Extract
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Nuclear receptors (NR) are a superfamily of
ligand-activated transcriptional factors that are involved in diverse
physiological functions[1,2]. Nuclear receptors share a
common protein structure, including a highly conserved
DNA-binding domain (DBD) responsible for binding to their
corresponding hormone response elements located in the
promoter region of their target genes, and a less-conserved
ligand-binding domain (LBD) responsible for hormone
binding, dimerization, and ligand-dependent activation. The
nuclear receptors are activated by ligands binding to the
hydrophobic ligand binding pockets in LBD, which triggers
a conformational change in the receptor proteins. Because
their activity can be modulated by small molecules that can
be easily modified, nuclear receptors have become
promising pharmacological targets for drug
development[3].
Numerous techniques and tools for the screening of small
molecular ligands have emerged over the past decade, but a
major challenge for traditional ligand screening methods has
been to express the protein of interest in soluble form and
purify it efficiently. Mass production and purification of
well-expressed and highly soluble proteins for traditional
screening is a major obstacle, because high-level expression
of recombinant proteins in E coli will always result in the
formation of insoluble inclusion bodies. Thus, there is a
significant need to develop a rapid protein expression and
purification approach for high-throughput screening.
Phage display is a method for the expression of peptides,
proteins or antibody fragments fused to the surface of
phage particles. The methodology combines the protein
expression and purification process with a subsequent rapid
selection procedure[4,5]. Therefore, it is a potential tool for
the production of proteins that could be used in the
screening of ligands. Lytic bacteriophages such as lambda, T4 or
T7 have been found to be useful for displaying foreign
proteins[6-8]. Using the lambda capsid protein pD appears to be
a particularly attractive option, because a variety of large
proteins or protein domains, such as
b-galactosidase, b-lactamase, and recombinant proteins encoded by cDNA have
been successfully displayed on the surface of lambda phage
as fusions to its N or C-terminus[9-12]. However, these
proteins are exclusively soluble when expressed in bacterial
systems, so the potential for lambda phage to display
proteins that can be aggregated, such as nuclear receptors, is
still unknown.
The peroxisome proliferator-activated receptors (PPAR)
are members of the nuclear receptor superfamily, and are
important in regulating lipid and glucose
homeostasis[3,13]. One isoform, PPARg, plays an important role in adipocyte
differentiation and lipid homeostasis, and is a drug target for
a variety of diseases, including obesity, diabetes,
atherosclerosis and cancer[3]. However, the existing
PPARg ligands on the market have been associated with hepatotoxicity,
which has resulted in the withdrawal of some of the PPAR
ligands[14]. Therefore, developing a superior
PPARg LBD model would be helpful in the search for more effective and
safe PPARg ligands that have the potential to treat human
diseases involving glucose and/or lipid disruption.
Similar to the other members in the nuclear receptor
superfamily, the production and subsequent purification of
large amounts of soluble PPARg protein are difficult because
of the hydrophobic nature of the ligand-binding pocket in
the LBD. Because pD, a protein of the lambda capsid, has
been described to have chaperone properties that can
increase the expression level of soluble heterologous proteins
in the cytoplasm of E coli[15], it could be used to express and
incorporate the pD-LBD fusion protein on the surface of
bacteriophage lambda.
In order to develop and implement phage surface display
technology for the ligand-binding domain of NR, the
PPARg LBD was expressed as fusion protein of LBD-g3p and
pD-LBD in E coli cells. The solubility characteristics of these
two systems were compared to determine the phage display
system most appropriate for PPARg LBD expression. Finally,
the PPARg LBD fused to the appropriate capsid protein was
characterized by Western blotting and phage capture assays.
Materials and methods
Plasmid construction A polymerase chain reaction (PCR)
fragment of the human PPARg2 LBD (amino acids 201-505,
GenBank accession No NM-015869) was amplified from plasmid pcDNA3.1-hPPARg2 (a gift from Dr Hitoshi
NISHIZAWA)[16] using the primers PP-Fwd1 (5¡¯-
AGGGA-TCCGTGGGGATGTCTCATAATGC-3¡¯ BamHI) and PP-Rev1
(5¡¯- ACGCGTCGACGTACAAGTCCTTGTAGAT-3¡¯
SalI). The pCGMT-LBD and pET-hPPGLBD expression vectors were
constructed by inserting the PCR fragment into the
BamHI and SalI sites of vector
pCGMT[17] and pET-21a, respectively.
The p171-LBD expression vector was constructed by
inserting a PCR fragment of human PPARg2 LBD, which was
amplified by the primers PP-Fwd2
(5¡¯-CGACTAGTGTGGGGATGTCTCATAATGC-3¡¯
SpeI) and PP-Rev2 (5¡¯-TGTTGCGGCCGCTACAAGTCCTTGTAGATC-3¡¯
NotI) from plasmid pcDNA3.1-hPPARg2, into the
SpeI and NotI sites of the p171Bio3 vector (provided by Dr Alfredo
NICOSIA)[10].
Protein expression and purification E coli
strain BB4
was transformed with pCGMT-LBD and p171-LBD and grown
to an OD600 of 0.6 in 50 mL LB media containing 1% (w/v)
glucose and 60 mg/L ampicillin at 37 °C. Afterwards, the
cells were induced with 1 mmol/L
isopropyl-D-thiogalacto-pyranoside (IPTG) for an additional 6 h at 30 °C, then the
cells were collected by centrifugation. After being washed
three times with sonication buffer [50 mmol/L Tris, pH 8.0,
0.15 mol/L NaCl, 1 mmol/L ethylenediamine tetraacetic acid
(EDTA)], the pellets were resuspended in 50 mL sonication
buffer again and half of them were disrupted by sonication
at 4 °C. After the lysate was centrifuged at
12000×g for 15 min, the resultant supernatant was recovered and the
resultant precipitate that had the insoluble fraction was
resuspended in 25 mL sonication buffer.
Polyclonal antibody preparation E
coli strain BL21(DE3) was transformed with pET-hPPGLBD, then grown to an
OD600 of 0.6 in LB media containing 60 mg/L ampicillin at 37 °C,
then the cells were induced with 1 mmol/L IPTG for another
6 h at 30 °C. The cells were harvested and disrupted by
sonication as described in the previous section. The human
PPARg2 LBD was expressed as inclusion bodies, whose
homogeneity was estimated to be greater than 90% by visual
inspection of Coomassie brilliant blue-stained sodium
dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) gels. The isolated inclusion bodies were used as
antigens to immunize mice.
The p171Bio3 vector containing lambda capsid gene gpD
was transformed into E coli BB4, which were then grown to
an OD600 of 0.6 in LB media and induced with 1 mmol/L IPTG
for 6 h at 30 °C. The cell extracts from sonication were
applied to a 12% SDS-polyacrylamide gel. After
electro-phoresis, the gels were soaked in 100 mL 0.3 mol/L KCl at
4 °C for 15 min. When the pD protein band in the gel became
white, the band was cut out, frozen and thawed. Proteins in
it were recovered with 1 mL 0.1 mol/L phosphate-buffered
saline (PBS) and used as antigens for immunization.
Polyclonal antibodies to human PPARg LBD (anti-LBD)
or lambda pD (anti-pD) were prepared by immunizing female
BALB/c mice with 50 µg of the recombinant protein
emulsified with an equal volume of Freund¡¯s complete adjuvant.
The mice were given booster immunizations three times
every 10 d with the same amount of antigen in Freund¡¯s
incomplete adjuvant. Ten days after the last immunization, blood
was collected for testing of antibody reactivity. Afterwards,
sera from the mice that contained the polyclonal antibodies
were collected.
Lambda lysogen preparation and lambda phage rescue
and titration Lysogenic BB4 was generated by infecting
E coli strain BB4 with lDam15 b538
cIts857 nin5 Sam100, then selecting lambda lysogens at 32 °C. The prophage contained
78.5% of the genome of the wild type phage and an amber
mutation in the gpD gene[18].
For lambda phage rescue, the lysogenic BB4 strain
transformed with p171Bio3 or p171-LBD was grown under
non-inducing conditions (below 38 °C) to an
OD600 of 0.3 at 32 °C in 50 mL LB media containing 0.2% maltose, 0.1% glucose
and 10 mmol/L MgSO4 with agitation, then induced at 42 °C
for 15 min to inactivate the temperature-sensitive
cIts857 repressor. IPTG was then added to the culture to a
concentration of 1 mmol/L and incubated at 38 °C for an additional 3 h with vigorous agitation. After 1 mL chloroform was added
to the culture to complete cell lysis, the culture was
incubated in a shaker for an additional 15 min. The released
phage particles in the culture were purified by two rounds of
standard PEG and NaCl precipitation, and the resultant phage pellets were resuspended in 5 mL SM buffer (0.1
mol/L NaCl, 10 mmol/L MgSO4, 50 mmol/L Tris, 0.01% gelatin, pH 7.5), and stored at 4 °C.
For lambda phage titer determination, lambda phage
samples were serially diluted in SM buffer, then mixed with
0.2 mL fresh cultured BB4 bacteria
(OD600 of 0.5) grown in LB medium containing 0.2%
(w/v) maltose and 10 mmol/L
MgSO4. After 30 min of incubation at 37 °C, infected cells
were mixed with 3 mL of molten LB top agar containing 0.2%
maltose, 10 mmol/L MgSO4 and poured immediately onto LB
plates, which were then incubated overnight at 37 °C. Phage
plaque number on the plate was counted and the titer of
lambda phage was calculated.
SDS-PAGE and Western blot analysis The protein
expression of pCGMT, pCGMT-LBD, p171Bio3 and p171-LBD
in the transformed BB4 strain was assayed. After cell lysis
by sonication, samples of the supernatant fraction and the
precipitated fraction were analyzed on a 10% SDS-PAGE gel.
For the expression comparison of PPARg LBD fused to g3p
or pD, equal amounts of total cell protein, supernatant
fraction and precipitated fraction after sonication were analyzed
by using standard SDS-PAGE. The bands were visualized
by using Coomassie brilliant blue staining and Western
blotting with anti-LBD polyclonal antibody. For phage
electro-phoresis, 1×109 lambda phage particles were mixed with the
loading buffer and boiled for 15 min, then applied to a 10%
SDS-PAGE gel and analyzed with anti-LBD or anti-pD
polyclonal antibody.
For Western blot analysis, separated proteins in the gels
were electrophoretically transferred onto a PVDF membrane
(Immobilon-P, Millipore) at 400 mA for 90 min. The
membrane was blocked in a blocking buffer [3% bovine serum
albumin (BSA) in Tris-buffered saline (TBS), 150 mmol/L NaCl,
pH 7.4] for 2 h at room temperature, and then incubated with
either a primary antibody (anti-LBD or anti-pD polyclonal
antibody, diluted 1:1000 in the blocking buffer) at 37 °C for 1 h. After three washes in TBST (0.1% Tween-20 in TBS
buffer, pH 7.4, 10 min for each wash), the blots were
incubated with a horseradish peroxidase (HRP)-conjugated goat
anti-mouse IgG secondary antibody (Calbiochem; at a
dilution of 1:1000 in the blocking buffer) at room temperature for
1 h. The blots were then washed in TBST (three times, 10
min for each wash), then stained with HRP substrate
diaminobenzidine (DAB).
Phage capture assay The plates were coated with the
serum containing anti-LBD polyclonal antibody in
carbonate buffer (50 mmol/L NaHCO3, pH 9.6) overnight at 4 °C (100
µL/well, n=6), and the control wells were coated with the
serum from the non-immunized mice (n=3). After discarding
the coating solution, each well was incubated with 200 µL
blocking solution (2% BSA in PBS, 0.05% Tween-20) for 2 h
at 37 °C. A total of 1×108 lp171Bio3 particles per well were
added to three wells coated with anti-LBD antibody, and the
same amount of lp171-LBD was added to the other three
wells coated with anti-LBD antibody and the three wells
coated with control serum, and incubated for 1 h at 37 °C
with gentle agitation. Afterwards, the plate was washed three
times with 200 µL washing solution (PBS, 0.05% Tween-20,
10 mmol/L MgSO4) and once with PBS (10 mmol/L
MgSO4). The lambda phages binding to each well were recovered by
directly adding 200 µL of fresh cultured BB4 cells. After 30
min of incubation at 37 °C, the phage titer of the mixture was
determined as described earlier. The statistical significance
of the differences between the captured phage titers was
assessed by using the paired Student¡¯s t-test, and the level
of statistical significance was set at P<0.05.
Results
Construction of p171-LBD, pCGMT-LBD and pET-hPPGLBD expression vectors
Plasmid p171-LBD (Figure 1) was constructed by inserting the PCR-amplified fragment of
the PPARg2 gene (coding amino acids 201-505) into the
SpeI-NotI site of p171Bio3, which contained the strong
tac promoter and the
lacIq gene, and thus could tightly control the
expression of the downstream lambda capsid gene
gpD. PPARg LBD was expressed as a fusion to the carboxyl
terminus of pD (approximately 11 kDa), which is one of the lambda
phage head proteins that form the protruding trimeric
structure essential for the stability of the capsid, and is also used
as a carrier protein for lambda phage
display[6,11].
The pCGMT-LBD plasmid (Figure 1) was constructed
similarly, except that different cloning sites were used. The
coding region of PPARg LBD was cloned into the 5¡¯ terminus
of g3 in phagemid pCGMT, which contained a
lac-promoter, a pelB signal sequence, and an amber codon between the fusion protein and the truncated g3p. PPARg LBD would be
expressed as a fusion protein in an amber suppressor strain,
such as BB4, to the N terminus of g3p, which is a minor coat
protein of phage M13[19], and the most commonly used
carrier protein for displaying large proteins in a filamentous
phage display system because it is less sensitive to the size
of protein inserts.
Plasmids of pET-hPPGLBD were constructed by cloning
the PCR fragment of the PPARg2 LBD into BamHI and
SalI sites in pET-21a, in an attempt to express a large amount of
PPARg LBD protein in E coli in order to obtain antigens for
generating mouse anti-LBD antibodies.
Lambda capsid protein pD was a more suitable carrier
protein for displaying PPARg LBD than g3p In order to
assess which of the 2 phage display systems (ie, the
filamentous phage system or the lytic lambda display system) was
more appropriate for displaying PPARg LBD, a comparison
between the expression of PPARg LBD fused to capsid
protein g3p or pD was firstly performed.
pCGMT-LBD and p171-LBD produced the LBD-g3p and
pD-LBD fusion proteins, respectively, when expressed in
the amber repressor bacterial strain BB4
(supE). The estimated molecular weights of these 2 proteins were 55 kDa
(the carboxyl-terminal PPARg protein is approximately 35 kDa,
whereas the truncated g3p accounts for approximately 20
kDa) and 46 kDa (pD is approximately 11 kDa), respectively.
The proteins in the induced total cells, the supernatant and
precipitated fractions of sonication lysate were analyzed with
Coomassie brilliant blue staining (Figure 2A) or Western
blotting with anti-LBD antibody (Figure 2B). A protein band
with a molecular weight of 55 kDa, corresponding to
LBD-g3p, was observed in the total cell lysate (lane 3), and a
35-kDa band corresponding to pD-LBD was observed as well
(lane 6), indicating that PPARg LBD could be expressed as a
fusion protein after induction. However, almost all of the
LBD-g3p protein was insoluble (lane 5), and no LBD-g3p
protein was found in the supernatant fraction (on the basis
of anti-LBD antibody detection; lane 4). Although
over-expression of pD-LBD protein could make this fusion
protein form insoluble inclusion bodies as well (lane 8), a
reasonable amount was expressed in a soluble form (lane 7).
Furthermore, by density analysis it was shown that the
soluble protein accounted for approximately 40% of the total
expressed LBD. These results show that the pD-LBD fusion
protein was partially soluble when expressed in
E coli, whereas the LBD-g3p fusion protein was detected only in
the insoluble fraction. Because solubility was a prerequisite
for displaying foreign proteins on the phage surface, the
lambda capsid protein pD seems to be a more appropriate
carrier protein for displaying PPARg LBD. Thus
PPARg LBD expressed with the lambda system rather than the
filamentous phage system was further characterized.
Part of PPARg LBD fused to pD was expressed in soluble
form in bacterial cytoplasm Additional studies for
assessing the solubility of expressed pD-LBD under induction and
noninduction conditions were performed. Crude protein
extracts from BB4 cells transformed with vector p171-LBD or
parent vector p171Bio3 were analyzed by SDS-PAGE (Figure
3A) and western blotting (Figure 3B). As shown in Figure
3B, a small amount of soluble pD-LBD (46 kDa) was detected
in the supernatant (Figure 3B, lane 6, without IPTG induction),
whereas no pD-LBD was detected in the precipitated
fraction (Figure 3B, lane 7). After IPTG induction (Figure 3B,
lanes 8 and 9), the expression of the pD-LBD fusion protein
increased markedly. Because the pD proteins could only
improve their solubility to some extent, most of the
overexpressed fusion protein under induction conditions
aggregated in an insoluble form (Figure 3B, lane 9), whereas
soluble pD-LBD increased moderately with IPTG induction
(Figure 3B, lane 8). The relative amount of pD-LBD fusion
protein in the supernatant and precipitated fraction was
estimated from the PVDF membrane by densitometry measurements, which indicated that approximately 30% of
total PPARg LBD fusion proteins were expressed in soluble
form. Although a reasonable number of them were expressed
as insoluble inclusion bodies, quite a few of the
hydrophobic PPARg LBD could maintain their solubility after being
fused to the lambda pD protein, which might be sufficient for
the protein to be displayed on the surface of lambda phage.
PPARg LBD could be incorporated into lambda phage
capsid Protein extracts from rescued lambda phage particles
of lp171Bio3 or lp171-LBD were probed with anti-LBD
(Figure 4A) or anti-pD (Figure 4B) polyclonal antibody,
respectively, after electrophoresis on a 10% SDS-PAGE gel,
and were transferred onto a PVDF membrane.
Western blotting analysis with an antibody against
PPARg LBD produced a prominent band of 46 kDa in the
lp171-LBD lane, whereas no corresponding band was detected in the control lane of lp171Bio3 (Figure 4A).
More-over, the result was validated by probing with anti-pD
polyclonal antibody. Two bands, with molecular weights of
approximately 11 kDa and 46 kDa, were detected (Figure 4B),
which represented protein pD from the gpD gene of the
integrated prophage genome, and the pD-LBD fusion protein
from the gpD gene of the plasmid, respectively. The ratio of
the proteins in the two bands represented the level of the
pD-LBD fusion protein incorporated into lambda phage.
Density comparisons of the two bands of lp171-LBD in Figure 4B indicated that the amount of fusion pD accounted for
nearly 28% of the total pD protein content on lambda phage.
Because there were 405 copies of protein pD on the capsid
of wild-type lambda, we could estimate that the average
number of PPARg LBD incorporated into the lambda phage capsid
was approximately 115 per phage particle. In summary, we
conclude that the ligand-binding domain of PPARg could be
efficiently incorporated into lambda phage particles.
Phage capture assay indicated that PPARg LBD was
expressed on the surface of lambda phage To identify the sites
displaying PPARg LBD on lambda phage, a phage capture
assay was performed. The titer of phage lp171-LBD was
compared to that of lp171Bio3 after binding to wells coated
with the anti-LBD antibody. From the results presented in
Figure 5, the titer of captured lp171-LBD phage with
incorporation of PPARg LBD was approximately four times more
than that of captured lp171Bio3 (P<0.01), indicating that
PPARg LBD incorporated in phage capsids is selectively
recognized by mouse antibodies. However, on the wells coated
with mouse normal serum rather than anti-LBD antibody
serum, the titer of lp171-LBD captured decreased markedly
(P<0.01), which indicated that only anti-LBD antibody could
capture lp171-LBD. It was clear that the PPARg LBD
incorporated in lp171-LBD phage exhibited specific binding to
the immobilized anti-LBD antibody. In summary, we
conclude from our results that the PPARg LBD was displayed on
the surface of the bacteriophage lambda capsid.
Discussion
Nuclear receptors are a large family of transcription
factors involved in many important metabolic processes. To
date, 48 members have been identified in the human genome,
and all the members of this family have a modular structure
composed of six domains (A-F)[2]. Endogenous ligands have
not been identified for all NR. NR are termed "orphan
receptors" if their endogenous ligands have not yet been
discovered, and "adopted" when their endogenous ligands
are identified. The identification of new ligands for NR not
only provides the opportunity to elucidate their function,
but can also bring about the discovery of potential
therapeutic agents for human
diseases[3,20].
Many in vitro high-throughput screening methods have
been applied in an effort to search for novel ligands for these
nuclear receptors; however, the production of large
quantities of highly soluble proteins as well as the subsequent
purification of these proteins is the main obstacle to
overcome in these high-throughput assays for novel ligands
using traditional screening systems. E
coli cells offer a convenient and inexpensive expression system for the production of human proteins; however, the high-level expression
of recombinant proteins in E coli often results in the
formation of insoluble inclusion bodies. The commonly used
approaches to address the solubility problems of recombinant
proteins have focused on optimizing expression conditions
or on fusion of protein partners, such as
glutathione-S-transferase (GST)[21], maltose binding protein
(MBP)[22] and thioredoxin
(Trx)[23]. However, these methods are not
always effective, especially for very hydrophobic proteins.
Phage display techniques can couple protein expression
and purification with the subsequent screening steps after
the protein is assembled on the phage surface, which can
circumvent the problems associated with protein
purification in conventional affinity screening methods. By affinity
binding with a given target, proteins can be isolated and
identified without consideration of protein purification and
yield, and vice versa. Previous reports have demonstrated
that large proteins can be displayed on the phage surface,
and this technique has been proven to be useful in
high-throughput screening for antagonists of the receptor and
other proteins[5]. However, these previous successfully
displayed proteins share the properties of soluble proteins when
expressed in E coli, such as antibody fragments (scFv),
enzymes[11,12], bacterial proteins (staphylococcal protein
A)[24] and virus capsid proteins (HIV-1 p24, and
HCV)[9]. Whether aggregation-prone proteins can be displayed on the phage
surface was not elucidated.
The four types of variant PPARg, which come from
alternative promoters and differential
splicing[4], have the same LBD and C-terminus. Because
its LBD and DBD function independently, as in the other nuclear receptors, it is
possible to express a truncated PPARg or an isolated
PPARg domain to study its functions and binding characteristics.
However, the ligand-binding pocket in the LBD makes
expression more difficult because of its hydrophobic
nature[25]. Our experiments indicated that nearly all the recombinant
PPARg LBD is in an insoluble form when expressed in a pET
system for antigen preparation (unpublished data).
Other reports have also indicated that the ability to
remain soluble is a prerequisite for a protein to be incorporated
into the phage surface. An improvement in protein
solubility after partner fusion or molecular chaperone coexpression
can increase the incorporation efficiency of fusion proteins
displayed on the phage surface[26,27]. However, unlike the
traditional affinity screening methods, which require the
purification of large quantities of soluble protein, phage
display only requires the protein to be moderately soluble, and
a small amount of soluble protein is sufficient to be expressed
on the phage surface. Furthermore, lytic bacteriophages such as lambda, T4 or T7 have been shown to be promising
systems for the of display foreign proteins, because the
encapsidation of the foreign fusion protein is an intracellular
event, thus making the secretion of the fusion protein a
less-demanding process and gaining an advantage over the
filamentous phage for displaying foreign
proteins[6-8]. Our results showed that protein pD improved the solubility of
pD-LBD to some extent, and that the fusion protein was soluble
enough for phage display of PPARg LBD. This might be a
useful approach for circumventing the expression and
purification problems in traditional screening methods.
Moreover, proteins as large as b-galactosidase have been
successfully displayed on lambda phage surfaces as fusions
to the amino or carboxyl terminus of protein
pD[11,12], which implies that the size limit for proteins in lambda phage
display systems is not very strict. Thus the pD lambda phage
system is superior to other phage display systems, and is
particularly appropriate for the expression of large proteins
that tend to form insoluble inclusion bodies.
The lambda display system based on protein pD is a
polyvalent display system, which is useful for the efficient
selection of ligands with either low or moderate binding
affinity. Previous studies indicate that the percentage of
fusion protein incorporated in the capsid can reach up to
90% of the total pD protein
content[24,28], which makes it difficult to select high-affinity binding ligands. However, the
efficiency of selection of high-affinity ligands can be
improved, because the expression of pD fusion protein can
be regulated by the promoter. The pD-LBD fusion protein
displayed on the lambda phage capsid represented nearly
one-third of the total pD protein content. By altering the
ratio of wild type pD to pD fusion protein, it is possible to
change the valency of fusion protein on the lambda surface.
Our results demonstrated that PPARg LBD fusion
protein was incorporated into the lambda phage capsid and
expressed on the surface of the lambda phage. Further studies
remain to be conducted to characterize the activity of the
displayed PPARg LBD, including binding assays with known
PPARg ligands, as well as pilot assays involving ligand
screening by directly panning phage-displayed fusion
proteins against immobilized molecules, or small molecule
competition binding between test compounds and
known complexes of phage-displayed PPARg LBD and its natural
ligand. This system may be a new alternative for expressing foreign
proteins that tend to be insoluble when using conventional
approaches, and quite possibly has great value for
downstream screening of novel PPARg ligands.
Acknowledgement
We gratefully acknowledge the gift of the E
coli strain and plasmid from Dr Alfredo NICOSIA (IRBM, Rome, Italy)
and Dr Hitoshi NISHIZAWA (Osaka University, Osaka,
Japan).
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