1B) and its presence reflects the background of expression as classically observed in the case of the tac promoter. This observation has also been reported by other groups ( Mérienne et al, 1997). Attempts to improve the periplasmic production of the recombinant fusion protein have been made using E. coli XL-1 blue and W3110 strain in various conditions. Namely, for the determination of the effect of IPTG concentration on the production of SAG1–AP, cultures in LB medium were induced with different IPTG concentrations (0.1, 0.25, 0.5, and 1.0 mM), then incubated overnight. The

Western blot pattern of periplasmic extracts from induced cultures showed the presence of a 78 kDa band corresponding to the SAG1–AP fusion protein in both E. coli strains ( Fig. 2). http://www.selleckchem.com/products/cobimetinib-gdc-0973-rg7420.html Optimal production was obtained in the W3110 strain with a 0.5 mM IPTG inducer concentration. Typical 1L recombinant bacteria culture in shaker flasks led to the production of about 1.5 mg recombinant soluble SAG1–AP conjugates. This yielded enough material for the evaluation of AP and T. gondii SAG1 activities of the recombinant protein and for an exploration of the immunoreactivity of the fusion protein with

human sera. Recombinant fusion protein was assayed for both AP and T. gondii SAG1 activities. Periplasmic extracts obtained previously were blotted onto nitrocellulose membrane under native PAGE condition and the AP activity was directly revealed using BCIP/NBT AP substrate (Fig. 3A,

lane 2). The periplasmic protein bands were stained Ipilimumab by the AP substrate. This result first indicates that the AP remains active within the fusion protein and second that the whole periplasmic extract can be directly used as a marker (Mousli et al., 2007 and Muller et al., 2001). Furthermore, AP activity was also determined using a biochemical colorimetric test in ELISA plates, with successive dilutions of crude periplasmic extracts in the presence of soluble pNPP AP substrate. Optical densities (O.D) were measured at 405 nm. SAG1–AP O.D values Meloxicam were calculated as the difference between O.D measures obtained with the induced and non-induced periplasmic extracts, respectively. As shown in Figure 3B, the SAG1–AP conjugate displays an enzymatic activity similar to that of free AP. This result is consistent with data published by Butera and co-workers (Butera et al., 2003). The specific activities were calculated as 18.5 OD405/μg to SAG1–AP and 21 OD405/μg to free AP. We investigated the bi-functionality of the recombinant fusion protein by ELISA using anti-T. gondii SAG1 Mab coated plates. The bound conjugate was directly revealed by the AP activity of the recombinant immunoconjugate preparation ( Fig. 4). The corresponding colorimetric signal increased in a dose-dependent manner with increasing amounts of SAG1–AP within the range tested.