Recombinant production of Annexin V protein for apoptosis detection to monitor cancer therapy

Introduction: Apoptosis or programmed cell death (PCD) plays an important role not only in physiology but also in pathology. One of the most prominent features of apoptosis is externalization of phosphatidylserine (PS), which in healthy cells are located in the inner leaflet of the plasma membrane. PS-externalization have made a well-explored phenomenon to image cell death for diagnostic and therapeutic purposes. Since many drugs induce a therapeutic effect through the activation of PCD in target cells, imaging of cell death offers a direct way to image therapy response. Moreover, failure of therapy is frequently a result of resistance against apoptosis. Therefore detection and quantification of apoptosis are some of the significant clinical value for diagnosis and assessment for  therapeutic efficacy. In this research we emphasize the expression and purification of recombinant polyhistidine-tagged human annexin V protein that binds to PS with high affinity and has been developed as a molecular imaging agent to measure cell death in vitro and  in vivo in animal models and in patients with cardiovascular diseases and cancer. We also describe conjugation of this protein to the Fluorescein isothiocyanate (FITC) fluorophore for the detection of apoptotic cells by flow cytometry or fluorescence microscopy. Methods and Results: In this project we transformed recombinant plasmid including annexin V gene into competent BL21 strain. after an overnight incubation at 37°C in LB medium,1 mL of that was used to inoculate 100 mL of TB culture. The expression of annexin V was induced with 1 mM IPTG (isopropyl-b-D-thiogalactopyranoside). The expression level of annexin V was evaluated by SDS-PAGE. Recombinant annexin V was expressed and purified to high yields. FITC as the fluorescent conjugate and a signal detector was used to attach to annexin V. we detected apoptotic cells in culture by annexin V-FITC produced probe in real time, using fluorescence microscopy and flow cytometry.  Conclusions: Annexin V staining is a simple and widely used method for detection of apoptosis in a rapid and highly quantitative manner for both early monitoring of therapy capability and assessment of disease development

Production and activity assay of recombinant micro-plasminogen in Pichia pastoris

Introduction: Plasmin is a trypsin like serine protease that can catalyze the hydrolytic cleavage of peptide bonds at the COOH sides of arginines and lysines in extracellular matrix (ECM) components including fibrin, laminin and fibronectin. Microplasminogen (µPlg) is the COOH-terminal portion of plasminogen from 531 to 791, and has molecular weight of about 30kDa. µPlg consists of the catalytic domain of plasminogen and can be converted by many plasminogen activators, for example, streptokinase (SK) into microplasmin, which shares the same catalytic properties as human plasmin. µPlg can be used as a therapeutic agent in vitreoretinopathies and thrombotic diseases. Methods and Results: The purpose of this study was production and activation of recombinant µPlg. After codon optimization and synthesis of the µPlg gene, it was inserted into expression vector pPICZα and this plasmid was transformed into Pichia pastoris X-33 cells by electroporation. The selected positive transforments were transferred into YPM (yeast extract-peptone-methanol) medium and were induced by methanol. The recombinant protein (33kDa) was successfully secreted into the supernatant of the induction medium and was purified by Ni-sepharose column. The presence of the recombinant µPlg was confirmed by SDS-PAGE. The activation of µPlg by streptokinase was performed at a 1:1 molar ratio of streptokinase to µPlg at 37°C for 2hr in Tris-HCl buffer, pH=7.8. Microplasminogen activity was measured by FDP (Fibrinogen Degradation Product) ELISA kit. In a blood sample the clot is broken down by plasmin. Some of These broken fibrin fragments are called d-dimers. In general, D-dimer elevation indicates increased fibrin turnover. In this procedure, 30 μL of active enzyme was added. And the rate of D-dimers was measured at 380 μg/mL. While the natural amount of D-dimers in the blood is less than 40 μg/mL. Conclusions: The availability of microplasminogen with lower molecular weight can be a valuable pharmaceutical tool for treatment of thrombotic diseases and vitreoretinopathies because the isolation of autologous plasmin is an expensive and time-consuming process. In this study we managed to produce microplasminogen and successfully turn it into active microplasmin. However more effort is needed to increase the yields of Protein Production

Recombinant production of Soluble Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (sTRAIL) as a therapeutic protein

Abstract:  Successfully cancer therapies aim to induce apoptosis in cancer cell lines. Recent advances in cancer therapy based on the use of some recombinant proteins such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL is a new member of the TNF superfamily. In this paper, we report the expression, purification, and  preparation of a recombinant form of the extracelluar domain of the TRAIL (sTRAIL) in Escherichia coli rosetta gami under the control of T7 promoter; which  may selectively induce apoptosis of tumor cells in vitro. To obtain recombinant sTRAIL protein, the encoding region for sTRAIL was cloned between Xho1 and BamHI in pET28a expression vector. The results showed that the recombinant sTRAIL was efficiently produced in Ecoli rosetta gami strain.                               Introduction: Apoptosis is an evolutionarily conserved and essential for maintenance of  tissue homeostasis and removal of unwanted cells. TRAIL belongs to the group of therapeutic agents selectively targeting a wide variety of cancer cells without affecting the normal cells .The therapeutic potential of TRAIL is attributed to its receptor expression in a variety of tissues; which initiates apoptosis in cancer cells through interaction with the death receptors DR4 and DR5. Due to its selective nature, it is considered as a significant therapeutic agent in cancer therapy. The purpose of this study was to produce recombinant human sTRAIL in Rosetta Gami2 E.coli strain and its functions on cancerous cells in vitro. Methods and results:  we optimized the coding sequence of this protein. The recombinant plasmid was transformed into Rosetta Gami2 E.coli strain for expression. The transformed bacteria which contain recombinant plasmid were cultured in 37ºC with 250 rpm in LB and in 20ºC in TB medium for 18 hours. TRAIL was purified by Ni sepharose column, and the presence of the recombinant protein was confirmed by SDS-PAGE. The concentration of purified protein was measured by Bradford assay. Our finding showed that the recombinant protein (34kD) has been successfully produced for next experiments, the purified protein was desalted and applied toward cancerous cells. Conclusions: In summary, TRAIL can be considered as a promising therapeutic agent for effective, targeted and less toxic agents for treatment of cancers

Biosynthesis of ternary NiCoFe2_2O4_4 nanoflowers: investigating their 3D structure and potential use in gene delivery

Multicomponent nanoparticle systems are known for their varied properties and functions, and have shown potential as gene nanocarriers. This study aims to synthesize and characterize ternary nickel–cobalt-ferrite (NiCoFe$_2$O$_4$) nanoparticles with the potential to serve as gene nanocarriers for cancer/gene therapy. The biogenic nanocarriers were prepared using a simple and eco-friendly method following green chemistry principles. The physicochemical properties of the nanoparticles were analyzed by X-ray diffraction, vibrating sample magnetometer, X-ray photoelectron spectroscopy, and Brunauer–Emmett–Teller. To evaluate the morphology of the nanoparticles, the field emission scanning electron microscopy with energy dispersive X-Ray spectroscopy, high-resolution transmission electron microscopy imaging, and electron tomography were conducted. Results indicate the nanoparticles have a nanoflower morphology with a mesoporous nature and a cubic spinel structure, where the rod and spherical nanoparticles became rose-like with a specific orientation. These nanoparticles were found to have minimal toxicity in human embryonic kidney 293 (HEK-293 T) cells at concentrations of 1 to 250 µg·mL–1. We also demonstrated that the nanoparticles could be used as gene nanocarriers for delivering genes to HEK-293 T cells using an external magnetic field, with optimal transfection efficiency achieved at an N/P ratio of 2.5. The study suggests that biogenic multicomponent nanocarriers show potential for safe and efficient gene delivery in cancer/gene therapy

Exploring the impact of taurine on the biochemical properties of urate oxidase: response surface methodology and molecular dynamics simulation

Abstract This paper investigates the impact of taurine as an additive on the structural and functional stability of urate oxidase. First, the effect of the processing parameters for the stabilization of Urate Oxidase (UOX) using taurine was examined using the response surface methodology (RSM) and the central composite design (CCD) model. Also, the study examines thermodynamic and kinetic parameters as well as structural changes of urate oxidase with and without taurine. Fluorescence intensity changes indicated static quenching during taurine binding. The obtained result indicates that taurine has the ability to preserve the native structural conformation of UOX. Furthermore, molecular dynamics simulation is conducted in order to get insights into the alterations in the structure of urate oxidase in the absence and presence of taurine under optimal conditions. The molecular dynamics simulation section investigated the formation of hydrogen bonds (H-bonds) between different components as well as analysis of root mean square deviation (RMSD), root mean square fluctuations (RMSF) and secondary structure. Lower Cα-RMSD and RMSF values indicate greater stabilization of the taurine-treated UOX structure compared to the free enzyme. The results of molecular docking indicate that the binding of taurine to the UOX enzyme through hydrophobic interactions is associated with a negative value for the Gibbs free energy

Isolation and identification of cutinase enzyme producing bacteria

Cutinase belongs to the family of serine hydrolases which are capable of hydrolysis of esters and small polyester such as cutin. The cutin of plants is composed of hydroxy and epoxy fatty acids and is substantially imperm-eable to water and only the plant pathogenic bacteria are capable of degrading it. Initially, in order to identify the extent of cutin in cucumber (Cucumis sativus' C. sativus'), golden apples (Golden Delicious apple 'Malus domestica'), red ap-ples (Red Delicious apple 'Malus domestica'') and tomatoes (Solanum lycopersicum Mill. Commun ' S. lycopersicum'), their fruits were obtained. Cutin was extracted from the fruit skin of each studied specimen and compared by chloro-form-methanol method. Then, isolation of enzyme-producing strains was performed by the use of the specific medium containing cutin and enzyme activity assay. DNA of the specimens was extracted, and PCR were performed with univ-ersal primers for 16s DNA. The remainder was devoted to bioinformatic analysis, identification and registration of samples in the gene bank. The results showed that the percentage of extracted cutin in red apple was more than other samples and as a result, red apple are expected to be more resistant against diseases and pests. Also, six cutinase-prod-ucer strains of Klebsiella and Enterobacter were isolated with the help of enzyme activity and special culture medium containing cutin, and their 16s DNA region coding sequences were recorded in the GenBank

Fabrication of a Novel and Ultrasensitive Label-Free Electrochemical Aptasensor Based on Gold Nanostructure for Detection of Homocysteine

The current attempt was made to detect the amino acid homocysteine (HMC) using an electrochemical aptasensor. A high-specificity HMC aptamer was used to fabricate an Au nanostructured/carbon paste electrode (Au-NS/CPE). HMC at high blood concentration (hyperhomocysteinemia) can be associated with endothelial cell damage leading to blood vessel inflammation, thereby possibly resulting in atherogenesis leading to ischemic damage. Our proposed protocol was to selectively immobilize the aptamer on the gate electrode with a high affinity to the HMC. The absence of a clear alteration in the current due to common interferants (methionine (Met) and cysteine (Cys)) indicated the high specificity of the sensor. The aptasensor was successful in sensing HMC ranging between 0.1 and 30 μM, with a narrow limit of detection (LOD) as low as 0.03 μM

A mononuclear diketone-based oxido-vanadium(iv) complex: structure, DNA and BSA binding, molecular docking and anticancer activities against MCF-7, HPG-2, and HT-29 cell lines

A mononuclear oxido-vanadium(IV) complex, [VO(L)(2)], has been prepared from the reaction of dibenzoylmethane (HL) and VO(acac)(2) in a 2 : 1 molar ratio, and fully characterized using elemental analyses, molar conductivity, FT-IR, and electronic spectroscopy. The structure of this compound was also confirmed by single crystal X-ray diffraction. It was found that in the title complex, the metal coordination geometry is described as a distorted square pyramid. DNA binding activities of this complex were investigated using electronic absorption titration, competitive fluorescence titration and cyclic voltammetry studies. The obtained results showed groove binding of the complex to salmon sperm DNA accompanied with a partial insertion of the ligand between the base stacks of the DNA with a binding constant of 2.3 x 10(3) M-1. In addition, the interaction of the complex with bovine serum albumin (BSA) was studied using electronic absorption and fluorescence spectroscopies at different temperatures indicating a good affinity of the complex for BSA. These experimental results were confirmed by the results of molecular docking. Finally, the in vitro cytotoxicity properties of the synthesized complex against MCF-7, HPG-2 and HT-29 cell lines were evaluated and compared with those of the ligand (HL). It was found that complexation improved the anticancer activity significantly. IC50 values for the V(IV) complex against MCF-7, HPG-2 and HT-29 cell lines were obtained as 7.8, 13.5 and 16.1 mu M, respectively

Synthesis, crystal structure and Hirshfeld surface analysis of copper(II) complexes: DNA- and BSA- binding, molecular modeling, cell imaging and cytotoxicity

In order to explore the structure–activity relationship of complexes, two copper(II) complexes, [Cu(dimethoxybpy)2](PF6)2 (1) and [Cu(dimethylbpy)2Cl]PF6 (2) (dimethoxybpy is 4,4′-dimethoxy-2,2′-bipyridine and dimethylbpy is 4,4′-dimethyl-2,2′-bipyridine), have been synthesized and characterized by several physicochemical techniques. The single-crystal X-ray structures of 1 and 2 exhibit distorted square-planar and distorted square-pyramidal structure, respectively. The Hirshfeld surface analysis and the associated 2D fingerprint plots of 1 and 2 have been also studied to evaluate intermolecular interactions. The DNA binding properties of 1 and 2 have been investigated by absorption and emission spectra, viscosity, cyclic voltammetry, circular dichroism and competitive DNA–binding studies, which indicate that the complexes interact with DNA through partial intercalation. The results of absorption and emission spectra, synchronous fluorescence and circular dichroism show that the complexes bind with BSA. The results exhibit that the complex 1 has stronger binding ability to DNA and BSA than the complex 2. Molecular docking technique has been used to evaluate and understand the interaction mode of 1 and 2 with DNA and BSA. The in vitro cytotoxicity of the complexes against MCF-7, A-549 and HT-29 cell lines has been assayed by MTT method. The complexes exhibit significant cytotoxicity in cell lines with IC50 values ranging from 1.5 to 53 μM. Based on the results of cytotoxicity, it would appear that the complex 2 has better cytotoxicity than the complex 1 under the same experimental conditions, suggesting that the hydrophobicity of methyl groups on the complex enhances the anticancer activity. The results of the microscopic analyses of cancer cells confirm the results of cytotoxicity