Soluble and insoluble expression of recombinant human interleukin-2 protein using pET expression vector in Escherichia coli.

Interleukin-2 has emerged as a potent protein-based drug to treat various cancers, AIDS, and autoimmune diseases. Despite its immense requirement, the production procedures are inefficient to meet the demand. Therefore, efficient production procedures must be adopted to improve protein yield and decrease procedural loss. This study analyzed cytoplasmic and periplasmic IL-2 expression for increased protein yield and significant biological activity. The study is focused on cloning IL-2 into a pET-SUMO and pET-28a vector that expresses IL-2 in soluble form and inclusion bodies, respectively. Both constructs were expressed into different E. coli expression strains, but the periplasmic and cytoplasmic expression of IL-2 was highest in overnight culture in Rosetta 2 (DE3). Therefore, E. coli Rosetta 2 (DE3) was selected for large-scale production and purification. Purified IL-2 was characterized by SDS-PAGE and western blotting, while its biological activity was determined using MTT bioassay. The results depict that the periplasmic and cytoplasmic IL-2 achieved adequate purification, yielding 0.86 and 0.51 mg/mL, respectively, with significant cytotoxic activity of periplasmic and cytoplasmic IL-2. Periplasmic IL-2 has shown better yield and significant biological activity in vitro which describes its attainment of native protein structure and function.

Improving Access to Anti-HDV Testing: Development and Validation of an Affordable In-House ELISA Assay.

In certain low-income nations, the hepatitis Delta virus and hepatitis B virus (HBV) pose a serious medical burden, where the prevalence of hepatitis B surface antigen (HBsAg) is greater than 8%. Especially in rural places, irregular diagnostic exams are the main restriction and reason for underestimation. Utilizing serum samples from a Pakistani isolate, an internal ELISA for the quick identification of anti-HDV was created, and the effectiveness of the test was compared to a commercial diagnostic kit. HDV-positive serum samples were collected, and a highly antigenic domain of HDAg antigen was derived from them. This antigenic HDAg was expressed in a bacterial expression system, purified by Ni-chromatography, and confirmed by SDS-PAGE and Western blot analysis. The purified antigen was utilized to develop an in-house ELISA assay for anti-HDV antibody detection of the patient's serum samples at very low cost. Purified antigens and positive and negative controls can detect anti-HDV (antibodies) in ELISA plates. The in-house developed kit's efficiency was compared with that of a commercial kit (Witech Inc., USA) by the mean optical density values of both kits. No significant difference was observed (a P value of 0.576) by applying statistical analysis. The newly developed in-house ELISA is equally efficient compared to commercial kits, and these may be useful in regular diagnostic laboratories, especially for analyzing local isolates.