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

Akmayan, Ilkgul; Ozturk, Abdullah Bilal; Ozbek, Tulin

doi:10.1080/10826068.2023.2299495

Cited by 1 publication

(6 citation statements)

References 67 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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].…”

Section: Expression and Purification Of Recombinant Proteinsmentioning

confidence: 78%

“…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.…”

Section: Bacterial Strains and Culture Conditionsmentioning

confidence: 99%

“…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.…”

Section: Genomic Dna Isolation Amplification and Cloningmentioning

confidence: 99%

“…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.…”

Section: Expression and Purification Of Recombinant Proteinsmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

See 4 more Smart Citations

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

Akmayan,

Oztav,

Coksu

et al. 2024

Nanotechnology

View full text Add to dashboard Cite

Safe and effective vaccine candidates are needed to address the limitations of existing vaccines against Brucellosis, a disease responsible for substantial economic losses in livestock. The present study aimed to encapsulate Omp25 and EipB proteins, knowledged antigen properties, into PLGA nanoparticles, characterize synthesized nanoparticles with different methods, and assessed their in vitro/in vivo immunostimulatory activities to develop new vaccine candidates. The rOmp25 and EipB proteins produced with recombinant DNA technology were encapsulated into PLGA nanoparticles by double emulsion solvent evaporation technique. The nanoparticles were characterized using SEM, Zeta-sizer, and FTIR instruments to determine size, morphology, zeta potentials, and polydispersity index values, as well as to analyze functional groups chemically. Additionally, the release profiles and encapsulation efficiencies were assessed using UV–Vis spectroscopy. After loading with recombinant proteins, O-NPs reached sizes of 221.2±5.21 nm, while E-NPs reached sizes of 274.4±9.51 nm. The cumulative release rates of the antigens, monitored until the end of day 14, were determined to be 90.39% for O-NPs and 56.1% for E-NPs. Following the assessment of the in vitro cytotoxicity and immunostimulatory effects of both proteins and nanoparticles on the J774 murine macrophage cells, in vivo immunization experiments were conducted using concentrations of 16 µg/ml for each protein. Both free antigens and antigen-containing nanoparticles excessively induced humoral immunity by increasing produced Brucella-specific IgG antibody levels for 3 times in contrast to control. Furthermore, it was also demonstrated that vaccine candidates stimulated Th1-mediated cellular immunity as well since they significantly raised IFN-gamma and IL-12 cytokine levels in murine splenocytes rather than IL-4 following to immunization. Additionally, the vaccine candidates conferred higher than 90% protection from the infection according to challenge results. Our findings reveal that PLGA nanoparticles constructed with the encapsulation of recombinant Omp25 or EipB proteins possess great potential to trigger Brucella-specific humoral and cellular immune response.

show abstract

Section: Expression and Purification Of Recombinant Proteinsmentioning

confidence: 78%

Section: Bacterial Strains and Culture Conditionsmentioning

confidence: 99%