Cloning, Expression and Characterization of Recombinant Human Fc Receptor Like 1, 2 and 4 Molecules

Document Type : Research Paper


1 Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, IR Iran and Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IR Iran

2 Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, IR Iran

3 Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran, IR Iran

4 Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IR Iran



Background: The Fc receptor like (FCRL) molecules belong to the immunoglobulin (Ig) superfamily with potentially immunoregulatory function. Among the FCRL family FCRL2 and 4 are predominantly expressed on memory B cells and FCRL1 is a pan- B cell marker. To date, no ligand has been identifid for the human FCRL1, 2 and 4 molecules. Objectives: Cloning, expression, purifiation and structural analysis of the extracellular domain of human FCRL1, 2 and 4 proteins. Materials and Methods: In this study, the extracellular part of human FCRL1, 2 and 4 were subcloned into prokaryotic expression vectors pET-28b (+) and transformed into BL21-DE3 E.coli strain. Protein expression was optimized by fie adjustments such as induction time, incubation temperature and expression hosts. Recombinant FCRL proteins were purifid by metal affity chromatography using Ni-NTA resin. Purifid FCRL proteins were further characterized by SDS-PAGE and immunoblotting using His-tag and FCRL specifi polyclonal antibodies. Results: Our results demonstrated that FCRL1, 2 and 4 were successfully expressed in pET-28b (+) vector. Optimization of the expression procedure showed that IPTG induction at OD600 = 0.9 and overnight incubation at 37˚C resulted in the highest expression levels of FCRL proteins ranging from approximately 15% (FCRL1) to 25% (FCRL2 and 4) of the total bacterial lysate proteins. Conclusions: These purifid recombinant proteins are potentially a valuable tool for investigating the immunoregulatory function of FCRL molecules and the production of specifi mAbs for immunotherapeutic interventions.


1. Background
Signaling through the B-cell Receptor (BCR) can induce
B cell activation, proliferation, diffrentiation or apoptosis. Thus, regulation of the BCR signaling is crucial for B
cell development and function (1). Balancing the competing activation and inhibitory signals is critical in regulating a BCR-mediated response to an antigenic stimulation.
In addition to classical BCR negative regulators (FcγRIIb,
CD22 and CD72) and positive regulators (CD19 and CD86),
it has been recently shown that Fc receptor like (FCRL)
molecules also have the potential to be important regulators of BCR signaling (2-4).
The human FCRL family is composed of six homologous
members which are exclusively expressed in immune
cells, particularly B cells. Each member possesses three to
nine Ig domains in its extracellular region, a cytoplasmic
tail together with consensus immunoreceptor tyrosinebased activating (ITAM) and/or inhibitory (ITIM) motifs
(2, 5). FCRL1, 2 and 4, which are the subject of this study,
have been found only on B lineage cells (3), with FCRL2
and 4 being predominantly expressed in memory B cells
(3, 4), whereas FCRL1 is a pan B cell marker (2). Due to their
unique expression on B lymphocytes, FCRLs have been
introduced as suitable targets for immunotherapy of Bcell malignancies such as chronic lymphocytic leukemia,
hairy cell leukemia and B-cell non-Hodgkin's lymphoma
(6, 7). FCRL1 has 2 ITAM-like motifs, a glutamic acid residue in its transmembrane region, and 3 extracellular Iglike domains (2). FCRL2 has four extracellular Ig domains,
an uncharged transmembrane segment and a cytoplasmic tail that contains a potential ITAM and two consensus ITIMs (3). FCRL4, on the other hand, has 3 ITIM-like
motifs, three acidic glutamic acid residues in its trans membrane region and 4 extracellular Ig-like domains (4,
8). Thus, FCRL1 and FCRL4 could operate as either activating or inhibitory receptors, whereas FCRL2 may play as a
bifunctional receptor due to possessing both ITAM and
ITIM motifs.
2. Objectives
To date, no ligand has been identifid for any of the human FCRL molecules, except FCRL6 which has recently
been shown to bind to HLA-DR, an MHC class II molecule
(9). Search for functionality of these molecules without
known ligand(s) needs extensive investigations using recombinant FCRL molecules and specifi polyclonal and
monoclonal antibodies (mAbs). In this study, we present data on the cloning, expression and purifiation of
extracellular part of human recombinant FCRL1, 2 and
4 molecules. The optimization of the expression protocol resulted in a high expression level in a prokaryotic
expression system with a potential implication for antibody production and functional and structural analyses
of FCRL molecules.
3. Materials and Methods
3.1. FCRL Plasmids Construction
The FCRL1-pCMV6-XL5 (SC123097, NM_052938, 2657bp),
FCRL2-pCMV6-XL5 (TC305281, NM_030764, 1600bp) and
FCRL4-pCMV6-XL5 (SC305326, NM_031282, 1600bp) DNA
ready to transfection have been purchased from ORIGENE
company (ORIGENE, MD, USA). These constructs were used
as template for subcloning the extracellular portion of
FCRL molecules into pET-28b(+) vector containing 2 Histags in the up and down stream of the multiple cloning
site (Novagen, Madison, WI, USA). The extracellular region
of FCRL1, 2 and 4 were amplifid with FCRL specifi primers harboring restriction enzymes cutting sites (Table 1).
Briefl, PCR amplifiation was performed with pfu taq DNA
polymerase (Promega, Madison, WI, USA) as follows: heating for 5 min at 95 ºC, 35 cycles of denaturation for 30 s at
95 ºC, an annealing process for 30 s at 57 ºC, and an extension for 2 min at 72 ºC. The amplifid genes were electerophoresed on a 1.5% Low Electroendosmosis (LE) agarose
gel, extracted and purifid by QIAquick gel extraction kit
(Qiagen, Hilden, Germany) according to the manufacturer’s protocol. In the next step, seven thymidine bases were
added to the 5' end of FCRL specifi primers (defied as TTFCRL primers) used as docking site for restriction enzymes.
Then the purifid PCR products were used as template for
amplifiation using the new set of TT-FCRL primers following the same PCR protocol described above. The new
PCR products were subsequently extracted and digested
with the corresponding restriction enzymes. After re-purifiation, the FCRL amplicons were ligated into digested
pET-28b(+) vector using T4 DNA ligase (Promega, Madison,
WI, USA) according to the manufacturer’s instruction. The
inserted genes were verifid by restriction enzyme digestion and DNA sequencing methods using universal T7 promoter and terminator primers (Applied Biosystems 3130
genetic analyzer, Foster City, CA, USA).

3.2. Expression of Recombinant FCRL Proteins
The competent BL21-DE3 Escherichia coli strain (Novagen,
Madison, WI, USA) was transformed with FCRL-pET-28b (+)
constructs by heat shock method. Briefl, 50 μL of competent BL21-DE3 glycerol stock was thawed on ice and mixed
gently. Then 1 μL of FCRL-pET28b (+) construct was added
directly to the bacterial suspension, stirred gently and incubated on ice for 5 min. The mixture was incubated for
exactly 70 s in a 42°C water bath and quickly placed on
ice for 2 min. For recovery, 100 μL of regular Luria–Bertani
(LB) broth (10 g bacto-tryptone (Sigma, St. Louis, USA),
5 g bacto-yeast extract (Sigma, St. Louis, USA), 5 g NaCl
(Roche, Germany), pH 7.0 was added on the transformed
cells and incubated at 37 °C for 1 h without shaking. The
transformed bacteria were cultured on LB agar plates
containing Kanamycin and selected by colony PCR using
T7 universal promoter primer (Novagen, Madison, WI,
USA) as forward and Not I-FCRL1, 2 and 4 as reverse primer
(Table 1). Single transformed colony was inoculated into
5 mL pre-heated LB broth medium supplemented with
50 µg.mL -1 Kanamycin while being shaked at 250 rpm
at 37 ºC. Three milliliters of the overnight starter culture
were added to 500 mL of LB media and let OD600 nm to
reach the desired point. Protein expression was induced
with 1 mM isopropyl-1-thio-β-D-galactoside (IPTG) (Sigma,
St. Louis, USA) and cells were grown for a further 14 - 16 h
in shaker incubator at 250 rpm at 37 ºC. One milliliter of

cultured induced bacteria was collected and centrifuged
at 4000 × g for 10 min. The pellet was dissolved in SDSPAGE sample buffr 6X (Tris-base 0.27 M, pH = 6.8, SDS 6%,
bromophenol blue 0.06%, glycerol 60%, DTT 0.1 M) and
heated in boiling water bath for 3 min. After centrifugation at 13000 rpm for 5 min, 20 µL of supernatant were
separated by 10% SDS-PAGE and stained by coomassie brilliant blue. Furthermore, the expression of recombinant
FCRL proteins in bacteria was confimed by immunoblotting technique.
3.3. Purifiation of Recombinant FCRL Proteins
After the confimation of the recombinant FCRL expression, the remaining amount of cultured bacteria
was centrifuged and the pellet was washed twice with
wash buffr (NaH2PO4 100 mM and Tris-Hcl 30 mM) and
stored at 70 ºC overnight. For cell lysing a ratio of 10 mL
of cold lysis buffr (pH = 8 containing NaH2PO4 100 mM,
Tris-Hcl 30 mM and NaCl 100 mM) to 1 g of wet weight
bacteria was applied and mixture was incubated for 1 h
on ice. Cells were lysed completely by sonication with
15 cycles (60 s on at 25% power, 60 s off at 0ºC) (HD2070
BANDELIN Sonopuls, Berlin). The lysate was centrifuged
at 10000 rpm for 10 min at 4ºC and the inclusion bodies in the pellet were dissolved in buffr A (NaH2PO4 100
mM, Tris-base 10 mM, urea 8 M, NaCl 100 mM, pH = 8)
containing imidazole 30 mM and centrifuged at 12000
rpm for 10 min. Ni–NTA agarose resin (Qiagen, Hilden,
Germany) was equilibrated with buffr A. The resin was
then mixed with transparent fitered lysate at a ratio of 1
mL of resin (50% slurry) with 1 mL of lysate and the mixture was shaken for 30 - 45 min on a rotator platform.
After coupling the FCRL-His-tag recombinant proteins
to Ni-NTA resin, unbound fraction was washed and removed with buffr A supplemented with 30 mM imidazole. Bound proteins were subsequently eluted by gradual increasing of imidazole molarity (80, 300 and 1000
mM) in buffr A. In order to remove urea, the eluted
recombinant proteins were dialyzed against phosphate
buffr saline (PBS). In addition, protein purity was determined by analyzing coomassie-stained SDS-PAGE as well
as immunoblotting.
3.4. Production of Polyclonal Antibodies to FCRL
The specifi peptides related to extracellular part of human FCRL1, 2 and 4 were designed (Table 2), purchased
from Thermo Electron Corporation (Thermo Electron
Corporation, GmbH, Ulm, Germany) and conjugated to
Keyhole Limpet Hemocyanin (KLH) (Sigma, St. Louis, USA)
according to standard protocol. Briefl, 1 mg of Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) in 200 µL
of Dimethyl Formamide (DMF) was added to the KLH solution (5 mg in 1 mL deionized water) and incubated at room
temperature for 2 h with gentle stirring. The MBS activated
KLH was dialyzed against large volumes of PBS overnight
and added to the FCRL peptide solution (5 mg in 1 mL PBS)
and the mixture was incubated for 4 h at room temperature. After overnight dialysis against PBS, the FCRL-KLH
conjugates were stored at -20 °C for later use. Six-monthold New Zealand white rabbits were immunized subcutaneously with the FCRL peptide-KLH in Freund’s adjuvant
fie times on weeks 0, 4, 7, 9, 11. The fist immunization
was performed using an emulsion of peptide-KLH (250
µg) and Freund’s complete adjuvant (Sigma, St. Louis, USA)
and the subsequent ones with peptide-KLH (125 µg) and incomplete Freund’s adjuvant (Sigma St. Louis, USA). Blood
samples were taken before every immunization for evaluation of anti-FCRL peptide antibody titration by ELISA. Sera
were obtained from the immunized rabbits 2 weeks after
the last immunization. The rabbits' IgG were purifid with
affity purifiation using protein-G Sepharose 4B columns (GE Healthcare, Uppsala, Sweden) and their purity
was checked by SDS-PAGE.

3.5. Immunoblotting of Recombinant FCRL Proteins Under Reduced and Non-Reduced Conditions
Purified FCRL recombinant proteins were suspended
in sample buffer, separated on a 10% SDS-PAGE under
reduced and non-reduced conditions and electrophoretically transferred onto polyvinylidene difluoride
(PVDF) membrane (Millipore Corporation, MA, USA).
The membrane was blocked with 5% skim milk in PBS
containing 0.05% Tween 20 (Sigma, St. Louis, USA)
(PBST) at 4 ºC overnight and subsequently immunoblotted with primary antibodies [polyclonal rabbit anti-His-tag (abcam, Cambridge, UK) at 1: 1000 dilution,
polyclonal anti-FCRL1 and FCRL4 peptides produced in
our lab at 10 - 15 µg.mL-1 concentration] for 1.5 h at room
temperature on shaker platform. After extensive washing with PBST, the membrane was next incubated with
the appropriate dilution of peroxidase-labeled sheep
anti-rabbit Ig (produced in our lab) for 45 min. The
bands were visualized using ECL chemiluminescence
detection system (GE Healthcare, Uppsala, Sweden)
and exposed by ECL Hyperfilm.
3.6. Transient Transfection of CHO Cell Lines
The transfection reagent, jetPEI (Polyplus, Paris, France),
was used to transfect the CHO cell line (National Cell Bank
of Iran, Tehran, Iran) based on the manufacturer’s recommendation. The CHO cells were seeded at 3 × 105 cells/well
in a 6-well plate a day before transfection. On the transfection day, 6 μL of jetPEI and 3 μg of constructs were diluted
with 100 μL of 150 mM NaCl separately and mixed gently.
The DNA solution was then added to the jetPEI solution,
and after 20 min of incubation at room temperature, 200
μL of the jetPEI/constructs mixture were added drop-wise
onto the cells grown in the serum containing medium.
The mixture was homogenized by gently swirling the
plate and the cells were incubated at 37 °C and 5% CO2 in a
humidifid atmosphere. Transfection experiments were
analyzed after 48 h by flw cytometry.
3.7. Inhibition of Binding of Anti-FCRL mAbs to FCRL
Transfected Cells by Recombinant FCRL Proteins
In this experiment 10 µg.mL-1 of commercial mAbs to
FCRL1 (1F9, human IgG4), FCRL2 (7G7, mouse IgG1), FCRL4
(1A3, mouse IgG2a) (generous gift from Prof. Polson, Genentech Inc., CA, USA), mAbs against recombinant FCRL2
( 3D8-G8, mouse IgG1) and FCRL4 (1A5-C10 mouse IgG2b)
(produced in our lab, data not presented) were separately
adsorbed to 10 and 40 µg.mL-1 of the corresponding purifid recombinant FCRL1, 2 and 4 proteins at room temperature overnight. Adsorbed and non-adsorbed mAbs were
used to stain CHO cells transiently transfected with the
corresponding FCRL genes. Stained cells were subjected
to flw cytometry.
The transient CHO-FCRL and CHO-pCMV6-Neo transfected cell lines were washed 2 times with wash buffr (PBS
containing 1% BSA) and stained with 5 µg.mL-1 of commercial mAbs (adsorbed and non-adsorbed) and 10 µg.mL-1 of
our FCRL2 and 4 mAbs (adsorbed and non-adsorbed) for 1
h at 4ºC. Cells were then washed 2 times with wash buffr
and stained with appropriate dilution of FITC-conjugated
goat anti-mouse Ig (Sigma, St. Louis, USA) or FITC-conjugated goat anti human polyvalent Ig. Stained cells were
subsequently analyzed by flw cytometry (FacsScan flw
cytometer, Becton-Dickinson, San Jose, CA, USA).
4. Results
4.1. FCRL Plasmids Construction
The extracellular region of FCRL1, 2 and 4 molecules was
amplifid from the full-length FCRL1-, FCRL2- and FCRL4-
pCMV6-XL5 constructs using PCR. Electrophoresis of the
PCR products verifid the amplifiation of the target
genes. The size of purifid FCRL1, 2 and 4 amplicons was
936 bp, 1216 bp and 1172 bp, respectively (data not presented). The amplifiation was repeated on the purifid
products using specifi TT-FCRL primers and the amplifid products were directly digested. The DNA fragments
encoding FCRL1, 2 and 4 were then cloned into prokaryotic expression plasmid pET-28 b(+), followed by restriction site analysis and DNA sequencing for fial approval.
DNA sequencing analysis showed that the sequences of
FCRL1, 2 and 4 were consistent with those deposited at
NCBI (FCRL1 GenBank accession no: NM_052938, FCRL2
GenBank accession no: NM_030764 and FCRL4 GenBank
accession no: NM_031282). These results clearly indicate
that the expression plasmids were constructed successfully with no mutation, frame shift or stop codons.
4.2. Optimization of Expression of the FCRL Constructs
The pET-28b (+)-FCRL1, 2 and 4 constructs were transformed into BL21-DE3 E.coli strains and cultured on LB agar
medium containing Kanamycin (50 µg.mL -1 ). Some single
growing colonies were checked by PCR using T7 promoter
as forward and Not I-FCRL1, 2 and 4 as reverse primers (Table 1). Positive colonies were selected and protein expression was induced by IPTG (1 mM) when OD600 reached
a 0.6 absorbance value and then were cultured at 25 ºC
overnight. Interestingly, SDS–PAGE analysis clearly indicated that all FCRL molecules were successfully expressed
in BL21-DE3 transformed cells (Figure 1 A). Theoretically,
the molecular weight of extracellular recombinant FCRL1
(354aa, PI: 6.43), FCRL2 (413aa, PI: 6.5) and FCRL4 (437aa, PI:
6.8) proteins was determined to be 38.5 KDa, 45.1 KDa and
48.8 KDa, respectively. The expressed FCRL1 and 4 molecules contain two His-tags at their C- and N-terminals,
whereas FCRL2 contains one His-tag in its C-terminal. In
order to clone FCRL2, the Nco I restriction site was chosen
in order to sublcone it into pET-28 b(+), upstream of the
N-terminal His-tag. Thus, the expressed FCRL2 contains
only one His-tag in its C-terminus. Immunoblotting of
the transformed BL21-DE3-FCRL extracts with polyclonal
anti-His-tag antibody showed that all FCRL proteins were
strongly expressed and exhibiting the expected molecular weight (Figure 1 B). The results obtained from our
extensive optimization experiments, using a variety of
bacterial strains, including [BL21(DE3)-pLysS (Novagen,
Madison, WI, USA), BL21-CodonPlus-DE3-RIL (Stratagene,

La. Jolla, CA, USA), NovaBlue-DE3 (Novagen, Madison, WI,
USA), Origami™B-DE3-pLacI (Novagen, Madison, WI, USA),
Rosetta-gami™-DE3-pLysS (Novagen, Madison, WI, USA)
and JM109 (Takara, Kyoto, Japan)] (data not presented),
and diffrent induction times (1, 2, 3, 4, 5, 6, and 14 h),
incubation temperatures (37ºC, 25ºC and 18 ºC) and induction points (OD600 : 0.5, 0.7, 0.9 and 1.1) showed that
induction at OD600 = 0.9 in BL21-DE3 incubated at 37 ºC
are the optimum conditions for all of the FCRL genes expression. Representative results are illustrated in Figure
2. The densitometry of the expressed protein bands in
comparison to that of the total cell extract in each lane
showed expression effiency of approximately 15%, 25%
and 25% for FCRL1, FCRL2 and FCRL4, respectively

4.3. Purifiation of Recombinant FCRL Proteins
Since all the expressed proteins were obtained from
pellets of sonicated lysates after centrifugation as inclusion bodies, so purifiation was performed under
denaturation condition using 8M urea. Large amounts
of purifid recombinant FCRL proteins were collected
following elution of Ni-NTA resin with a buffr containing 300 mM and 1 M imidazole. Representative results
obtained for FCRL1, 2 and 4 purifiation are shown in
Figure 3. The purity of FCRL2 and FCRL4 was higher than
that of FCRL1 protein. The purifid FCRL proteins were
detected with polyclonal anti-His-tag antibody by immunoblotting (Figure 4). Although FCRL1 and 2 displayed
a single band on their SDS-PAGE, FCRL4 transformed
BL21-DE3 bacteria expressed FCRL4 in two diffrent sizes
(~47 and 36 KDa) (Figures 1, 3 and 4). The FCRL4 predicted size is 47 KDa, however, the smaller band was seen
both in SDS-PAGE and western blotting with polyclonal
anti-His-tag antibody (Figure 1). This band could also be
detected with polyclonal anti FCRL4-2 peptide (Figure 5
F) and the commercial anti FCRL4 mAbs (1A3) (data not
presented). These results confimed that both bands belong to FCRL4. FCRL4 protein has 4 Ig domains in its extracellular segment. The polyclonal antibody to FCRL4-2
peptide was designed to a segment of the 4th Ig domain
of FCRL4 proximal to membrane. Thus, it seems that the
small molecule represents the extracellular FCRL4 protein truncated at the N-terminal. This truncated protein
might have been generated due to enzymatic digestion.
The smaller fragment of FCRL4 has no effct on mAbs
production against this recombinant protein, because
it is a part of FCRL4. Further investigations should be
performed to identify whether it corresponds to the Nterminal or C-terminal of the native protein and whether
it displays any functional activity. Basically, the untruncated 47 KDa FCRL4 protein could be electro-eluted from
the SDS-PAGE gel for functional and structural studies.

4.4. Analysis of the Recombinant FCRL Proteins in
Reduced and Non-Reduced Conditions
To demonstrate the presence of disulphide bridges in
the recombinant FCRL proteins, all purifid recombinant
FCRL proteins were electrophoresed under reduced and
non-reduced conditions. When the recombinant FCRL1
and 2 proteins were run in non-reduced conditions in
SDS-PAGE, no band could be detected after staining the

gel with coomassie blue. Both proteins were polymerized and mostly retained in stacking gel and stackingresolving gels interface (Figure 5 A and C). Immunoblotting with polyclonal anti-His-tag antibody showed a
faint band in the stacking and resolving gels interface
(data not presented). This fiding implied that the polymerized FCRL1 and 2 recombinant proteins were not
transferred from gel to the PVDF membrane because of
their big size. To confim this assumption, the recombinant proteins were reduced with diffrent concentration
of DTT and run in a 8% resolving gel without a stacking
gel. These experiments showed that, by increasing the
concentration of DTT, the polymerized FCRL1 and 2 recombinant proteins were completely reduced to monomer structure (Figure 5 A-D). These results indicate
that the cysteine residues in FCRL1 and 2 proteins bind
to each other to form highly polymerized molecules.
Similar results were obtained for FCRL4 recombinant
protein, except that this protein was not totally polymerized in non-reduced conditions, and a band corresponding to monomer FCRL4 protein with approximately 36
KDa molecular weight, could be observed in non-reduced
conditions (Figure 5 E). Thus we decided to use only one
concentration of DTT for its reduction. The SDS-PAGE
and WB results showed that the reduced monomer band
seems to have a molecular weight higher than the non-reduced monomer, indicating linearization of the reduced
FCRL molecule resulting in a shorter migrated distance
in the gel (Figure 5 E and F). The diffrence between the
upper bands of Figure 5 E and Figure 5 F could be related
to the diffrence of electrophoresis running times for
the two experiments. The Coomassie Blue stained proteins shown in Figure 5 E were not transferred and blotted directly. This gel was used just to show the SDS-PAGE
pattern. We did run the samples again at the same conditions for the purpose of immunoblotting. Obviously,
there will be some variations between the two electrophoresis patterns, despite the fact that the same conditions were applied.
4.5. Identifiation of Purifid Recombinant FCRL
Proteins by FCRL-Specifi Polyclonal Antibodies
To further characterize the purifid FCRL proteins, we
separately raised polyclonal antibodies to 6 specifi peptides spanning two diffrent parts of the extracellular region of each FCRL molecule (Table 2). Sera collected from
all 6 immunized rabbits, each immunized with a single
peptide, reacted with the immunizing peptides at dilutions higher than 1/2500 by ELISA. However, only two of
the antisera produced to FCRL1-1 and FCRL4-2 peptides
could specifially recognize the target proteins by western blot (WB) technique (results not presented). IgG fractions purifid from serum of these two rabbits together
with a mAb to specifi for FCRL1 (clone 1H7-F6) (produced
in our lab) were employed to perform immunoblotting
on un-purifid FCRL proteins. The results showed specifi
reactivity of these antibodies with their corresponding
FCRL proteins without any cross-reactivity with other
FCRL proteins or other proteins from the lysate of nontransformed BL21-DE3 bacteria (Figure 6).
4.6. Reactivity of Anti-FCRL Peptide Polyclonal Antibodies with Mammalian FCRL Proteins
To test the reactivity of the polyclonal anti FCRL peptide
antibodies with the native FCRL proteins, anti-FCRL1-1 and
FCRL4-2 antibodies were applied in WB using cell extracts
from peripheral blood or tonsil; as well as a Burkitt's
lymphoma B-cell line (Ramos) known to endogenously
express FCRL1 protein. The polyclonal anti-FCRL1-1 recognized native FCRL1 protein in normal PBMCs, tonsil
mononuclear cells (MNCs) and Ramos B-cell line, however, the enriched T cells did not react with this antibody
(Figure 7). The polyclonal anti-FCRL4-2 could not detect
any band related to FCRL4 protein in PBMCs or tonsil
MNCs (data not shown). The restricted expression of
FCRL4 on memory B cells could explain these fidings.
Thus, the anti-FCRL4-2 peptide polyclonal antibody was
applied in WB on FCRL4 transfected CHO lysate. However,
no specifi band was detected (data not presented). Altogether, our results indicate that with the exception of
anti-FCRL1-1 peptide antibody, the polyclonal anti–FCRL4-
2 peptide antibody does not react with the native FCRL4
protein expressed in mammalian cells.

4.7. Inhibition of Binding of Anti-FCRL mAbs to
FCRL Transfected Cells by Recombinant FCRL Proteins
To confim the activity of our recombinant FCRL proteins,
we investigated whether these proteins could inhibit the

binding of FCRL-specifi mAbs to FCRL-transfected cells.
Our results showed that the commercial FCRL4 specifi
mAb adsorbed with FCRL4 recombinant protein could no
longer bind to the native FCRL4 protein expressed on the
surface of transfected CHO cells (Figure 8 C). However, no
inhibition was observed for the adsorbed FCRL1 and FCRL2
specifi commercial mAbs (Figure 8 A and Figure 8 B). Interestingly, our anti FCRL2 and 4 mAbs were blocked with
recombinant FCRL2 and 4, respectively (Figure 8 D and E).
This fiding implied that the FCRL4 specifi mAb recognizes an epitope on the FCRL protein expressed in transfected
mammalian cells. However, the epitopes recognized by
the commercial FCRL1 and 2 specifi mAbs were either not
expressed on the prokaryotic recombinant FCRL proteins
or lost due to denaturation of these proteins by 8M urea. It
is worth noting that the commercial mAbs against FCRL1,
2 and 4 were produced against recombinant FCRL proteins
expressed in eukaryotic cells (10).

5. Discussion
The discovery of the FCRL molecules has considerably
expanded the network of lymphocyte coreceptors and
unraveled an unexpected layer of biological complexity
(11). The human FCRL1–6 encode type I transmembrane
glycoproteins with 3 - 9 extracellular Ig domains and cytoplasmic tails containing ITAM and/or ITIM (5, 11). FCRL
immunoregulatory potential is implicated by the presence of consensus ITAM or ITIM in their cytoplasmic
tails. All of them except FCRL6, are expressed on B cells
at diffrent stages of diffrentiation (5). Although, FCRL

5. Discussion
The discovery of the FCRL molecules has considerably
expanded the network of lymphocyte coreceptors and
unraveled an unexpected layer of biological complexity
(11). The human FCRL1–6 encode type I transmembrane
glycoproteins with 3 - 9 extracellular Ig domains and cytoplasmic tails containing ITAM and/or ITIM (5, 11). FCRL
immunoregulatory potential is implicated by the presence of consensus ITAM or ITIM in their cytoplasmic
tails. All of them except FCRL6, are expressed on B cells
at diffrent stages of diffrentiation (5). Although, FCRL

molecules have homology with classical immunoglobulin Fc receptors, but none of the FCRL family members
have been shown to bind Igs (3). Up to now just murine
FCRL5 and human FCRL6 ligands have been identifid (9,
12). Schreeder et al. showed that human FCRL6 which is
selectively expressed on cytotoxic T and NK cells directly
binds to HLA-DR, an MHC class II molecule (9). Campbell
et al. reported that the orthopoxvirus MHC class I-like
protein encoded by monkeypoxvirus and cowpoxvirus
is the fist known virally encoded ligand of FCRL5 (12).
Due to the lack of natural ligand(s) for FCRL molecules,
direct inspection of the signaling capacity of these molecules has not so far been possible. However, a model of
co-ligation of a chimeric FcγRIIb/FCRL (consisting of extracellular domain of FCγRIIb and cytoplasmic domain
of FCRL) and the BCR has recently been employed which
allows pairing of the chimeric FCRL with the ITAM- and/
or ITIM-containing Igα/β of the BCR complex (3). Production of recombinant proteins from diffrent domains of
the FCRL molecules will pave the way for more functional
and structural analyses of these molecules. In this study,
we constructed the pET-28b (+) - FCRL1, 2 and 4 plasmids
and successfully expressed His-tagged FCRL proteins in
BL21-DE3. The extracellular parts of FCRL1, 2 and 4 have 3,
4 and 4 Ig-like domains, respectively. The digestion and
DNA sequencing results showed that the cloned genes in
pET vector had the correct size and sequence as predicted
for FCRL1 (936 bp), FCRL2 (1216 bp), and FCRL4 (1172 bp).
The constructs were transformed into BL21-DE3 E. coli
strain and the expressed recombinant FCRL proteins containing His-tag were purifid successfully using conventional Ni-NTA system. The extracellular portions of FCRL1,
2 and 4 proteins were predicted to have 354, 413 and 437
amino acids, respectively. The predicted amino acids content matched the size of the purifid FCRL1 (38.5 kDa),
FCRL2 (45.1 KDa) and FCRL4 (48.8 KDa) detected by SDSPAGE and immunoblotting techniques. The recombinant
FCRL proteins were purifid in denaturing condition using urea 8M and gradual increasing in imidazole’s molarity. After solubilization of a protein with high concentrations of denaturing agents, in our case 8M urea, refolding
should be performed by controlled gradual removal of
excess denaturant. Thus, the purifid proteins need to be
equilibrated under a continuous and step-wise decreasing concentration of urea to allow gradual removal of
the reducing agent. The recombinant FCRL proteins were
produced as inclusion bodies and thus were solubilized
in denaturing condition using 8M urea. Renaturation of
such proteins requires gradual and step-wise removal of
excess urea during Ni-NTA purifiation process using a
continuous gradient mixer. However, since this process
might result in signifiant decrease of protein recovery,
in the current study we did not use this approach and
the recombinant proteins were dialyzed directly against
PBS 1X (0.14 M). At this condition cysteine residues are
exposed leading to extensive polymerization of proteins
like FCRL. This limitation regarding the process of purifcation and dialysis of the purifid recombinant proteins
usually leads to protein aggregation and polymerization. Our results presented in fiure 5 may explain this
observation. In addition to our study, production of recombinant FCRL protein in prokaryotic system has been
reported in two other studies (13, 14). Falini et al. cloned
extracellular portion of FCRL4 into pGEX 3X vector and
produced the recombinant protein in BL21 E.coli strain.
They used purifid FCRL4 for mAb production in their
study (13). Won et al. generated mAb against FCRL3 using His-tagged recombinant protein consisting of D1-D2
and D3-D4 extracellular Ig domains of FCRL3 cloned into
pET24b (14). In some studies the recombinant FCRL proteins were also expressed in eukaryotic cells mainly for
production of mAbs (2, 6, 10, 12, 15, 16). However, detailed
data regarding the process of cloning, expression and
purifiation of FCRL molecules are not available and have
not been reported in any of these studies. Our results
showed that the human FCRL1, 2 and 4 proteins could be
produced with pET-28b (+) in BL21-DE3 E.coli strain system. Compatible with this fiding, it has been reported
that the combination of the bacteriophage T7 promoter
with BL21-DE3 host cells appears to be the most frequently used expression system (17, 18). However, it should be
kept in mind that the prokaryotic expression system has
some advantages and some drawbacks. Recombinant
proteins produced in bacteria are often insoluble and
inactive and need burdensome refolding procedure, as
opposed to the eukaryotic system which yields bioactive
soluble proteins (19). Indeed, our results as presented in
fiure 8, indicate that some epitopes expressed in native
FCRL molecules might be lost due to the denaturation
of the recombinant FCRL proteins. The other advantage
of eukaryotic system is post-translational modifiation
(N- and O-linked glycosylation, fatty acid acylation, phosphorylation) which is not available in prokaryotic systems (19, 20). On the other hand, protein production in
eukaryotic host systems is a cumbersome procedure and
frequently results in low protein yields. In this regard,
gene expression in bacteria is straightforward and has
the potential to produce large quantities of recombinant
proteins (17).
In summary, human FCRL1, 2 and 4 genes were cloned
and expressed in BL21-DE3 E.coli strain. The recombinant
proteins were purifid using Ni-NTA and characterized
using His and FCRL specifi polyclonal antibodies. These
recombinant proteins are potentially useful tools for the
identifiation of the natural ligand(s) of FCRL molecules
and also production of a panel of mAbs recognizing different domains of each FCRL molecule for signaling
studies and targeted immunotherapeutic interventions.
Searching for ligands of these molecules needs appropriately folded proteins. Thus our purifid recombinant
FCRL proteins need to have their native structure to
achieve this purpose.

We thank Dr. Sima Rafati, Dr. Fereidoun Mahboubi, Ms
Atousa Aliahmadi, and Ms Farnaz Zahedifard for their
invaluable and helpful discussions about protein expres
sion and purifiation methods. We also thank Dr. Andrew
G. Polson from Genentech, Inc. for providing the mAbs
specifi to FCRL1, 2 and 4 proteins.
MS performed the experiments, analyzed data and
wrote the manuscript. AH, MZ, and JK performed experi
ments, MJT, HR and ZA provided consultation and super
vised the study, FS designed and supervised the study,
analyzed data and wrote the manuscript.
Financial Disclosure
The authors declare that they have no competing inter
ests to disclose.
This work was partly supported by a grant from the
Food and Drug Administration of the Ministry of Health,
Treatment and Medical Education of Iran (grant number

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