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HYBRIDOMAVolume 25, Number 6, 2006© Mary Ann Liebert, Inc.

Preparation and Characterization of a Monoclonal AntibodySpecific to Plasmodium falciparum TATA Binding Protein

OMAR-KAYYHAN RUVALCABA-SALAZAR, HÉCTOR ROMERO-RAMÍREZ, LEOPOLDO SANTOS-ARGUMEDO, MIGUEL VARGAS, and ROSAURA HERNÁNDEZ-RIVAS

ABSTRACT

PfTBP is a transcriptional factor required by all three types of RNA polymerases in eukaryotic cells. In or-der to obtain a specific monoclonal antibody (MAb) against PfTBP, a DNA fragment of 684 base pairs (bp)that contained the complete PfTBP gene was amplified by polymerase chain reaction (PCR) and inserted intothe pGEX prokaryotic expression vector. The recombinant protein (GST-PfTBP) was expressed in Esche-richia coli, purified, and used as antigen to immunize mice. MAbs against PfTBP were obtained and hy-bridomas were screened by enzyme-linked immunosorbent assay (ELISA). Western blotting and immunoflu-orescence assays showed that MAb Pf.r1 recognized the PfTBP protein in nuclear extracts from Plasmodiumfalciparum as well as a native protein in the nuclei of this parasite. This MAb will be a helpful tool for theidentification of the TBP associated factors (TAFs), which are apparently highly divergent with other eu-karyotes. This information could help to identify new candidate gene products to develop novel drugs or vac-cines.

INTRODUCTION

PLASMODIUM FALCIPARUM is responsible for the most severeform of human malaria. A rigorously regulated gene ex-

pression pattern controls the differentiation of the parasite,from one stage to another, during its life cycle, in which theregulation at the transcriptional level is important for the tem-poral expression of the genes required in each stage of de-velopment.(1) Therefore, the identification and characteriza-tion of cis- and trans-acting elements is a necessary step tounderstand the molecular mechanisms that control the tran-scriptional gene expression in this parasite. Transcription fac-tors from P. falciparum are apparently highly divergent fromthose of other eukaryotes due to the high content of A�T inthe P. falciparum genome, which makes their identificationby sequence homology very improbable. Currently, a fewtranscription associated TAPs proteins and general transcrip-tion factors have been identified in the P. falciparum genomeand unexpected TBP is the only known component of theTFIID complex that has been characterized in this para-site.(2,3) In eukaryotic cells, TBP associates with several sub-sets of proteins called TATA-binding protein-associated fac-tors (TAFs) that form the multimeric TFIID complex.(4)

Analysis of the molecular organization of yeast TFIID

(yTFIID) has revealed that more than half (9/14) of the yeastTAFs contain a histone fold motif, and that they specificallyassemble into five histone-like pairs.(5) Recently, putative or-thologous of TAF1, TAF2, TAF7, and TAF10 were also pre-dicted in the P. falciparum genome.(6,7) but, no candidatesfor TAFs with classic histone fold domain (HDF) have beenidentified yet; this suggests an unusual architecture of theTFIID complex in this parasite.(7)

We have a long-standing interest in the possible role ofPfTBP in the gene expression and the dynamics of TBP in theintraerythrocytic developmental cycle of P. falciparum. In thepresent study, a monoclonal antibody (MAb) against PfTBP de-noted Pf.r1 was prepared and examined by Western blot andimmunofluorescence. Pf.r1 MAb was able to identify the PfTBPprotein in nuclear extracts from P. falciparum and a native anti-gen in the nuclei of this parasite.

MATERIALS AND METHODS

Cell culture

P. falciparum FCR3 strain parasites were grown as previ-ously described at 5% hematocrit and parasitemia between

Departmento de Biomedicina Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México, D.F., México.

RUVALCABA-SALAZAR ET AL.368

5%–10% in RPMI-1640 medium supplemented with 20 mM L-glutamine, 25 mM HEPES, 50 �g mL�1 gentamycin, and 0.5%Albumax II (Gibco-BRL, Carlsbad CA).(8)

DNA extraction and polymerase chain reaction

DNA from asynchronous parasite cultures was extracted us-ing GenElute™ Mammalian Genomic DNA minipreparationKit (Sigma, St. Louis MO) as specified by the manufacturer.

Cloning and expression of fusion protein GST-PfTBP

A 684 base pair (bp) DNA fragment of PfTBP gene was ob-tained by polymerase chain reaction (PCR) using 100 ng ofFCR3 DNA. Oligonucleotide primers were designed using thesequence of PfTBP previously reported.(9) The sequences of direct and reverse oligonucleotides were: PfTBPEco 5�-CGGAATTCATGAATTTTTTAGAACAAGACCAA-3�and PfTBPXho 5�-CCGCTCGAGCATTTTTGTATTGAAT-TAACACATTG-3�, respectively.

The PCR reaction was carried out in a 100 �L reaction vol-ume using the buffer provided by the manufacturer (Amersham,Uppsala, Sweden), 10 pmol of each primer, 100 ng of the P.

falciparum genomic DNA template, 2.5 mM deoxynucleotidestriphosphates (Pharmacia, La Jolla, CA) 1.5 mM MgCl2 and2.5 U of Taq DNA polymerase (Amersham). Conditions forPCR were 35 cycles of denaturation at 94°C for 30 seconds,primer annealing at 50°C for 20 seconds, and extension 60°Cfor 1 minute; at the end of the amplification reaction, supple-mentary incubation was performed for 5 minutes at 72°C. PCRfragments were digested, retrieved and inserted into the EcoRI-XhoI–digested pGEX-4T1 vector (Amersham) fused to a GST-tag. The resulting construct was called GST-PfTBP and it wasfurther characterized by sequencing it with 5� and 3� primersto the pGEX cloning site (Amersham). GST-PfTBP was ex-pressed in Escherichia coli DH5�. Expression of the GST fu-sion protein was induced with 0.5 mM IPTG at 30°C for 4hours. GST fusion protein was purified with glutathione-sepharose (Amersham) by standard methods and analyzed bysodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). Purity and size of GST-PfTBP was verified byWestern blot with anti-GST tag specific antibodies. Purified fu-sion protein was used as immunogen and as screening antigenin an enzyme-linked immunosorbent assay (ELISA) for identi-fication of MAbs.

FIG. 1. Cloning and expression of PfTBP A: Schematic representation of the PfTBP protein motifs: N-terminal region (whitebox), direct repeat sequences DR1 and DR2 (black boxes) B, basic region (hatched box). The arrow indicates the primers usedto amplify the PfTBP gene. PfTBP product obtained with the PfTBP specific primers were separated on 1.5% agarose gel. B:Positive clones were identified by colony polymerase chain reaction (PCR) using 5� and 3� primers to the pGEX cloning site. C:Total proteins from DH5� expressing PfTBP before (lane 1) and after IPTG induction (lane 2) were separated by sodium dode-cyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue. Arrow indicates recombinant GST-PfTBP thatwas purified (lane 3).

A B

C

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Generation of polyclonal antibodies against PfTBP protein

The 52-kDa molecule corresponding to GST-PfTBP fusionprotein was purified by electroelution. BALB/c mice at 6 to 8weeks of age were used in all experiments. They were producedat the Centro de Investigación y de Estudios Avanzados (CINVESTAV, México) animal facility, and the Animal Careand Use Committee of CINVESTAV approved all experiments.Six mice were intraperitoneally inoculated with 100 �g of pu-rified GST-PfTBP fusion protein five times with intervals of 15days. Initial immunization was performed with complete Fre-und’s adjuvant. Subsequent challenges were performed with in-complete Freund’s adjuvant. Animals were bled 7 days afterthe last boost.

Immunofluorescence staining

All manipulations were carried out at room temperature. Thinblood smears were made on glass slides, air-dried, and fixedwith methanol. Intracellular parasites were fixed with 4% para-formaldehyde for 20 minutes. Following fixation, slides werebriefly rinsed in phosphate-buffered saline (PBS). Cells werethen permeabilized in acetone for 6 minutes at �20°C and thenblocked with 3% bovine serum albumin (BSA) for 60 minutesat 37°C. Slides were incubated for 2 hours with anti-PfTBP an-tisera (diluted 1:25), or hybridoma supernatants in PBS (con-taining 0.1% BSA). The preparations were washed and incu-bated for additional 60 minutes in fluorescein isothiocyanate(FITC)-labeled goat anti-mouse antibodies diluted in PBS con-taining 0.1% BSA. In order to contrast the nucleus, slides wereincubated for 30 seconds in 4�, 6-diamidino-2-phenylindole(DAPI), washed briefly, mounted in Vectashield (Vector,Burlingame, CA), and kept at 4°C. Images were examined withan Olympus BX63 microscope equipped with a Pixera 6000camera. Adobe PhotoShop (Adobe Systems, Mountain View,CA) was used for image processing.

Cell fusion

Hybridomas were produced by fusing spleen cells from theimmunized BALB/c mice with the myeloma cell line P3/x63.Ag8at 10:1 ratio in polyethylene glycol 1300–1600 (Sigma) accord-ing to standard procedure.(10) The hybridomas were selected inRPMI-1640 medium (Gibco-BRL) supplemented with 20% (v/v)fetal calf serum (Gibco-BRL) and 5 � 10�3 M hypoxanthine,2 � 10�5 M aminopterin, and 8 � 10�4 M thymidine (HAT,Sigma). After 10–15 days, supernatants of the growing hybrido-mas were screened for the presence of anti-PfTBP antibodies byindirect ELISA. Selected hybridomas were subcloned four timesby limiting dilution. The isotype of the MAb was determined us-ing a commercial ELISA kit (Zymed, South San Francisco, CA)according to the supplier’s instructions.

ELISA

Polyestyrene 96-well plates were coated with purified GST-PfTBP at 5 �g/mL or GST at the same concentration in 0.05M bicarbonate buffer pH 9.6. The plates were incubatedovernight at 4°C. After incubation the plates were washed threetimes with PBS containing 0.05% Tween 20 (USB, Cleveland,OH) binding sites were blocked with 5% skimmed milk at roomtemperature for 2 hours. The hybridoma supernatants were

added to each well (50 �L/well) and incubated at 37°C for 2hours. The plates were washed with Tween/PBS, and thenhorseradish peroxidase (HRP)-labeled goat anti–mouse im-munoglobulin G (IgG) was added (50 �L/well). The plates werefurther incubated at room temperature for 1 hour. Peroxidaseactivity was measured with 0.5 mg/mL OPD substrate solution(100 �L/well). After 5 minutes at room temperature, the reac-tion was stopped with 2 M H2SO4 (50 �L/well). Optical den-sity (OD) absorbance was determined using a Sunrise mi-croplate reader (Tecan, Salzburg, Austria).

Preparation of nuclear extracts

Nuclear preparations were made as described by Lanzer etal.(11) Briefly, 5 � 109 parasites were isolated from infectederythrocytes by saponin lysis and resuspended in 1 mL of ly-sis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM eth-ylenediaminetetraacetic acid [EDTA], 0.1 mM ethylenegly-coltetraacetic acid [EGTA], 1 mM dithiothreitol [DTT], 0.5 mMphenylmethyl sulfonyl fluoride [PMSF], and 0.65% NP-40).Nuclei were collected by centrifugation and nuclear protein ex-tracted using 100 �L of extraction buffer (20 mM HEPES pH7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and1 mM PMSF). After 15 minutes of vigorous shaking at 4°C,

FIG. 2. Monoclonal antibody (MAb) Pf.r1 identifies the de-natured and native form of PfTBP protein. A: Enzyme-linkedimmunosorbent assay (ELISA) for the specificity of MAb Pf.r1with PfTBP, GST, and preimmune antiserum. B: Total proteinsfrom Escherichia coli and Plasmodium falciparum nuclear andcytoplasmic extracts were transferred to nitrocellulose mem-brane and incubated with MAb Pf.1r raised against recombi-nant PfTBP.

B

A

the extract was cleared by centrifugation. The supernatant con-tained nuclear proteins.

Western blot

SDS-PAGE was performed using standard methods.(12) Pu-rified fusion protein and nuclear extracts from P. falciparumwere separated by SDS-PAGE and transferred to nitrocellulose.Western blot analysis was carried out using 10%–15% poly-acrylamide gels under reducing conditions with (0.2%) �-mer-captoethanol in the loading samples. After electrophoresis, theproteins were transferred to Hybond ECL nitrocellulose (Amer-sham) according to Towbin.(13) The membrane was incubatedwith hybridoma supernatants overnight at 4°C.

RESULTS

Cloning and expression of PfTBP

A DNA fragment (684 bp) corresponding to the open read-ing frame (ORF) of PfTBP was amplified and sequenced (Fig.1A) Then, this fragment was cloned into the expression vec-tor pGEX 4T-1 in frame with a GST tag. Recombinant plas-mid pGEX4-1-PfTBP was used to transform E. coli DH5�.Positive clones were identified by colony PCR using 5� and3� primers for the pGEX cloning site (Pharmacia) (Fig. 1B).GST-PfTBP fusion protein was overexpressed and purified

on glutathione-sepharose beads, eluted, and separated onSDS-PAGE gel. The purified GST-PfTBP fusion proteinshowed the expected molecular weight of approximately 52kDa in which 26 kd corresponds to GST and 26 kDa to PfTBP(Fig. 1C).

Preparation of anti-PfTBP MAb

GST-PfTBP or GST proteins were used in the ELISA screen-ing. GST alone was obtained in this work and used to elimi-nate antibodies that recognize this protein. One stable hy-bridoma, called Pf.r1, was obtained after cell fusion andsubcloning. Pf.r1 was identified as IgM producing-hybridoma.ELISA results indicated that Pf.r1 mainly recognized PfTBPhaving low reaction with GST alone (Fig. 2A). Immunoblotanalysis showed that Pf.r1 recognized the 52-kDa fusion pro-tein and the 26-kDa native PfTBP molecule contained in thenuclear extracts from P. falciparum (Fig. 2B). No cross-reac-tion was observed when Pf.r1 monoclonal antibody was incu-bated with total proteins from E. coli, which demonstrated thespecificity of this antibody.

These results show that TBP protein is present in nuclear ex-tracts from P. falciparum. Immunofluorescence assays wereperformed in order to determine the subcellular localization ofthis protein. Differential interference contrast image showedthat both infected and noninfected red blood cells were presentin the preparation. The immunofluorescence signal of Pf.r1 wasdetected in infected erythrocytes with P. falciparum, which in-

RUVALCABA-SALAZAR ET AL.370

FIG. 3. Nuclear localization of PfTBP. Plasmodium falciparum FCR3 strain parasites were fixed, permeabilized, and incubated with monoclonal antibody (MAb) Pf.r1. PfTBP was detected by MAb Pf.r1 and there after by fluorescein isothio-cyanate (FITC)-labeled goat anti-mouse immunoglobulin (Ig) while nuclei were stained with 4�,6-diamidino-2-phenylindole(DAPI). Differential contrast image (DIC) shows infected and noninfected red blood cells.

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dicates that this antibody specifically recognized a protein pres-ent in this parasite (Fig. 3). In order to confirm whether thissignal was located in the nucleus or cytoplasm, parasite nucleiwere stained with DAPI. Figure 3 shows that DAPI and Pf.r1immunofluorescence signals colocalized. Together, these re-sults demonstrate that PfTBP is located in the nucleus of par-asites and that Pf.r1 could be used to detect PfTBP by meansof Western blot and immunofluorescence.

DISCUSSION

The initiation of gene transcription involves the ordered as-sembly of a multiprotein complex on proximal promoter ele-ments such as TATA box, located upstream of the transcriptionstart codon. Transcription factor class II family proteins (TFII)are required for the initiation of the transcription in addition toRNA polymerases. The first step in the formation of this initi-ation complex is the stable binding of TFIID to the TATAbox.(14) General transcription factor TFIID, consisting ofTATA-binding protein (TBP) and TBP-associated factors(TAFs), plays a central role in both positive and negative reg-ulation of transcription.(3) TBP is an essential component ofthis basal transcriptional machinery and nucleates formation ofthe transcription initiation complex.(3) P. falciparum is thecausative agent of the most severe form of human malaria. Theparasite displays a complex life cycle that requires differentialand coordinated gene expression in which the transcriptioncould be playing a central role. There are several lines of evi-dence that indicate that Plasmodium spp. promoters have a bi-partite structure. However, the extreme A�T content in theirintergenic regions have made it difficult to identify the func-tional TATA box.(15) To date, the TATA binding protein(PfTBP) is the only trans-acting factor that has been isolatedand its corresponding cis-acting sequences have been identifiedin two P. falciparum promoters.(2) The TBP protein and manyTBP associated factors (TAFs) form the multimeric TFIID com-plex. Currently, some TAF have been identified (i.e., TAF1,TAF2, TAF7, and TAF5) in P. falciparum. However, no HFD-containing TAFs could be found, except for the putative or-tholog of TAF10.(7) We propose that the Pfr-1 antibody couldbe used to immunoprecipitate TAFs with classic histone folddomain (HDF) that are apparently highly divergent from othereukaryotes due to the high A�T content in P. falciparum.(7)

This information could help to identify new candidate geneproducts to develop novel drugs or vaccines. The identificationand characterization of cis-acting elements and transcriptionfactors is necessary to underlay the molecular mechanisms thatcontrol the timing of gene expression correctly in this parasite.

ACKNOWLEDGMENTS

We thank Benito Gutierrez Castañeda for critical commentsand advice, and the staff at Juarez Hospital for the red bloodcells provided. This work was supported by a grant fromCONACyT (México contract numbers P45687-Q)

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Address reprint requests to:Rosaura Hernández-Rivas, Ph.D.

Departamento de Biomedicina MolecularCentro de Investigación y de Estudios Avanzados del IPN

Apartado Postal 14-740, 07360, México, D.F.México

E-mail: rohernan@cinvestav.mx

Received for publication July 28, 2006. Accepted for publica-tion August 24, 2006.