Ecos competent e coli bl 21

The expression plasmids of glutathione S- transferase (GST)-fused proteins were constructed by recombining the cDNA sequences into pGEX-4T-3 (GE Healthcare Life Sciences, Pittsburgh, PA). The inserted DNA fragments were ligated in frame to pGEX-4T-3 using the Ligation Convenience Kits (Nippon Gene, Toyama, Japan). Ligation mixtures were used to transform ECOS™-competent E. coli BL-21 (Nippon Gene), and appropriate recombinants were confirmed by DNA sequencing as well as protein expressions. Treating the transformed E. coli with 0.1 mM IPTG for 3 h induced the expression of the GST-fusion proteins. The GST-fused recombinant proteins were purified by Glutathione Sepharose column chromatography according to the manufacturer's instructions (GE Healthcare Life Sciences) and dialyzed against phosphate-buffered saline as described in previous studies [20 –22 ].

Recombinant GST-fusion proteins were constructed by different methods depending on whether the EcoRI and XhoI sequences were present in the sequence of the identified antigen. If they were not present, the plasmid DNA was double digested with EcoRI and XhoI enzymes. The digested cDNA fragments were electrophoresed on an agarose gel and purified using GeneElute™ Minus EtBr SPIN COLUMNS (Sigma-Aldrich). Then the purified cDNA fragments were ligated into the pGEX vector double-digested with EcoRI and XhoI using the DNA Ligation Kit (Takara). On the other hand, if the EcoRI and XhoI sequences were present in the sequence of the identified antigen, the inserted cDNA fragments were ligated into pGEX vector with the In-Fusion HD Cloning system (Takara Bio). Then the pGEX vector was transformed into ECOS™ Competent E. Coli BL21 (NIPPON GENE, Tokyo, Japan). Positive transformants were selected on LB plates containing 50 μg/ml ampicillin and incubated at 37°C. The plasmid DNA was purified and the appropriate recombinants were confirmed by DNA sequencing. Expression of the GST-fusion proteins was induced by incubating the transformed E. Coli BL21 with 0.5 mM IPTG for 4 h at 25°C. The GST-fusion proteins were purified over the GSTrap FF column (GE Healthcare, Chicago, IL, USA) and the buffer was exchanged with PBS using the Amicon Ultra 15 filter (Merck Millipore, Billerica, MA, USA).