Expression and purification of soluble and functional fusion protein DAB₃₈₉IL‐2 into the E. coli strain Rosetta‐gami (DE3)

Abstract

DAB389IL‐2 (Denileukin diftitox) is considered an immunotoxin, and it is the first immunotoxin approved by Food and Drug Administration. It is used for the treatment of a cutaneous form of T‐cell lymphoma. This fusion protein has two disulfide bonds in its structure that play an essential role in toxicity and functionality of the immunotoxin. Escherichia coli (E. coli) strain BL21 (DE3) is not capable of making disulfide bonds in its reductive cytoplasm, but the E. coli strain Rosetta‐gami (DE3) is a proper strain for the correct expression of the protein due to mutations in glutaredoxin reductase and thioredoxin reductase. In this study, a pET21a vector with the His6‐tag fused at the N‐terminus of DAB389IL‐2 was used to express the soluble immunotoxin in E. coli Rosetta‐gami (DE3). After the purification of the soluble protein by two‐step column chromatographies, the structure of DAB389IL‐2 was analyzed using the Native‐PAGE and circular dichroism methods. In the following, the nuclease activity of soluble DAB389IL‐2 and its cytotoxicity activity were determined. It is concluded that the soluble recombinant protein expressed in the E. coli Rosetta‐gami (DE3) has an intact structure and also functional; hence, this form of immunotoxin could be competitive with its commercial counterparts.

Designing and Expression of Recombinant Chimeric Spike Protein from SARS-CoV-2 in Escherichia coli and Its Immunogenicity Assessment

“…The S1/S2 cleavage was used as a linker between RBD and FP. There are 3 to 5 epitope regions available in the RBD sequence for vaccine design ( 36 ). Due to the optimization performed on the RS gene and the RBD epitope, our sequence contained S1/S2 cleavage and FP region epitopes to stimulate the immune system.…”

“…The spike protein has multiple disulfide bonds ( 41 ). Therefore, the E. coli Rosetta-gami (DE3) was selected, as it is a proper host for the spike expression due to uncommon codons ( 42 ) and the formation of target protein disulfide bonds in the bacterial cytoplasm ( 36 ).…”

Identification and structural analysis of a thermophilic β-1,3-glucanase from compost

“…The β-1,3-glucanase gene was codon optimized, synthesized, and cloned into the expression vector pCold II (Tsingke). The construct was transformed into the E.coli Rosetta (DE3) competent cells (Novagen) which are proper bacterial strains for the intact formation of disulfide bonds in cytoplasm (Zarkar et al 2019 ). The protein was induced at 15 °C with 0.5 mM IPTG after 24 h cultivation in the minimal media, which could slow down protein folding and prevent formation of inclusion body during protein expression (Törnkvist et al 1996 ).…”

Site-Specific PEGylation of Recombinant Immunotoxin DAB389IL-2: Structural and Functional Assessment

“…It is used for the treatment of cutaneous T cell lymphoma. DAB 389 IL-2 contains two distinct portions: Catalytic domain of Diphtheria Toxin (DT) and IL-2 [17][18][19]. DT and IL-2 are considered as cell-killing and cell-binding, respectively [20].…”

“…In cancer cells, DT exerts its cytotoxic role after ADP-ribosylation of Elongation Factor 2 (EF-2) and eventually, inhibition of protein synthesis and cell death [21]. Also, the DT-moiety of immunotoxin has a nuclease activity by which it can induce cellular death through DNA degradation [19,22]. DAB 389 IL-2 with 521 amino acids has a highly reactive free cysteine residue in the IL-2 portion along with two disulfide bonds in 202-187 and 446-493 that play an important role in its toxicity and function.…”