title: Ascaris Suum and Trichuris Suis IgG antibody profiling with Ascaris Suum protein microarray
status: Public on Jun 08 2023
contact.city: St. Louis
contact.first: Bruce
contact.middle: A
contact.last: Rosa
contact.email: barosa@wustl.edu
contact.state: MO
contact.address: 4444 Forest Park Ave
contact.department: Medicine
contact.country: USA
contact.institute: Washington University in St Louis
contact.phone: 5178997546
organism: Ascaris suum
manufacture_protocol: Cloning and expression was performed using standard techniques. All sequences were PCR amplified from cDNA using sequence specific forward and reverse primers that encompassed the mature protein and contained Sac I (forward) and Xho I (reverse) restriction sites for downstream subcloning. Translation stop sites were incorporated into all reverse primers. Amplified sequences were first cloned into pCR2.1-TOPO by TA cloning then transformed into DH5α cells for sequence verification. Validated sequences were restriction enzyme digested and subcloned into the pSUMO bacterial e xpression vector (Life Sensors) and transformed into BL21 cells for protein production. Expression was performed using overnight cultures to spike 500ml of LB medium containing ampicillin (100 µg/ml) which was induced at OD = 0.7 for 5 hrs. with isopropyl β-D-1-thiogalactopyranoside (0.3 mM final). Pelleted cells were lysed with 1 mg/ml lysozyme, frozen overnight then sonicated. Because all clones formed inclusion bodies during production, the sonicated pellets were first washed 3X with 2% Triton X-100, solubilized in 8M Urea (made fresh) and batch purified by affinity chromatography using 2 ml of Ni-NTA. All mixtures were added to columns, washed with 6M urea (3X), then wash buffer containing 50 mM sodium phosphate, pH 8.0, 300 mM sodium chloride and 20 mM imidazole. Recombinant proteins were eluted with 5 ml of wash buffer containing 500 mM imidazole and 12 mM sodium lauryl sarkosine. Briefly, the clone library was created through an in vivo recombination cloning process with PCR-amplified coding sequences from cDNA, and a complementary linearized expressed vector transformed into chemically competent E. coli cells was amplified by PCR and cloned into the pXI vector using a high-throughput PCR recombination cloning method. The cloning methodology is described in detail elsewhere (Davies et al., 2005). All the clones were sequenced (Retrogen, Inc., San Diego, CA), and the results matched the correct target for the selected genes.  From each clone, the corresponding protein was expressed using an in vitro transcription and translation (IVTT) system, the E. coli cell-free rapid translation system (RTS) kit (Biotechrabbit, Berlin, Germany), as previously described (Davies et al., 2005). Each expressed protein includes a 5′ polyhistidine epitope tag and a 3′ hemagglutinin (HA) epitope tag. After expressing the proteins according to the manufacturer's instructions, translated proteins were printed onto nitrocellulose-coated glass AVID slides (Grace Bio-Labs, Inc., Bend, OR) using an ArrayJet Marathon Argus robotic microarray non-contact printer (ArrayJet, Roslin, UK). Each slide contained 16 nitrocellulose pads on which the expressed proteins along with controls were printed (this allowed sixteen samples to be probed per slide using sealed chambers that isolate the arrays). Microarray chip printing and protein expression were quality checked by probing random slides with anti-His and anti-HA monoclonal antibodies with fluorescent labeling.