After purification, the PCR products were inserted into the Gateway® Lazertinib expression vector pDEST17, as previously described [41]. The inserts were then this website sequenced to rule out any mutations. The ligation mixtures were transformed into E. coli DH5α competent cells. Transformants were selected on LB plates containing 100 μg/ml ampicillin, and the positive clones were confirmed by colony PCR with the Fg and Rg primers. Plasmid DNA was isolated from positive clones from overnight

cultures using a Midi plasmid purification kit (Qiagen). Fifty ng of plasmid DNA was transformed into E. coli BL21 (DE3), and cells containing the recombinant plasmids were grown in 17 ml of LB broth (containing 100 μg/ml ampicillin and

20 μg/ml chloramphenicol) to an optical density at 600 nm (OD600) of 0.3. Protein expression was induced by 0.5 mM IPTG (isopropyl-D-thiogalactopyranoside, Sigma-Aldrich). Once the OD600 had reached 1, the bacteria were pelleted by centrifugation and further subjected to SDS-PAGE as described by Laemmli [42] and western blot to evaluate the expression and antigenicity of the expressed recombinant proteins. The two proteins were found to be expressed in inclusion bodies. The expression protocol was specifically designed to increase the quantity of expressed recombinant proteins in order to facilitate further purification. Bacterial growth was monitored by measuring absorbance at 600 nm. No toxic effect due to the over-expressed recombinant proteins was observed on E. coli cells. Purification of recombinant rAtpD and rP1-C proteins For large-scale production of recombinant S3I-201 proteins, 2l of culture of E. coli cells expressing rAtpD and rP1-C were grown and induced with 0.5 mM IPTG. After induction, the bacterial pellet was obtained by centrifugation at 5,251 × g for 6 min at 4°C and resuspended in 60 ml of lysis buffer (20 mM Tris HCl, pH8, 0.5

M sucrose, 100 mM EDTA, pH8, 2 mg/ml lysozyme, Bay 11-7085 1 mM phenylmethylsulfonyl fluoride (PMSF)). After incubation on ice for 45 min, the tubes were centrifuged at 15,557 × g at 4°C for 10 min. The pellets were frozen at -20°C until purification. The cells were then sonicated three times with a 20 s pulse at 1-min intervals on ice in a sonication buffer (8 M urea, 20 mM triethanolamine, pH8, 500 mM NaCl, 25 mM imidazole, 1 mM PMSF). The cells were harvested by centrifugation at 15,557 × g for 45 min with a buffer containing 20 mM triethanolamine, pH8, 500 mM NaCl and 0.25 M imidazole and then subsequently using buffers with the same composition containing 1 M and 8 M urea. These three “”wash steps”" were used to eliminate the majority of E. coli contaminants before purification. The supernatant of the final step containing the protein of interest was filtered and loaded onto a HisTrap column (GE Healthcare) at 4°C.