Recombinant proteins production in Escherichia coli BL21 for vaccine applications: a cost estimation of potential industrial-scale production scenarios

Construction of recombinant Omp25 or EipB protein loaded PLGA nanovaccines for Brucellosis protection

“…The purified proteins were analyzed with SDS-PAGE and Western Blot technics, which were detected in a molecular weight of approximately 41 kDa for rOmp25 and ~35 kDa for rEipB, as reported in our study by Akmayan et al The endotoxin values of the rOmp25 and rEipB proteins were determined to be 1.125 EU and 0.725 EU, respectively. These values are suitable for vaccine purposes, as they are below the acceptable level of 20 EU ml −1 as stated in the literature [19].…”

“…Endemic strain of Brucella abortus biovar 3 (smooth) was cultivated in Blood Agar and Trypticase Soy Broth by incubation at 37 • C in 10% CO 2 for 48-72 h. Escherichia coli BL21 strain was grown in Luria-Bertani Broth (LB) and Terrific Broth (TB) media supplemented with ampicillin (100 µg ml −1 ) [19,26,27]. The bacterial strains in this study are from the culture collection of the Microbiology Laboratory at the Department of Molecular Biology and Genetics, Yildiz Technical University.…”

“…Thermal cycler conditions consisted of an initial denaturation at 98 • C for 30 s; 35 cycles of denaturation, at 98 • C for 10 s, 58 • C for 30 s and 72 • C for 30 s and a final extension at 72 • C for 10 min with Phusion High-Fidelity DNA Polymerase (Thermo scientific, F530L). After transferring the ligation product into E. coli BL21 cells using the heat shock method [28], the cells were spread onto LB agar supplemented with ampicillin (100 µg ml −1 ) and incubated overnight at 37 • C [19]. After sequence analysis of the colonies confirmed by colony PCR, the sequence data were aligned a bioinformatics tool (Clustal Omega) with the EipB gene sequence.…”

“…The hydrophobic outer membrane protein Omp25 was cloned into the Thiredoxin-containing pET102/D-TOPO vector to solubilize the protein [22], while the hydrophilic periplasmic protein EipB was cloned into the His-tag containing pLATE51 vector [19]. Both proteins were successfully expressed in E. coli BL21 (DE3) cells, and then purified by using affinity chromatography.…”

“…Especially, proteins manufactured via recombinant DNA technology offer numerous advantages in today's vaccine industry. These include inert properties, known compositions, high homogeneity, lack of infection risk, and the capacity to trigger antigen-specific immune responses, along with the flexibility of incorporating adjuvants [19]. Consequently, they are increasingly favored as antigens.…”