amino acids (130) completely eliminated adhesion, indicating all
binding activity was present within that portion of the molecule.
Combined mutation and deletion analyses of this region revealed the
involvement of dual
4
1 binding sites. Synthetic peptides for both regions in OPN, ELVTDFPTDLPAT (131) and SVVYGLR (162), were found to block
4
1-dependent adhesion. The
first peptide when coupled to Sepharose bound the
4
1 integrin directly whereas a mutated
ELVTEFPTELPAT peptide showed a dramatically reduced ability to bind.
These data collectively demonstrate that dual
4
1 integrin binding sites are present in
a 38 amino acid domain within the N-terminal thrombin fragment of OPN.
 |
INTRODUCTION |
Integrins are a family of transmembrane heterodimeric cell
adhesion receptors (1). The
4
1 integrin
(VLA-4) is predominantly expressed on leukocytes (2-4). It is capable
of existing in multiple activation states (5) to mediate cell-cell and
cell-extracellular matrix interactions (6-14). Known binding sites for
4
1 include LDVP (8-10), IDAPS (15), REDV
(16), QIDSPL (17-20), and under certain activating conditions, RGD
(21). The
4
1 integrin is intricately
involved in trafficking of mononuclear leukocytes into tissues during
the normal inflammatory response as well as in pathological situations
(22), such as encephalomyelitis (23), diabetes (24), and graft
rejection (25). Also, increased expression of
4
1 is observed on smooth muscle cells
within atherosclerotic plaques (26), and
4
1 plays a role in tumor metastasis (12, 27). Collectively, the
4
1 integrin
appears to play a pathogenic role in inflammation, wound repair and
tumor progression.
Osteopontin (OPN)1 is an
extracellular matrix protein originally isolated from bone (28), and
much evidence has accumulated as to its role in bone physiology (29).
OPN is also secreted by many epithelial surfaces (30), and one early
study supported a role for OPN in host-response to bacterial infection
(31). Accumulating evidence also indicates OPN secretion is involved in
inflammation and tumor progression. OPN has previously been found to be
up-regulated in a variety of inflammatory, cardiovascular, and
infectious diseases (32-37) and is a major secreted product of
macrophages in inflammatory settings (32-36). It is also associated with tumors (38-41), particularly at the tumor-host interface (42). Recent data from knockout mice show that OPN facilitates wound healing
(43), aids in host defense against viral (44) and bacterial infections
(44, 45), and is involved in granuloma formation (44). Other studies
have suggested OPN may facilitate tumor cell metastasis (46) and
decrease complement-mediated tumor cell destruction (47). Based on
these data, the presence of OPN in the wound environment likely plays
an important role in regulating disease progression in inflammatory and
other conditions. The molecular domains in OPN that mediate its effects
in these phenomena remain to be defined.
The parallels between expression of the
4
1 leukocyte integrin and expression of
OPN in wounds prompted a previous study by our laboratory to define
osteopontin as a ligand for the
4
1 integrin (14). Here, we define two binding sites for the
4
1 integrin in the recombinant N-terminal
thrombin fragment of human OPN using deletion and mutation analyses.
 |
EXPERIMENTAL PROCEDURES |
Preparation of Recombinant Osteopontin; Cloning Strategy--
A
full-length cDNA clone of the human osteopontin gene was obtained
from the American Type Culture Collection (ATCC, Manassas, VA) (48),
and the sequence is shown in Fig.
1A. Sequences encoding the
wild-type N-terminal thrombin fragment of osteopontin
(rOPN-(17-168)) were amplified by polymerase chain
reaction using the primers 5'-TAGGATCCATACCAGTTAAACAGGCTGATTCTGGAAG-3'
and 5'-GTAAGCTTTTACCTCAGTCCATAAACCACACTATCACCTCGGCCA-3'(Genosys, The
Woodlands, TX). Sequences encoding a mutated N-terminal fragment ([Glu161]rOPN-(17-168)) in which the
single RGD sequence at residues 159-161 was changed to RGE were
obtained by substituting
5'-TAAAGCTTTTACCTCAGTCCATAAACCACACTTTCACCTCGGCCA-3' for the
downstream primer used to generate the wild-type fragment. Restriction
digests of the polymerase chain reaction products and the
pQE30 vector (Qiagen) were carried out overnight with BamHI and HindIII (Life Technologies, Inc.).
Digested vector and insert were purified, quantitated, and ligated at
an insert/vector ratio of 4.5:1 overnight at 14 °C (Roche Molecular
Biochemicals). These constructs encode a modified version of
rOPN-(17-168) and [Glu161]rOPN-(17-168)
where the sequence RGSHHHHHHS replaces MRIAVICFCLLGITCA at the N
terminus of wild-type osteopontin. All positive clones were confirmed
by sequence analysis at Lone Star Labs (Houston, TX). All subsequent
constructs studied contained the RGE mutation at amino acid 161.

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Fig. 1.
Production of the N-terminal thrombin
fragment of human OPN. A, amino acid sequence reported
for the N-terminal thrombin fragment of OPN (48). Bracketed
signal sequence representing amino acids 1-16 is not present on
constructs generated here. This sequence is replaced by RGSHHHHHHS. The
underlined sequences represent regions of OPN with affinity
for 4 1. B, combined truncation
and mutation analyses generated various constructs listed by amino acid
number. Asterisk indicates mutation of Asp to Glu.
|
|
Additional recombinant constructs of OPN (Fig.
1B) were produced using the following primer sets:
rOPN-(55-168),
5'-TAAAGCTTTTACCTCAGTCCATAAACCACACTTTCACCTCGGCCA-3' and
5'-AGGGATCCCTAGCCCCACAGAATGCTGTGTCC-3'. The remaining constructs were
constructed with a common upstream primer,
5'-TAGGATCCATACCAGTTAAACAGGCTGATTCTGGAAG-3' and different downstream
primers: rOPN-(17-164), 5'-AGAAGCTTTTAAACCACACTTTCACC-3'; rOPN-(17-129), 5'-AGAAGCTTTTAAGATTCATCAGAATGGTGAGAC-3';
rOPN-(17-135), 5'-CGAAGCTTTTAATCAGTGACCAGTTCATCAG-3';
rOPN-(17-138), 5'-AGAAGCTTTTACGTGGGAAAATCAGTGACC-3'; [Glu135]rOPN-(17-138),
5'-AGAAGCTTTTACGTGGGAAACTCAGTGACCAGTTC3'; rOPN-(17-142), 5'-AGAAGCTTTTATGCTGGCAGGTCCGTGGGAAAATC-3';
[Glu139]rOPN-(17-142),
5'-AGAAGCTTTTATGCTGGCAGCTCCGTGGGAAAATC-3'; rOPN-(17-150), 5'-AGAAGCTTTTAGACAACTGGAGTGAAAACTTCGGTTGC-3'.
Cloning experiments were performed as described above, and positive
clones were confirmed by sequence analysis at Lone Star Labs (Houston, TX).
Production and Characterization of Recombinant Wild-type and
Mutated N-terminal Thrombin Fragments of
Osteopontin--
Escherichia coli strain RY2840 [MC4100
lacIQ1 lac+ slyD Kmr] (49) was
transformed with plasmids encoding the His6-tagged OPN derivatives. 2 ml of overnight cultures were inoculated into 200 ml of
Luria-Bertani media (Life Technologies, Inc.) containing 50 µg/ml
ampicillin (Sigma). Cultures were grown to an
A600 of 1.0 (~2.5 h) before induction with 0.5 mM isopropylthiol-
-D-galactoside (IPTG-Life
Technologies, Inc.). Cultures were allowed to incubate for 3.5-4 h at
37 °C in a shaking incubator before being placed on ice for 15 min.
Bacteria were pelleted, supernatants removed, and pellets frozen at
80 °C. Pellets were thawed at 25 °C for 10 min, resuspended on
ice in 20 ml Hepes buffered saline, pH 8.1 containing 25 mM
Hepes, 150 mM NaCl and 1 mM
4-(2-aminoethyl)benzene sulfonylfluoride, HCl (CalBiochem).
Bacteria were lysed and debris pelleted (20,000 × g at
4 °C for 20 min) before adding supernatants to 2 ml of TALON metal
ion affinity column (CLONTECH) equilibrated with
Hepes buffer. Columns were incubated for a minimum of 20 min at 4 °C
before washing with 20-column volumes of Hepes buffer. His-tagged
proteins eluted with 0.2 M imidazole (Sigma) in Hepes buffer, and fractions were dialyzed (Mr cutoff
7,500) against 8 liters of phosphate-buffered saline. The purity of
recombinant proteins was assessed by SDS-PAGE and Western blot
analysis. Protein concentration was estimated according to the method
of Pace et al. (50). Yields were ~6 mg per 200 ml of culture.
Cell Adhesion Assays--
Cell adhesion assays were
performed to determine the ability of OPN to promote leukocyte
adhesion. Polystyrene microwells (Corning-Costar, Cambridge, MA) were
coated with 50 µl of bovine OPN purified as previously described (51)
or recombinant fragments of OPN at a concentration of 20 µg/ml in TBS
overnight at 4 °C. After blocking with 100 µl of 10 mg/ml BSA
(Sigma, St. Louis, MO) in TBS, wells were rinsed with PSA (Life
Technologies, Inc., Grand Island, NY). HL-60 promyelocytic leukemia
cells and Ramos cells (ATCC) were grown in RPMI 1640 (Life
Technologies, Inc.) and 10% fetal calf serum. Human umbilical vein
endothelial cells were grown in M199 (Life Technologies, Inc.)
supplemented with heparin (Sigma), bovine brain extract (52), and 20%
fetal calf serum (Life Technologies, Inc.). Leukocytes were rinsed and
resuspended in PSA at a density of 100,000 cells/well and endothelial
cells at 35,000 cells per well. Media for adhesion in all leukocyte experiments contained a final concentration of 100 µg/ml BSA with physiological doses of CaCl2 (2 mM) and
MgCl2 (1 mM). HL-60 cells were activated with
the
1-activating antibody, 8A2 (53) at a concentration
of 1 µg/ml and a phorbol ester, 12-0-tetradecanoyl phorbol
13-acetate at a concentration of 50 ng/ml. Endothelial cells were
allowed to attach in the presence of 100 µg/ml BSA with 1.5 mM CaCl2 and 1.5 mM
MgCl2. After plating, cells were allowed to adhere for
30-60 min at which time they were rinsed and fixed with formalin.
Plates were stained with 0.1% Amido Black for 5 min and rinsed and
solubilized with 2 N NaOH to obtain an absorbance reading at 595 nm,
which corresponds directly to the number of cells stained in each well
(54).
Peptide Synthesis and Adhesion Blockade--
To confirm the
findings of the truncation studies, the SVVYGLR peptide (corresponding
to C-terminal amino acids 162-168) was synthesized (Sigma-Genosys).
Also generated were the wild-type peptide ELVTDFPTDLPATK and aspartate
mutant ELVTEFPTELPATK, representing amino acids 131-143. The molecular
weight of each peptide was confirmed by mass-spectral analysis
(Sigma-Genosys). The synthetic peptides SVVYGLR, ELVTDFPTDLPATK, and
ELVTEFPTELPATK were preincubated with cells at 250, 500, and 500 µg/ml, respectively under activating conditions in the presence of
divalent cations for 15 min. Following the incubation period, cells
were seeded, and the assay was performed as described above.
Direct Integrin Binding using Affinity Chromatography--
To
illustrate the integrin-binding capacity of osteopontin, the synthetic
peptides ELVTDFPTDLPATK and ELVTEFPTELPATK were coupled to
cyanogen-bromide 4B (Sigma) at 5 mg/ml according to the manufacturer's
instructions. Ramos cells (ATCC) were surface biotinylated as described
(55), and a 50-µl pellet of cells was extracted with 1 ml of TBS
containing 3% octylglucoside (ICN, Irvine, CA) in 1.5 mM
Mg2+, 1.5 mM Mn2+, and
10
3 M phenylmethane sulfonic acid. The HL-60
cell extracts were agitated at 5-10 min intervals with Sepharose
columns (0.5 ml) over a 2-h period at 0 °C. The columns were washed
with 5 ml of TBS containing 3% octylglucoside, 1.5 mM
Mg2+, and 1.5 mM Mn2+. This was
followed with a 15-ml wash in TBS containing 1% octylglucoside, 1.5 mM Mg2+, and 1.5 mM
Mn2+. Integrins were eluted with 2 ml of TBS with 1%
octylglucoside + 10 mM EDTA (0.25-ml fractions). 40 µl of
each fraction were loaded and run under nonreducing conditions on a 7%
acrylamide gel and transferred to polyvinylidene difluoride membrane
(Millipore). The membrane was blocked overnight at 4 °C with 5%
milk in 0.1% Tween 20 saline containing 2.5 mM Tris-HCl,
pH 7.5. Blots were washed and streptavidin alkaline phosphatase (Sigma)
was added (1:1000) to 1% BSA in Tween 20 saline and incubated for 30 min followed by a 30-min wash in Tween 20 saline. The alkaline
phosphatase activity was developed using alkaline phosphatase
development kit (Bio-Rad) and stopped with water.
Integrin Immunoprecipitation--
Integrins that bound to the
OPN-Sepharose column were identified using immunoprecipitation.
Sepharose beads conjugated with goat anti-mouse IgG (Sigma) were rinsed
and suspended 1:1 with 0.5% Triton X-100 in TBS. In 1.5-ml
microcentrifuge tubes, 200 µl of the bead mixture was added to 5 µg
of monoclonal antibodies against several human integrin subunits
including
4 (HP2/1, Immunotech) (56),
1
(mAb13, Becton-Dickinson) (57), and
5 (IIA1, PharMingen) (58). These mixtures were then combined with 280 µl of pooled EDTA
eluate from OPN-Sepharose and 700 µl of 0.5% Triton X-100 in TBS.
This mixture was rotated continuously at 4 °C overnight after which
time tubes were centrifuged and rinsed six times with 1 ml of 0.5%
Triton X-100 in TBS. 75 µl of 2× sample buffer was added to the
beads, and this mixture was boiled for 5 min. 30-µl samples were run
on 7% SDS-PAGE under nonreducing conditions, and blots were developed
as described above.
 |
RESULTS |
To rule out the involvement of the RGD site in
4
1-dependent adhesion to OPN,
the wild-type, RGD-containing N-terminal thrombin fragment
(rOPN-(17-168)) and an RGE mutant
([Glu161]rOPN-(17-168)), where Asp161
was mutated to Glu161, were produced. Each clone is
described as rOPN followed by the amino acids coded for in the
construct (e.g. 17-168) and finally the mutation
incorporated into the clone (e.g. Glu161 for
Asp161 mutated to Glu161). Clones were
sequenced to confirm successful mutation, and recombinant proteins
(>95% purity) were analyzed using SDS-PAGE (not shown). Western
blotting experiments using a monoclonal antibody directed to the
N-terminal histidine tag revealed a pattern exactly matching staining
results (not shown). Proteins were tested for their ability to promote
v
3-dependent attachment (Fig.
2A). Wild-type
rOPN-(17-168) promoted endothelial cell attachment dose
dependently, whereas no attachment occurred to the RGE mutant. This
confirmed successful functional mutation of the RGD site in OPN. The
ability of both recombinant OPN constructs to promote
4
1-dependent adhesion was
compared using the HL-60 promyelocytic cell line in the presence of
physiologic divalent cations (2 mM Ca2+, 1 mM Mg2+). As shown in Fig. 2B, no
differences were observed in the ability of either construct to promote
HL-60 cell attachment. Additionally, both rOPN-(17-168) and
[Glu161]rOPN-(17-168) were comparable with bovine
OPN with respect to their ability to promote
4
1-dependent cell attachment
(Fig. 2C). Adhesion to both native OPN and recombinant
constructs was completely inhibited by the
4
1-specific LDV peptide. The control peptide, LEV, had lesser effects compared with control (no peptide). Minimal adhesion was observed to the BSA substrate. Collectively, these
data indicate that the RGD site in the N terminus of OPN is not
involved in
4
1-dependent
adhesion to recombinant OPN, as rOPN-(17-168) and
[Glu161]rOPN-(17-168). Consequently, subsequent
constructs described contain the RGE mutation (Glu161) to
rule out any influence from the RGD site in OPN, although the presence
of this mutation is not reflected to simplify nomenclature.

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Fig. 2.
Characterization of recombinantly
produced N-terminal thrombin fragment of OPN. A,
dose-response curves comparing the ability of wild-type
rOPN-(17-168) (17-168) and
[Glu161]rOPN-(17-168) (17-168E161)
mutant to promote RGD-dependent attachment of human
endothelial cells. B, dose-response curves comparing the
ability of wild-type rOPN-(17-168) and
[Glu161]rOPN-(17-168) mutant to promote
4 1-dependent attachment of
HL-60 cells. C, peptide-blocking data indicating HL-60 cell
adhesion to native and recombinantly produced OPN occurs through the
4 1 integrin. Experiments were conducted
in the presence of 250 µg/ml of the LDV and LEV peptides. Experiments
were performed as described under "Experimental Procedures."
bOPN, bovine OPN; BSA, control substrate. The
data shown are representative experiments (n = 3)
performed in triplicate wells, and values shown are mean
absorbance ± S.D.
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Various deletions of the OPN molecule were introduced in an attempt to
localize
4
1 binding sites. SDS-PAGE
analysis of the recombinant proteins revealed ~95% purity as
visualized by Coomassie Blue staining, and all proteins contained an
N-terminal histidine tag by Western blot analysis (data not shown). The
rOPN-(55-168), rOPN-(17-129), rOPN-(17-138),
rOPN-(17-142), rOPN-(17-164), and rOPN-(17-168)
constructs were tested for the ability to promote
4
1-dependent attachment of
HL-60 cells (Fig. 3). These experiments revealed that removal of the first 39 amino acids
(rOPN-(55-168)) did not reduce cell binding compared with the
entire thrombin fragment, rOPN-(17-168). All binding activity
was removed by truncation of amino acids 130-168
(rOPN-(17-129)), with partial activity returning in the
presence of amino acid residues 130-142 (rOPN-(17-138) and
rOPN-(17-142)). In addition, decreased adhesion occurred by deletion of the last 4 amino acids (rOPN-(17-164)), which was previously shown to eliminate the
9
1
integrin binding site in OPN (59).
A more detailed analysis of binding activity was conducted with the
constructs shown in Fig. 1B. Dose-response curves
illustrating the ability of constructs to promote HL-60 cell attachment
are shown in Fig. 4. No binding was
observed with truncation of the last 38 amino acids
(rOPN-(17-129)), and identical results were observed with the
rOPN-(17-135) construct. Partial activity was restored with the
addition of amino acids 130-138 (rOPN-(17-138)) indicating the
presence of a potential binding site. Activity remained at similar
levels with the addition of amino acids 139-164 (rOPN-(17-142), rOPN-(17-150), rOPN-(17-164)).
Only in the presence of the last 4 amino acids did full activity return
(rOPN-(17-168)). Thus, deletion of the last 4 amino acids on
the C terminus of the thrombin fragment of OPN partially eliminated the
ability of
4
1-dependent cell
attachment to occur. The remainder of binding activity was completely
removed with further truncation of the molecule by ending at amino acid
residue 129. These data strongly support the concept that two binding
sites exist within residues 130-168. To examine in more detail whether
the Asp135 and Asp139 residues located within
the upstream binding region identified were important in
4
1-dependent cell attachment,
additional constructs incorporating mutations were generated (Fig. 4).
The [Glu135]rOPN-(17-138) exhibited reduced
adhesion compared with rOPN-(17-138). The same was true for
[Glu139]rOPN-(17-142) versus
rOPN-(17-142). Interestingly, neither of the mutations
completely abolished adhesion, indicating that the conservative
substitution of glutamate for aspartate did not remove all activity.
Collectively, these data indicate that there are dual
4
1 binding sites in the N-terminal
thrombin fragment of OPN.
As further evidence for direct interaction of amino acids 131-143 in
OPN with the
4
1 integrin, affinity
chromatography experiments were performed using surface-labeled Ramos
cell extracts (Fig. 6A).
Wild-type ELVTDFPTDLPATK-Sepharose, aspartate mutant
ELVTEFPTELPATK-Sepharose and blank-Sepharose beads were incubated with
labeled extracts, and EDTA elutions were collected and analyzed
(E1-E4). Results show strong binding of the
4
1 integrin to the wild-type peptide, whereas minimal binding occurred to the aspartate mutant. No binding of
the
4
1 integrin was observed using
blank-Sepharose. Immunoprecipitation of fractions from both wild-type
and mutated peptide columns revealed the presence of
4
and
1 integrin subunits (Fig. 6B), although much greater binding occurred to the wild-type peptide. Mutation of
aspartate residues resulted in a reduced ability of
4
1 to bind but did not completely
eliminate binding activity. In both experiments, control integrin
antibodies failed to immunoprecipitate integrins. Similar results were
observed using surface-labeled HL-60 cell extracts (data not
shown).
Using sequential truncation analysis of the N-terminal thrombin
fragment of OPN, we observed that dual
4
1
integrin binding sites exist in a 38-amino acid C-terminal domain.
Synthetic peptides encompassing either of these regions interfered with
the ability of
4
1-dependent
attachment to occur, whereas a control peptide had lesser effects.
Also, using affinity chromatography we were able to demonstrate direct
binding of the
4
1 integrin to the wild-type synthetic peptide coupled to sepharose, whereas a mutated peptide bound considerably less well. These results show that dual
binding sites exist for the
4
1 integrin
in the N-terminal thrombin fragment of OPN.
Additionally, we present evidence for the direct involvement of
aspartate residues in the affinity of
4
1
for ELVTDFPTDLPAT based on evidence that a mutant synthetic peptide,
containing glutamate substituted for aspartate residues, was less
effective at blocking cell adhesion. It also minimally bound the
4
1 integrin in affinity chromatography
experiments. Conservative substitution of glutamate for aspartate
residues did not remove 100% activity in either peptide blocking
studies or direct integrin binding experiments. These results are
consistent with previous data from our laboratory where the control LEV
peptide also had slight effects (Ref. 14 and Fig. 2C). These
results correlate with previous evidence that the
4
1 integrin recognizes a wide variety of
motifs in FN, VCAM-1, OPN and denatured proteins (10, 15-21). As was previously suggested, this integrin shows a broader ligand binding specificity than most other members of the integrin family (13).
An interesting feature of the above findings is that all three known
integrin binding sites are located within a very limited 38-amino acid
region of the thrombin fragment of OPN. The ELVTDFPDLPAT (shown here),
RGD (51, 60-62, 67) and SVVYGLR sites (shown here and in Refs. 59, 72)
are all localized to a 38-amino acid region just proximal to the
thrombin cleavage site (Fig. 7). These sequences are highly conserved,
particularly in large species of mammals, whereas they are less
conserved or absent in rodents, particularly concerning the upstream
binding site. The thrombin fragment appears to have altered biological
activity compared with intact OPN and contains matricryptic sites (62, 65, 67, 73, 75). Localization of these integrin binding sites directly
adjacent to the thrombin cleavage site strongly implicate the
physiological importance of this region of osteopontin in inflammatory
and wound repair responses.
We would like to thank Dr. Nicholas Kovach
for the kind gift of 8A2 antibody, Dr. Doug Struck for assistance in
production and purification of recombinant proteins, and Dr. Ry Young
for the kind gift of slyD-deficient E.coli strain.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M011392200
The abbreviations used are:
OPN, osteopontin;
TBS, Tris-buffered saline;
PSA, Puck's Saline A, BSA,
bovine serum albumin;
Tween 20, polyoxyethylene sorbitan monolaurate;
PAGE, polyacrylamide gel electrophoresis.
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