Pgex 6p 1

The cDNA encoding J1-1 was cloned into pGEX-6P-1 (Amersham Biosciences, Freiburg, Germany) between EcoRI and XhoI, creating an in-frame fusion with the sequence encoding glutathione-S-transferase (GST). The primers used were a forward primer (5′-GGAATTCCTTATGGCTGGCTTTTCCAAAG-3′) and a reverse primer (5′-CCCTCGAGGGATTAAGCACAGGGCTTCGT-3′). The GST fusion protein was then expressed in E. coli strain BL21 and purified according to the manufacturer's instructions. The protein concentration was determined using the Bradford method. Following purification, the antifungal activities of the GST/J1-1 fusion protein were examined against C. gloeosporioides. The fungal growth was monitored by microscopic examination on cover glass with 5×10² spores in sterile water containing various concentrations of GST/J1-1 recombinant protein or heated protein obtained by incubating at 90°C for 10 min. The spores were treated with the proteins for 24 hours at 26°C, and then counted for germination and appressorium formation in at least five microscopic fields. The experiment was conducted in triplicate.

To facilitate raising antibodies against ORF5, EcoRI and XhoI sites were engineered into the PCR oligonucleotides used for amplification of the full-length ORF5 gene according to the archived PCV2 Yangling strain nucleotide sequence (S1 Fig) using the following primers: forward, 5ˊ-GCGAATTCATGTACACGTCATTGTGGGG-3ˊ, and reverse, 5ˊ-GGCCTCGAGTCAGTAGATCATCCCAGGGCAGC-3ˊ. PCR products were then inserted into the prokaryotic expression vector pGEX-6p-1 (Amersham). The recombinant plasmid (pGEX-ORF5) was sequenced and transformed into Escherichia coli BL21 cells (Invitrogen). These cells were induced to express ORF5 using isopropylthio-β-D-galactoside (Amersham). The purified GST-ORF5 protein was injected into SPF BALB/c mice to induce polyclonal antibody production as previously described [24 (link)]. The reactivity of the anti-ORF5 pAb was confirmed via western blot and indirect immunofluorescence assay (IFA), performed on either ORF5-transfected- or wPCV2-infected PK-15 cells.

For retroviral transduction of cells, cDNA encoding full-length Foxp3 (with HA or FLAG tag when necessary), Foxp3 deletion mutants, negative control GFP or HDACs 1, 2, 3 and 7 were cloned into the bicistronic MLV-based retroviral plasmid m6p co-expressing a GPI-linked extracellular part of ratCD8a (rCD8a) [56 (link)]. For in vitro translation, cDNA encoding Foxp3 was cloned into the pCDNA3.1(+) vector (Invitrogen). For expression in E. coli, cDNA encoding HDAC1, 2 and 3 was cloned into pGEX-6P-1 (Amersham).

Constructs were generated with Gateway cloning technology (Life Technologies). Human Rad6B and human Rad18 cDNA sequences from entry constructs were recombined into a modified pGEX-6P-1 (Amersham) destination vector bearing GST and FLAG tags with a Gateway cassette via the LR Clonase II reaction (Invitrogen). Proteins were overexpressed in the E. coli strain BL21-CodonPlus (DE3)-RIL (Agilent). The proteins were purified with removal of the GST moiety, as above.

To determine if LamB is a functional amylase, the lamB gene was cloned into the IPTG-inducible GST-fusion expression vector pGEX-6p-1 (Amersham) and expressed in E. coli BL21 using primers listed in Table S1. Additionally, residues within the predicted catalytic pocket were substituted to alanine using inverse PCR using primers listed in Table S1. E. coli cultures (5 ml) harboring either the empty vector, lamB, or the various catalytic inactive mutants were grown in LB broth at 37 °C with shaking until the OD_600nm reached 0.8. The cultures were spilt and one half was induced with 0.1 mM IPTG for 2.5 h at room temperature. One ml of each culture was pelleted by centrifugation and subjected to lysis with 0.5 ml buffer (0.1% v/v Triton X-100, 150 nM NaCl, 10 mM Tris pH7.5), containing protease inhibitors. Insoluble material was pelleted by centrifugation (16000 × g, 10 min, 4 °C) and the resulting supernatant was retained. Expression of fusion proteins was similar in all cultures (see Supplementary Fig. S3). To measure amylase activity, 25 μl of supernatant was analysed using an Amylase Assay Kit (Sigma), following the manufacturer’s instructions. This kit utilizes an artificial substrate, ethylidene-pNP-G7, which when cleaved by an amylase generates a colorimetric product detectable at 405 nm.