Figure 5

Far-Western blot of E. coli BL21 (1), E. coli BL21 transformed with pET30a (Novagen) (2), and E. coli BL21 transformed with pET30-AR1 (3) (see below), probed with rat F _c fragments conjugated to alkaline phosphatase (Jackson Immunoresearch). All strains were grown in LB to log-phase and induction of plasmid expression was achieved using IPTG as per the manufacturer’s instructions (Novagen). Construction of pET30a-AR1: The B. henselae groEL ORF was amplified using a PCR incorporating primers BhgroELF (AAG GAG AGG AAG AAA TGG CTG CTA AAG AAG T) and BhgroELR (TCA AGG GCT TAG AAA TCC) then cloned into pCR2.1-TOPO and used to transform E. coli TOP10 cells according to the manufacturer’s instructions (Invitrogen). The resulting plasmid, pAR1-TOPO, was purified then digested with Bam HI and Xho I to yield a 1600 bp fragment that included the groEL ORF. This fragment was cloned into compatible sites of pET30a (Novagen) to generate pET30-AR1, which was used to transform E. coli XL10 cells (Stratagene). pET30-AR1 was recovered from these cells and subcloned into E. coli BL21 cells.