Investigation of the effect of different culture conditions on recombinant protein production

Abstract

After the COVID-19 pandemic, vaccine production technologies have become the focus of attention of researchers. As a matter of fact, recombinant protein-based antigen production, which is one of them, has taken its place in the first place. Proteins obtained by recombinant DNA technology are used in many industrial areas, especially vaccine applications, due to their reliability. Therefore, it is very important to produce targeted recombinant proteins in large quantities. This study, for the high amounts production of Omp25 protein, which is used as a vaccine candidate against brucellosis, in laboratory conditions, is aimed to reveal the effects of conditions that are the pre-culturing process, inoculation in LB or TB media, denatured or native purification, culturing with/without IPTG. All the results were analyzed by SDS-PAGE, confirmed Western Blot, and the total protein amounts were measured Bradford method. According to the results, Omp25 protein could not be obtained under native purification conditions in both cultures without induction, but it was observed under denatured conditions. This result can be explained that the protein in the cell is either misfolded or incorporated into the membrane. The amount of protein appears to be much higher in the presence of the inducer in both media inoculated with the starter pre-culture compared to the overnight pre-culture; 8.79 mg and 39.4 mg from 1 L culture, respectively. Additionally, as expected, the addition of IPTG increased the amount of protein, approximately one-and-a-half-fold for LB and about three-fold for TB. Finally, it was observed that TB medium provided higher protein production than LB, which can be explained by the presence of glycerol and high yeast extract in the medium. Although our study contains results that will attract the attention of vaccine industry, it should be kept in mind that all process should always be optimized depending on the structure of the targeted protein and thus the production amount can be further increased.

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

“…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.…”

Proteomics and metabolic burden analysis to understand the impact of recombinant protein production in E. coli

“…There are several cellular and environmental factors that affect the RPP in the host system. The type of media used for bacterial growth and choice of protein expression system are among the major parameters that influence the RPP in a very significant manner 3 . The promoter used for facilitating the expression of RPP is another factor that makes a large difference.…”