foreskin fibroblasts (HFFs) as previously described [51]. For each T. gondii transfection, 30–40 mg of DNA was electroporated into 16107 extracellular parasites suspended in DMEM with 1% BCS. Electroporation was performed using a Harvard Apparatus BTX- BCM630 electroporator (Holliston, MA,) with the electroporation voltage set at 1480 V, capacitance set at 50 mF, and resistance set at 25 V. Construction of TgICMAP1 expression plasmids 1. T. gondii expression plasmids. For the construction of pmin-eGFP-TgICMAP1, the TgICMAP1 coding region was amplified from the RH cDNA library (obtained as previously described [6]) using S124 and A125 primers (Table S1) digested with BamHI and AflII, and subcloned into pmin-eGFP-TgDLC plasmid [6] replacing the TgDLC/BglII-AflII fragment. The BamHI and BglII ligation created a sequence that cannot be re-cut by either BglII or BamHI. To introduce a unique BamHI site between the eGFP and TgICMAP1 for subsequent subcloning, a BamHI site downstream of the TgICMAP1 coding sequence on pmin-eGFP-TgICMAP1 was first removed by replacing the fragment between AflII and NotI sites with a PCR product amplified from pmin-eGFP-TgICMAP1 using PCR mutagenesis by overlap extension with primers S555, A555, S556, and A556 (Table S1). This resulted in the plasmid pmin-eGFP-TgICMAP1- minus-BamHI. A BamHI site is then created between eGFP and TgICMAP1 by replacing the fragment between NheI and NcoI sites in pmin-eGFP-TgICMAP1-minus-BamHI with a PCR product amplified from pmin-eGFP-TgICMAP1-minus-BamHI using PCR mutagenesis by overlap extension with primers S574, A574, S575, and A575 (Table S1). This resulted in the plasmid pmin-eGFP-TgICMAP1-BamHI-linker. The construction of pmin-eGFP-TgICMAP1-minus-BamHI and pmin-eGFP- TgICMAP1-BamHI-linker was carried out by Biomeans Inc. (Sugar Land, TX). pmin-eGFP-TgICMAP11–474 was constructed by replacing eGFP-TgDLC/NheI-AflII in pmin-eGFP-TgDLC with eGFP-TgICMAP11–474/NheI-HindIII from pc22-eGFP- TgICMAP11–474 (see below). AflII and HindIII sites were filled- in by Klenow fragment before the ligation. pmin-eGFP- TgICMAP1455–1231 was constructed by replacing TgICMAP1/ BamHI-AflII in pmin-eGFP-TgICMAP1-BamHI-linker with TgICMAP1455–1231/BamHI-AflII from pQE30-His-FLAG- eGFP-TgICMAP1455–1231 (see below). 2. Bacterial expression plasmids. To generate a bacterial expression plasmid that has compatible sites with T. gondii expression plasmids, AflII and NheI sites were introduced into pQE30-DIP13 (a kind gift from Dr. Wolfgang Mages, Universita¨t Regensburg, Germany) in two steps. First, an AflII site was introduced downstream of DIP13 by replacing the fragment between HindIII and PvuII in PQE30-DIP13 with a PCR product amplified from pQE30-DIP13 using PCR mutagenesis by overlap extension with primers S552, A552, S553, A553, S554, and A554 (Table S1). This resulted in the plasmid pQE30-DIP13_AflII. Second, a NheI site was introduced upstream of DIP13 by replacing the fragment between EcoRI and HindIII in pQE30- DIP13_AflII with a PCR product amplified from pQE30- DIP13_AflII using PCR mutagenesis by overlap extension with primers S552, A552, S576, and A576 (Table S1). This resulted in the plasmid pQE30-DIP13_AflII-NheI. The construction of pQE30-DIP13-AflII and pQE30-DIP13_AflII-NheI was carried out by Biomeans Inc. pQE30-6xHis-eGFP-TgICMAP11–474 was constructed by replacing DIP13/NheI-HindIII in pQE30- DIP13_AflII-NheI with eGFP-TgICMAP11–474/NheI-HindIII from pmin-eGFP-TgICMAP1-BamHI-linker. pQE30-6xHis- eGFP-TgICMAP1 was constructed by replacing DIP13/NheI- Characterization of TgICMAP1 AflII in pQE30-DIP13_AflII-NheI with eGFP-TgICMAP1/NheI- AflII from pmin-eGFP-TgICMAP1-BamHI-linker. A FLAG tag was then introduced by amplifying a portion of the pQE30-6xHis- eGFP-TgICMAP11–474 plasmid with primers pQE30 Forward and His-FLAG-NheI-AS (Table S1). The PCR product was digested with EcoRI and NheI and ligated into pQE30-His-eGFP- TgICMAP11–474_EcoRI-NheI to form pQE30-6xHis-FLAG- eGFP-TgICMAP11–474. To construct pQE30-His-FLAG-eGFP- TgICMAP1455–1231, TgICMAP1455–1231 was first amplified by PCR using pQE30-6xHis-eGFP-TgICMAP1 as a template and primers BamHI-TgICMAP1C-S and AflII-TgICMAP1C-AS (Table S1). The PCR product and pQE30-His-FLAG-eGFP- TgICMAP11–474 were digested with BamHI and AflII restriction enzymes and ligated such that TgICMAP11–474 was replaced with TgICMAP1455–1231. pBAD24-6xHis-TgICMAP11–474 was constructed by ligating the 6xHis-TgICMAP11–474 EcoRI-HindIII region from pQE30-6xHis-TgICMAP11–474 into the EcoRI and HindIII sites of pBAD24 (A kind gift from Dr. Pat Foster, Indiana University, Bloomington). 3. Mammalian cell expression plasmids. pc22-eGFP- TgICMAP1 was constructed by replacing the eGFP-TubA1 NheI-AflII fragment in pc22-eGFP-TubA1 (a kind gift from Dr. John Murray, University of Pennsylvania) with the eGFP- TgICMAP1 NheI-AflII fragment from pmin-eGFP-TgICMAP1 (see above). To construct pc22-eGFP-TgICMAP11–474, pc22- eGFP-TgICMAP1 was digested with HindIII and AflII, filled in by Klenow fragment, and religated. Protein expression and purification 1. Expression and purification of 6xHis-eGFP- TgICMAP1. Single BL21(DE3)pLysS bacterial colonies contain- ing pQE30-6xHis-eGFP-TgICMAP1 plasmid were grown over- night in 50 ml LB containing 100 mg/ml of ampicillin, 50 mg/ml chloramphenicol (LB-amp-cap) at 37uC. Cultures were diluted 1:25 into 750 ml LB-amp-cap until the OD600 reached ,0.6. Cultures were induced with 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG) and grown overnight (,18 hrs) at 16uC. Cells were then pelleted at 15,000 rpm for 5 minutes, resuspended in 40 ml of cold lysis buffer (8 mM Tris-Ac pH 7.5, 3 mM Trisbase, 100 mM KAc, 1 mM MgAc) containing 1% TX-100, 1.2 g of cell lytic express (,1 vial, Cat#C1990, Sigma), 1 mM TAME (Cat#T4626: Sigma) and 1 mM PMSF (Cat#P7626:Sigma), and incubated at 4uC for 15 minutes. Cells were sonicated five times for 30 seconds each with 1 minute cooling between each cycle, then centrifuged at 15,000 rpm for 10 minutes at 4uC. 0.5 ml of Talon resin (Cat#635501, Clontech) equilibrated with lysis buffer was then added to the supernatant and gently mixed at 4uC for 2 hours. The resin was loaded on a gravity flow column (Cat#635513: Clontech) and then washed with 30 ml of wash buffer (lysis buffer containing 10 mM imidazole). Proteins were eluted with 250 ml of elution buffer (50 mM Tris-Cl pH 7.5, 250 mM imidazole, 300 mM KCl). Proteins were stored on ice and used within one week of purification. 2. Expression and purification of recombinant 6xHis- TgICMAP11–474 for antibody production. Single BL21 (DE3)pLysS bacterial colonies containing pBAD24-6xHis- TgICMAP11–474 plasmid were grown and lysed as described above with the following exceptions. Cultures were grown in LB- amp-cap containing 0.2% glucose and protein expression was induced with glucose-free LB-amp-cap containing 0.2% arabinose for four hours at 37uC [52]. After lysis and centrifugation the supernatant was discarded and insoluble proteins were solubilized using 6 M guanidine hydrochloride, 50 mM Hepes (pH 7) and 25 mM DTT (6 M guanidine HCl buffer) at 4uC for 1 hour. PLoS ONE | www.plosone.org 9 October 2009 | Volume 4 | Issue 10 | e7406