Background/Objectives: Immune checkpoint blockade, particularly targeting the programmed cell death 1 (PD-1) receptor, is a promising strategy in cancer immunotherapy. The interaction between PD-1 and its ligands, PD-L1 and PD-L2, is crucial in immune evasion by tumors. Blocking this interaction with monoclonal antibodies like Nivolumab can restore anti-tumor immunity. This study aims to develop a stable expression system for Nivolumab-based anti-PD-1 in the Chinese Hamster Ovary (CHO) DG44 cell line using two different expression vector systems with various signal sequences. Methods: The heavy chain (HC) and light chain (LC) of Nivolumab were cloned into two expression vectors, pOptiVEC and pcDNA3.3. Each vector was engineered with two distinct signal sequences, resulting in the creation of eight recombinant plasmids. These plasmids were co-transfected into CHO DG44 cells in different combinations, allowing for the assessment of stable antibody production. Results: Both pOptiVEC and pcDNA3.3 vectors were successful in stably integrating and expressing the Nivolumab-based anti-PD-1 antibody in CHO DG44 cells. This study found that the choice of signal sequence significantly influenced the quantity of antibodies produced. The optimization of production conditions further enhanced antibody yield, indicating the potential for large-scale production. Conclusions: This study demonstrates that both pOptiVEC and pcDNA3.3 expression systems are effective for the stable production of Nivolumab-based anti-PD-1 in CHO DG44 cells. Signal sequences play a critical role in determining the expression levels, and optimizing production conditions can further increase antibody yield, supporting future applications in cancer immunotherapy.

New proteins as molecular targets in development of therapies are discovered every day. However, study of their interactions with other proteins or binding partners in complex cellular environments has its limits. Therefore, high-yield production of these proteins in heterologous systems is a valid necessity, while obtaining these proteins linked to suitable fluorescent markers represents a step ahead in protein-protein interaction studies and cellular or subcellular localization. In this study, we present production of human SMAC/Diablo recombinant protein with EGFP as a fusion partner. High-yield expression of the fusion protein was carried out in E. coli Rosetta™(DE3)pLysS strain, and an acceptable purity of the protein was obtained after affinity chromatography purification and gel filtration. The obtained protein can be further used in protein-protein interaction studies, whereas our method represents a cost-effective and efficient production method for EGFP-fused proteins, applicable for a number of therapeutically important polypeptides.

Crotoxin is a heterodimeric β-neurotoxin isolated from the venom of Crotalus durissus terrificus, consisting of two, non-covalently bound subunits, crotapotin (CA) and crotoxin B (CB). Both subunits present four different isoforms, consequently there are up to 16 different crotoxin complexes, each with different activity levels. The biological activities usually associated with crotoxin include neurotoxicity, myotoxicity and cardiotoxicity, however several other important biochemical and pharmacological effects of crotoxin have been observed, such as the antibacterial, antiviral, anti-inflammatory, antitumoral and analgesic activity. In this study we present the production of recombinant crotoxin B (isoform C). Expression of the protein was carried out using E. coli RosettaTM (DE3)pLysS strain, and the obtained protein was separated and purified using Ni2+-affinity chromatography. To the best of our knowledge the developed method represents the first reported method for obtaining the crotoxin B (isoform C) through heterologous expression using an efficient and cost-effective procedure.

Human proteins expressed in prokaryotic systems tend to form inclusion bodies. Proteins in inclusion bodies are inactive and the refolding of these densely packed protein molecules is affected by several factors depending on the applied refolding technique. To obtain the active form of protein the most common technique is denaturation of the protein aggregates followed by refolding of inclusion proteins. Conventional denaturants for solubilization are urea, guanidine hydrochloride and sodium dodecyl sulphate (SDS), while refolding can be achieved by several techniques found in the literature. In our study, the recombinant GST-tagged XIAP (X-linked Inhibitor of Apoptosis protein) construct was expressed as inclusion bodies. The protein was solubilized with high efficiency using N-Lauroylsarcosine (ionic detergent). A chromatography-based method using different ratios of detergents was investigated for the refolding process. Batch mode affinity purification was successfully executed using Glutathione Sepharose 4B resin and TritonX-100, n-octyl β-D-thio-glucopyranoside (OTG) and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate hydrate (CHAPS) detergents in the appropriate ratio. Finally, the refolded protein was purified by size-exclusion chromatography and investigated by western blot analysis.

Apoptotic regulation has been implicated in many human diseases, including cancer, autoimmune disease, inflammation and neuro degradation. Mapping up critical apoptosis regulators is a strategy for the development of new therapies [1, 2]. Present work highlights optimization of heterologous expression conditions for the X-linked inhibitor of apoptosis protein (XIAP). Genes of target protein containing pGEX-4T vector was transformed in chemically competent E. coli Rosetta™(DE3)pLysS cells. The recombinant construct contained a glutathione S-transferase (GST) fusion partner, which assured the purification of the protein by affinity chromatography. In the next step, we examined the growth dynamics of the expression culture in M9 minimal medium, meanwhile we also determined the appropriate time of induction. Following this we carried out the optimization of expression, examining the expression’s effectiveness under different conditions. On the basis of these fermentation experiments the target protein expression was the most prominent at 18 °C with 0.2 mM IPTG induction for 12 hours. During large scale fermentation experiments, we followed the optical density (OD), dry cell weight and substrate utilization. Finally, recombinant protein expression inhancement in the presence of 3% ethanol was successfully achieved in bioreactor. In this case, the target protein was expressed in inclusion bodies, therefore solubilisation and refolding is necessary.