Affinity purification/immobilization of poly histidine-tagged proteins by nickel-functionalized porous chitosan membranes.

Although immobilized metal ion affinity chromatography (IMAC) is one of the most effective methods for purifying his-tagged proteins, it has limitations such as expensive commercial resins and non-specific binding of unwanted proteins to the nickel immobilized on the resin. In this study, biocompatible chitosan and porous chitosan membranes as alternative resins were synthesized for protein immobilization and purification, but finally porous chitosan membrane was selected due to its higher porosity and consequently higher nickel adsorption. Once the membrane was functionalized with nickel ions and its metal adsorption confirmed by EDS and ICP methods, it was used to immobilize and purify recombinant ß-NGF as a protein model with his-tag tail in batch-fashion. Protein binding and purification were also approved by FTIR and UV-Vis spectroscopy and SDS-PAGE technique. Our results indicated that the protein of interest could bind to the nickel-functionalized porous chitosan membrane with high efficiency at pH=7. Furthermore, for protein purification, the pH value of 6 and an imidazole concentration of 750 mM were suggested for the final elution buffer. In conclusion, nickel-functionalized porous chitosan membrane could be a suitable alternative to IMAC for low cost and specific protein immobilization and purification.

Comparison of the efficiency of the Sec and Tat secretory pathways in the secretion of recombinant neurturin protein using de novo designed signal peptides.

Since cytoplasmic expression of heterologous proteins with disulfide bonds leads to the formation of inclusion bodies in E. coli, periplasmic production is preferable. The N-terminal signal peptide attached to the secreted protein determines the type of secretory pathway through which the target protein is secreted; Sec, Tat, or SRP. The aim of this study was to design and compare two novel signal peptides for the secretion of recombinant neurturin (as a model) via the Sec and Tat pathways. For this purpose, we aligned the natural signal peptides from E. coli and Bacillus subtilis to identify the conserved amino acids and those with the highest repetition. The SignalP4.1 and TatP1.0 software were used to determine the secretion efficiency of the new signal peptides. The efficiency of new signal peptides was then evaluated and compared experimentally with two naturally used signal peptides. Quantitative analysis of Western blot bands showed that approximately 80% of the expressed neurturin was secreted into the periplasmic space by new signal peptides. Circular dichroism spectroscopy also confirmed the correct secondary structure of the secreted neurturin. In conclusion, these novel signal peptides can be used to secrete any other recombinant proteins to the periplasmic space of E. coli efficiently.

Engineering a CEACAM1 Variant with the Increased Binding Affinity to TIM-3 Receptor

Background: T-cell immunoglobulin domain and mucin domain-3 (TIM-3) is an inhibitory receptor expressed in a variety of cells, including dendritic cells, T-helper 1 lymphocytes, and natural killer cells. Binding of this protein to its ligand, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), causes T-cell exhaustion, a specific condition in which effector T cells lose their ability to proliferate and produce cytokines. Blocking this inhibitory receptor is known to be an effective strategy for treating cancer and other related diseases. Therefore, in this study, in order to block the inhibitory receptor of TIM-3, we designed and produced recombinantly a protein with a high binding affinity to this receptor. Methods: The extracellular domain of CEACAM1 involved in binding to TIM-3 was mutated using R script to obtain a variant with the increased binding affinity to TIM-3. The binding energy of the mutant protein was calculated using the FoldX module. Finally, after recombinant production of the most appropriate CEACAM1 variant (variant 39) in E. coli, its secondary structure was determined by CD spectroscopy. Results: The binding free energy between variant 39 and TIM-3 decreased from -5.63 to -14.49 kcal/mol, indicating an increased binding affinity to the receptor. Analysis of the secondary structure of this variant also showed that the mutation did not significantly alter the structure of the protein. Conclusion: Our findings suggest that variant 39 could bind to TIM-3 with a higher binding affinity than the wild-type, making it a proper therapeutic candidate for blocking TIM-3.

Magnetically purification/immobilization of poly histidine-tagged proteins by PEGylated magnetic graphene oxide nanocomposites.

Carbon-based nanomaterials have many applications in biomedicine due to their unique mechanical, chemical, and biological properties. Among them, graphene has received special attention due to its very high specific surface area, high flexibility, and chemical stability. In this study, graphene oxide was first functionalized with amine groups (GO-NH2) and then Fe3O4 nanoparticles were deposited on it using the hydrothermal method. In addition, polyethylene glycol (PEG) was attached to the magnetic graphene nanoparticles to increase their stability and solubility. Finally, PEGylated magnetic graphene nanocomposites were functionalized with nickel-nitrilotriacetic acid (NTA-Ni+2) to bind to the poly-histidine tag in recombinant proteins. The resulting nanocomposites (MG-PEG-NTA-Ni+2) were then used for magnetic immobilization and purification of recombinant ß-NGF as a protein with his-tag sequence. Binding and purification were confirmed by FTIR and SDS-PAGE techniques, respectively. Importantly, differentiation of the PC12 cell line into neurons demonstrated that the purified ß-NGF was fully functional. Our results suggest that MG-PEG-NTA-Ni+2 nanocomposites may be a suitable alternative to commercial resins for rapid and specific protein immobilization and purification.

In silico engineering a CD80 variant with increased affinity to CTLA-4 and decreased affinity to CD28 for optimized cancer immunotherapy.

CD80 or cluster of differentiation 80, also known as B7-1, is a member of the immunoglobulin super family, which binds to CTLA-4 and CD28 T cell receptors and induces inhibitory and inductive signals respectively. Although CTLA-4 and CD28 receptors belong to the same protein family, slight differences in their structures leads to CD80 having a higher binding affinity to CTLA-4 (-14.55 kcal/mol) compared with CD28(-12.51 kcal/mol). In this study, we constructed a variant of CD80 protein with increased binding affinity to CTLA-4 and decreased binding affinity to CD28. This variant has no signaling capability, and can act as a cap for these receptors to protect them from natural CD80 proteins existing in the body. The first step was the evolutionary and alanine scanning analysis of CD80 protein to determine conserved regions in this protein. Next, complex alanine scanning technique was employed to determine CD80 protein hotspots in CD80-CTLA-4 and CD80-CD28 protein complexes. This information was fed into a computational model developed in R for in silico mutagenesis and CD80 variant library construction. The 3D structures of variants were modeled using the Swiss model webserver. After modeling the 3D structures, HADDOCK server was employed to build all protein-protein complexes, which contain CTLA-4-CD80 variant complexes, Wild type CD80-CD28 complexes and CD28-CD80 variant complexes. Protein-protein binding free energy was determined using FoldX and the variant number 316 with mutations at 29, 31, 33 positions showed increased binding affinity to CTLA-4 (-21.43 kcal/mol) and decreased binding affinity to CD28 (- 9.54 kcal/mol). Finally, molecular dynamics (MD) simulations confirmed the stability of variant 316. In conclusion, we designed a new CD80 protein variant with potential immunotherapeutic applications.

Identification of new potent agonists for toll-like receptor 8 by virtual screening methods, molecular dynamics simulation, and MM-GBSA.

Toll-like receptor 8 (TLR8), as an endosomal transmembrane receptor, plays a crucial role in the innate immune response to neoplasia and viruses. Previous studies have shown that TLR8 agonists e.g. Motolimod can be used to treat patients with last-stage cancer. In this study, in order to find new suitable ligands for TLR8, 16 PBD codes related to TLR8 complexes were collected to design the pharmacophore models using the Pharmit server. Then the PubChem, and ZINC databases were screened by them. Subsequently, the ADME-Tox features of the compounds were detected using FAF-Drugs4 and the selected compounds were docked to TLR8 (PDB: 3w3j). Molecular dynamics simulation was used to compare compounds with the best docking scores, with Motolimod in complex with TLR8. Finally, two compounds were identified, PubChem: 124126919 (A) and PubChem: 18559540 (B), each with advantages over Motolimod. As the RMSD results showed that compound A has very good flexibility, in terms of energy calculated using the MM-GBSA method, complex B and TLR8 showed the lowest energy level compared to the rest of the complexes. These observations suggest that these two compounds could be used as TLR8 agonists with the desired pharmacological features in future experimental studies.Communicated by Ramaswamy H. Sarma.

Engineering a variant of IL-17RA with high binding affinity to IL-17A for optimized immunotherapy.

Immunotherapy is one of the most recently used treatments for numerous cancer types and also some autoimmune and inflammatory diseases. One of the valuable targets for immunotherapy is Interleukin-17A (IL-17A) or its receptor (IL-17RA) because overexpression of IL-17A as a pro-inflammatory cytokine is associated with several inflammatory, autoimmune and cancer diseases. In this study, the extracellular domain of IL-17RA involved in binding to IL-17A was mutated by using R software to achieve a variant with increased binding affinity to IL-17A. The ∆∆G value of -30.89 kcal/mol was calculated for the best variant (385) with point mutations of R265N, N91T, and W31K using the FoldX module. Also, the KD for its interaction with IL-17A was calculated 0.06 nM by surface plasmon resonance (SPR) technique. Our results indicated that variant 385 could bind to IL-17A with higher binding affinity than wild-type one, so, it can be a good therapeutic candidate for blocking IL-17A.

Denovo designing: a novel signal peptide for tat translocation pathway to transport activin A to the periplasmic space of E. coli.

OBJECTIVES: The twin-arginine translocation (Tat) pathway is one of the bacterial secretory strategies which exports folded proteins across the cytoplasmic membrane. RESULTS: In the present study, we designed a novel Tat-signal peptide for secretion of human activin A used as a recombinant protein model here. In doing so, Haloferax volcanii, Halobacterium salinarum, and Escherichia coli Tat specific signal peptides were aligned by ClustalW program to determine conserved and more frequently used residues. After making the initial signal peptide sequence and doing some mutations, efficiency of this designed signal peptide was evaluated using a set of well-known software programs such as TatP, PRED-TAT, and Phobius. Then the best complex between TatC as an initiator protein in Tat secretory machine and the new designed signal peptide connected to activin A with the lowest binding energy was constructed by HADDOCK server, and ΔΔG value of - 5.5 kcal/mol was calculated by FoldX module. After that, efficiency of this novel signal peptide for secretion of human activin A to the periplasmic space of E. coli Rosetta-gami (DE3) strain was experimentally evaluated; to scrutinize the activity of the novel signal peptide, Iranian Bacillus Licheniformis α-Amylase enzyme signal peptide as a Sec pathway signal peptide was used as a positive control. The quantitative analysis of western blotting bands by ImageJ software confirmed the high secretion ability of the new designed signal peptide; translocation of 69% of the produced recombinant activin A to the periplasmic space of E. coli. Circular Dichroism (CD) spectroscopy technique also approved the proper secondary structure of activin A secreted to the periplasmic space. The biological activity of activin A was also confirmed by differentiation of K562 erythroleukemia cells to the red blood cell by measuring the amount of hemoglobin or Fe2+ ion using ICP method. CONCLUSIONS: In conclusion, this novel designed signal peptide can be used to secrete any other recombinant proteins to the periplasmic space of E. coli efficiently.

Enhanced periplasmic expression of human activin A in Escherichia coli using a modified signal peptide.

Activin A is a member of the transforming growth factor-beta (TGF-ß) protein superfamily, which acts as a hormone in regulating cell proliferation and differentiation. Structurally, activin is a dimer of two subunits linked by a disulfide bond. Since the correct folding of this protein is essential for its function, we aimed to use a modified signal peptide to target the expressed recombinant protein to the periplasm of Escherichia coli as an effective strategy to produce correctly-folded activin A. Therefore, the coding sequence of native Iranian Bacillus licheniformis α-amylase signal peptide was modified and its efficiency was checked by SignalP bioinformatics tool. Then its final sequence was cloned upstream of the activin A mature cDNA. Protein expression was done using 1 mM of isopropyl thio-ß-D-galactoside (IPTG) and a post-induction time of 8 hr. Additionally, following purification of recombinant activin A, circular dichroism spectroscopy was used to determine the accuracy of secondary structure of the protein. Importantly, differentiation of K562 cells to the red blood cell was confirmed by measuring the amount of Fe+2 ions after treatment with recombinant activin A. The results indicated that the produced recombinant activin A had the same secondary structure as the commercial human activin A and was fully functional.

Improved Production of Recombinant Human ß-NGF in Escherichia coli - a Bioreactor Scale Study.

Human nerve growth factor ß (ß-NGF) is considered a major therapeutic agent for treatment of neurodegenerative diseases. We have previously reported the optimized conditions for ß-NGF overproduction in Escherichia coli in a shake-flask culture. In this study the optimal %DO (dissolved oxygen) and post induction temperature values for improved production of ß-NGF were found in the bioreactor scale using response surface methodology (RSM) as the most common statistical method. Also, for further enhancement of the yield, different post-induction periods of time were selected for testing. In all experiments, the productivity level and bacterial cell growth were evaluated by western blotting technique and monitoring of absorbance at 600 nm, respectively. Our results indicated that %DO, the post-induction time and temperature have significant effects on the production of ß-NGF. After 2 hours of induction, the low post induction temperature of 32°C and 20% DO were used to increase the production of ß-NGF in a 5-l bioreactor. Another important result obtained in this study was that the improved ß-NGF production was not achieved at highest dry cell weigh or highest cell growth. These results are definitely of importance for industrial ß-NGF production.

Evaluation of Anticancer Activity of Extracted Flavonoids from Morus Alba Leaves and its interaction with DNA

ABSTRACT Morous Alba, known as white mulberry contains many oxidative flavonoids, widely used in the treatment of many diseases like hyperglycemia, inflammation, fever and cancer. In the present study we investigated the interaction of extracted flavonoids from Iranian Morus Alba leaves with DNA as a main target for anticancer drugs. Various spectroscopic techniques (UV/Vis, CD and Fluorescence Spectroscopy) were used to detect the interaction. In vivo studies also were done to confirm the effectiveness of the extracted flavonoids. The spectroscopic results showed that the extracted flavonoids bind to DNA especially to the sugar-phosphate backbone and making DNA conformational changes upon this binding. Experiments on the cancerous mice with solid tumors indicated that the treatment of mice with these extracted flavonoids increased significantly the life span but they did not have any effects on the tumor size reduction. These data suggest that Morus Alba flavonoids may use as an effective natural anticancer drug in the near future.

Optimization of recombinant ß-NGF expression in Escherichia coli using response surface methodology.

Human nerve growth factor a member of the neurotrophin family can be used to treat neurodegenerative diseases. As it has disulfide bonds in its structure, periplasmic expression of it using appropriate signal sequence is beneficial. Therefore, in this work ß-nerve growth factor (ß-NGF) was expressed in Escherichia coli using pET39b expression vector containing DsbA signal sequence. In an initial step, the effect of isopropyl ß-D-1-thiogalactopyranoside (IPTG) and lactose concentration as inducer on protein production was investigated using response surface methodology. Then the effect of different postinduction time and temperature on protein production was studied. Our results indicated that the highest ß-NGF production was achieved with 1 mM IPTG and low concentrations of lactose (0-2% w/v), low cultivation temperature of 25°C and postinduction time of 2 hr. Also following ß-NGF purification, bioassay test using PC12 cell line was done. The biological activity of the purified ß-NGF showed a similar cell proliferation activity with the standard recombinant human ß-NGF. In conclusion, the results indicated an optimized upstream process to obtain high yields of biologically active ß-NGF.

The effect of mitoxantrone as an anticancer drug on hepatocytes nuclei and chromatin: Selective release of histone proteins.

OBJECTIVES: Mitoxantrone is an anticancer drug widely used in the treatment of various cancers. In the present study the effect of mitoxantrone on chromatin proteins of intact hepatocytes nuclei was investigated and compared with soluble chromatin. MATERIALS AND METHODS: UV/Vis spectroscopy, SDS polyacrylamide gel electrophoresis, and western bolting were used. RESULTS: The results show that exposure of intact nuclei to various concentrations of mitoxantrone resulted in the release of histone H1 family proteins, H1 and H1°, in a dose-dependent manner but not core histones and high mobility group proteins. Western blot analysis using antiserum against histones H1 and H1° revealed cross-reactivity and confirmed the result. Spectroscopy results showed that mitoxantrone binds to nuclear components and reduces the absorbances at 608 and 400 nm. The binding isotherms revealed cooperative binding with one binding site. CONCLUSION: From the results it is suggested that mitoxantrone binds to intact nuclei and chromatin with different affinities and linker DNA can be considered as a main binding site for mitoxantrone at the nuclei level.

Interaction of mitoxantrone, as an anticancer drug, with chromatin proteins, core histones and H1, in solution.

In the present study, for the first time we have investigated the interaction of anticancer drug mitoxantrone with histone H1 and core histone proteins in solution using fluorescence, UV/Vis, CD spectroscopy and thermal denaturation techniques. The results showed that mitoxantrone reduced the absorbencies of H1 and core histone proteins at 210 nm (hypochromicity) and fluorescence emission intensity was decreased in a dose dependent. Binding of mitoxantrone changed secondary structures of the proteins as circular dichroism analysis confirmed it. Also, mitoxantrone increased the melting temperature of core histones at the final step of denaturation. The results suggest higher affinity of mitoxantrone to histone H1 compared to core histones providing histone proteins as a new target for mitoxantrone action at the chromatin level.

The effect of vinca alkaloid anticancer drug, vinorelbine, on chromatin and histone proteins in solution.

Vinorelbine (navelbin) belongs to vinca alkaloid anticancer drugs family with a broad spectrum of selective activity against mitotic microtubules. The present study is the first report demonstrating chromatin components as a novel target for vinorelbine in hepatocytes. The interaction was carried out in solution, employing fluorescence, UV spectroscopy and thermal denaturation techniques. Fluorescence emission spectra represented quenching of DNA chromospheres with drug and decreased fluorescence emission intensity in a dose-dependent manner. Binding of vinorelbine to chromatin induced very high hypochromicity and shifted DNA melting temperature to lower Tm. Vinorelbine binds to histone proteins with very high affinity when compared with the interaction of DNA intercalator anticancer drug, daunomycin, and the globular domain of the histones is considered as a main drug binding site. The results also showed that in the presence of vinorelbine, the absorbance of chromatin at 260 nm was decreased and the binding pattern was similar to daunomycin-chromatin complex. The results for the first time suggest that apart from tubulins, chromatin components can also be considered as a new target for this anticancer drug.

Studies on the binding affinity of anticancer drug mitoxantrone to chromatin, DNA and histone proteins.

Mitoxantrone is a potent antitumor drug, widely used in the treatment of various cancers. In the present study, we have investigated and compared the affinity of anticancer drug, mitoxantrone, to EDTA-soluble chromatin (SE-chromatin), DNA and histones employing UV/Vis, fluorescence, CD spectroscopy, gel electrophoresis and equilibrium dialysis techniques. The results showed that the interaction of mitoxantrone with SE-chromatin proceeds into compaction/aggregation as revealed by reduction in the absorbencies at 608 and 260 nm (hypochromicity) and disappearance of both histones and DNA on the gels. Mitoxantrone interacts strongly with histone proteins in solution making structural changes in the molecule as shown by CD and fluorescence analysis. The binding isotherms demonstrate a positive cooperative binding pattern for the chromatin- mitoxantrone interaction. It is suggested higher binding affinity of mitoxantrone to chromatin compared to DNA implying that the histone proteins may play an important role in the chromatin- mitoxantrone interaction process.