Bacterial production and biophysical characterization of a hard-to-fold scFv against myeloid leukemia cell surface marker, IL-1RAP.

BACKGROUND: Interleukin-1 receptor accessory protein (IL-1RAP) is one of the most promising therapeutic targets proposed for myeloid leukemia. Antibodies (Abs) specific to IL-1RAP could be valuable tools for targeted therapy of this lethal malignancy. This study is about the preparation of a difficult-to-produce single-chain variable fragment (scFv) construct against the membrane-bound isoform of human IL-1RAP using Escherichia coli (E. coli). METHODS: Different approaches were examined for refolding and characterization of the scFv. Binding activities of antibody fragments were comparatively evaluated using cell-based enzyme-linked immunosorbent assay (ELISA). Homogeneity and secondary structure of selected scFv preparation were analyzed using analytical size exclusion chromatography (SEC) and circular dichroism (CD) spectroscopy, respectively. The activity of the selected preparation was evaluated after long-term storage, repeated freeze-thaw cycles, or following incubation with normal and leukemic serum. RESULTS: Strategies for soluble expression of the scFv failed. Even with the help of Trx, ≥ 98% of proteins were expressed as inclusion bodies (IBs). Among three different refolding methods, the highest recovery rate was obtained from the dilution method (11.2%). Trx-tag substantially enhanced the expression level (18%, considering the molecular weight (MW) differences), recovery rate (˃1.6-fold), and binding activity (˃2.6-fold increase in absorbance450nm). The produced scFv exhibited expected secondary structure as well as acceptable bio-functionality, homogeneity, and stability. CONCLUSION: We were able to produce  21 mg/L culture functional and stable anti-IL-1RAP scFv via recovering IBs by pulse dilution procedure. The produced scFv as a useful targeting agent could be used in scheming new therapeutics or diagnostics for myeloid malignancies.

Discovery of Leptulipin, a New Anticancer Protein from theIranian Scorpion, Hemiscorpius lepturus.

Cancer is one of the leading causes of mortality in the world. Unfortunately, the present anticancer chemotherapeutics display high cytotoxicity. Accordingly, the discovery of new anticancer agents with lower side effects is highly necessitated. This study aimed to discover an anticancer compound from Hemiscorpius lepturus scorpion venom. Bioactivity-guided chromatography was performed to isolate an active compound against colon and breast cancer cell lines. 2D electrophoresis and MALDI-TOF were performed to identify the molecule. A partial protein sequence was obtained by mass spectrometry, while the full-length was deciphered using a cDNA library of the venom gland by bioinformatics analyses and was designated as leptulipin. The gene was cloned in pET-26b, expressed, and purified. The anticancer effect and mechanism action of leptulipin were evaluated by MTT, apoptosis, and cell cycle assays, as well as by gene expression analysis of apoptosis-related genes. The treated cells displayed inhibition of cell proliferation, altered morphology, DNA fragmentation, and cell cycle arrest. Furthermore, the treated cells showed a decrease in BCL-2 expression and an increase in Bax and Caspase 9 genes. In this study, we discovered a new anticancer protein from H. lepturus scorpion venom. Leptulipin showed significant anticancer activity against breast and colon cancer cell lines.

Mild and catalytic electrocyclizations of heptatrienyl anions.

We report the synthesis of functionalized cycloheptanes by thermal electrocyclization of heptatrienyl anions under mild conditions. In addition, we disclose the first examples of this electrocyclization manifold conducted under catalytic conditions. Previously, electrocyclization of heptatrienyl systems required formation of anions with a strong base, resulting in limited functional group compatibility. We demonstrate that polarization of heptatrienyl anions using strategically positioned electron-withdrawing groups lowers the energy landscape of the reaction by stabilizing both the acyclic heptatrienyl anion and cycloheptadienyl product. Divergent reactivity is observed between aliphatic and aromatic substrates, with the latter requiring only catalytic amounts of base for complete conversion. This can be rationalized by the relative stability of the acyclic and cyclic anions and their ability to participate in a chain reaction process.

Magnetic carboxymethyl cellulose-silk fibroin hydrogel: A ternary nanobiocomposite exhibiting excellent biological activity and in vitro hyperthermia of cancer therapy.

In this work, a magnetic nanobiocomposite scaffold based on carboxymethylcellulose (CMC) hydrogel, silk fibroin (SF), and magnetite nanoparticles was fabricated. The structural properties of this new magnetic nanobiocomposite were characterized by various analyses such as FT-IR, XRD, EDX, FE-SEM, TGA and VSM. According to the particle size histogram, most of the particles were between 55 and 77 nm and the value of saturation magnetization of this nanobiocomposite was reported 41.65 emu.g- 1. Hemolysis and MTT tests showed that the designed magnetic nanobiocomposite was compatible with the blood. In addition, the viability percentage of HEK293T normal cells did not change significantly, and the proliferation rate of BT549 cancer cells decreased in its vicinity. EC50 values for HEK293T normal cells after 48 h and 72 h were 3958 and 2566, respectively. Also, these values for BT549 cancer cells after 48 h and 72 h were 0.4545 and 0.9967, respectively. The efficiency of fabricated magnetic nanobiocomposite was appraised in a magnetic fluid hyperthermia manner. The specific absorption rate (SAR) of 69 W/g (for the 1 mg/mL sample at 200 kHz) was measured under the alternating magnetic field (AMF).

Computational and Experimental Evaluation of Linker Peptides and Thioredoxin Fusion Tag in CD20-rituximab Specific Interactions.

Background: Overexpression of CD20 protein on the surface of B cells in lymphoma can be targeted by several anti-CD20 molecules. The development of accessible interactive epitopes is more favorable than the full-length transmembrane CD20 in the affinity assessment of anti-CD20 monoclonal antibodies (mAbs). Methods: The sequence of these epitopes was extracted, and the effects of different linker peptides and the location of histidine (His)-tag were computationally analyzed. The impact of thioredoxin (Trx)-tag on the folding of the selected construct and its interaction with rituximab was further investigated. The two final expression cassettes were expressed in Escherichia coli after optimization of culture conditions for incubation temperature, post-induction time, optical density at the induction time, and concentration of the inducer. ELISA evaluated the binding affinity of rituximab towards the recombinant proteins. Results: By homology modeling studies, C-terminal His-tagged structures represented more desirable folded structures. Validation of the models revealed that CD20 extracellular domain linked by the G4S polypeptide had better stereochemical quality and structural compatibility. It was selected due to its more effective interaction with rituximab showing the highest dissociation constant of 5.8E-09M, which improved after the fusion of Trx-tag (7.1E-10M). The most influential parameters in the expression of the two selected proteins were post-induction temperature and optical density at the induction time. Homemade ELISA assays revealed a slightly higher affinity of rituximab towards the Trx-CD20 protein than the CD20/G4S molecule. Conclusions: Experimental in vitro studies confirmed the computationally calculated affinity of rituximab towards the two designed CD20 constructs. Also, the cell-based binding assessment of anti-CD20 mAbs could be substituted by the engineered extracellular domain of human CD20 protein.

Fabrication of a magnetic alginate-silk fibroin hydrogel, containing halloysite nanotubes as a novel nanocomposite for biological and hyperthermia applications.

In this study, the main focus was on designing and synthesizing a novel magnetic nanobiocomposite and its application in hyperthermia cancer treatment. Regarding this aim, sodium alginate (SA) hydrogel with CaCl2 cross-linker formed and modified by silk fibroin (SF) natural polymer and halloysite nanotubes (HNTs), followed by in situ Fe3O4 magnetic nanoparticles preparation. No important differences were detected in red blood cells (RBCs) hemolysis, confirming the high blood compatibility of the treated erythrocytes with this nanobiocomposite. Moreover, the synthesized SA hydrogel/SF/HNTs/Fe3O4 nanobiocomposite does not demonstrate toxicity toward HEK293T normal cell line after 48 and 72 h. The anticancer property of SA hydrogel/SF/HNTs/Fe3O4 nanobiocomposites against breast cancer cell lines was corroborated. The magnetic saturation of the mentioned magnetic nanobiocomposite was 15.96 emu g-1. The specific absorption rate (SAR) was measured to be 22.3 W g-1 by applying an alternating magnetic field (AMF). This novel nanobiocomposite could perform efficiently in the magnetic fluid hyperthermia process, according to the obtained results.

Production of an Antibody Fragment (scFv) Targeting PcrV Protein of Pseudomonas aeruginosa in Fed-Batch Cultivation Mode

Background: Pseudomonas aeruginosa is one of the opportunistic pathogens causing frequent hospital-acquired life-threatening infections in mechanically ventilated patients. The most significant virulence factor of P. aeruginosa is type III secretion system (T3SS). PcrV is an important structural protein of the T3SS. Methods: In the current investigation, a recombinant single-chain fragment variable (scFv) mAb against the PcrV protein was expressed in EnBase® (fed-batch) cultivation mode. The pETiteTM N-His SUMO Kan vector, including anti-PcrV scFv gene, was transformed into Escherichia coli (BL21) cells. The expression and solubility of anti-PcrV scFv protein were investigated at two different temperatures (25 °C and 30 °C) and at different induction times (4, 6, 8, 12, and 24 hours). Results: : Increased efficiency was achieved by EnBase® compared to Luria­Bertani broth; owing to the slow release of glucose, the maximum level of solubility and total protein expression was observed in EnBase® cultivation system at 30 °C and 24 h post induction. Furthermore, IC50 for anti-PcrV scFv protein was determined to be approximately 7 µg/mL. Conclusion: : Anti-PcrV scFv produced in this study showed promising in vitro results, protecting RBC from lysis by P. aeruginosa (exoU+).

Characterization of a novel mCH3 conjugated anti-PcrV scFv molecule.

Pseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. The stereochemical properties of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular interactions towards Pseudomonas aeruginosa PcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within the E. coli BL21-DE3 strain. After Ni-NTA affinity chromatography, the biological activity of the proteins in inhibition of PA induced hemolysis as well as cellular cytotoxicity was assessed. In silico analysis revealed the satisfactory stereochemical quality of the models as well as common residues in their interface with PcrV. The structural differences of proteins through circular dichroism spectroscopy were confirmed by NMR analysis. Both proteins indicated inhibition of ExoU positive PA strains in hemolysis of red blood cells compared to ExoU negative strains as well as cytotoxicity effect on lung epithelial cells. The ELISA test showed the longer serum stability of the YFL001 molecule than YFL002. The results were encouraging to further evaluation of these two scFv molecules in animal models.