Molecular Cloning, Characterization and Predicted Structure of a Putative Copper-Zinc SOD from the Camel, Camelus dromedarius

Superoxide dismutase (SOD) is the first line of defense against oxidative stress induced by endogenous and/or exogenous factors and thus helps in maintaining the cellular integrity. Its activity is related to many diseases; so, it is of importance to study the structure and expression of SOD gene in an animal naturally exposed most of its life to the direct sunlight as a cause of oxidative stress. Arabian camel (one humped camel, Camelus dromedarius) is adapted to the widely varying desert climatic conditions that extremely changes during daily life in the Arabian Gulf. Studying the cSOD1 in C. dromedarius could help understand the impact of exposure to direct sunlight and desert life on the health status of such mammal. The full coding region of a putative CuZnSOD gene of C. dromedarius (cSOD1) was amplified by reverse transcription PCR and cloned for the first time (gene bank accession number for nucleotides and amino acids are JF758876 and AEF32527, respectively). The cDNA sequencing revealed an open reading frame of 459 nucleotides encoding a protein of 153 amino acids which is equal to the coding region of SOD1 gene and protein from many organisms. The calculated molecular weight and isoelectric point of cSOD1 was 15.7 kDa and 6.2, respectively. The level of expression of cSOD1 in different camel tissues (liver, kidney, spleen, lung and testis) was examined using Real Time-PCR. The highest level of cSOD1 transcript was found in the camel liver (represented as 100%) followed by testis (45%), kidney (13%), lung (11%) and spleen (10%), using 18S ribosomal subunit as endogenous control. The deduced amino acid sequence exhibited high similarity with Cebus apella (90%), Sus scrofa (88%), Cavia porcellus (88%), Mus musculus (88%), Macaca mulatta (87%), Pan troglodytes (87%), Homo sapiens (87%), Canis familiaris (86%), Bos taurus (86%), Pongo abelii (85%) and Equus caballus (82%). Phylogenetic analysis revealed that cSOD1 is grouped together with S. scrofa. The predicted 3D structure of cSOD1 showed high similarity with the human and bovine CuZnSOD homologues. The Root-mean-square deviation (rmsd) between cSOD1/hSOD1 and cSOD1/bSOD1 superimposed structure pairs were 0.557 and 0.425 A. The Q-score of cSOD1-hSOD1 and cSOD1-bSOD1 were 0.948 and 0.961, respectively

A novel strategy for the periplasmic production of heterologous proteins in E. coli

Die Produktion disufidhaltiger Proteine in ihrer nativen Form in Escherichia coli ist eine Herausforderung für die wissenschaftliche Forschung. Durch Sekretion solcher Proteine in das oxidierende Periplasma kann die Bildung der natürlichen Struktur erreicht werden. In der Untersuchung wurden humanes Pepsinogen und humanes Proinsulin als Modellproteine verwendet. Humanes Pepsinogen wurde an den C-Terminus von Ecotin, den E. coli trypsin inhibitor, fusioniert. Ecotin ist ein sehr stabiles homodimeres periplasmatisches Protein (16 kDa). Nach Fermentation und Produktion von Ecotin-Pepsinogen unter Kontrolle des trc-Promotors wurde das Fusionsprotein zur Homogenität aufgereinigt. Die Ausbeute für natives Pepsinogen betrug 7,6 mg pro L Fermentationsmedium. Für die Quantifizierung von Pepsinogen wurde ein neuartiges Fluoreszenz-Nachweissystem entwickelt, mit EGFP (enhanced green fluorescent protein) als Substrat. Die Eignung von Ecotin als Fusionspartner für die periplasmatische Proteinproduktion wurde mit humanem Proinsulin als weiterem Modellprotein überprüft. Proinsulin unterscheidet sich von Pepsinogen in Größe, Struktur, Muster der Disulfidverbrückung, und Funktion. Proinsulin wurde an den C-Terminus von Ecotin fusioniert und unter der Kontrolle des trc-Promotors in E. coli exprimiert. Nach Fermentation im Hochzelldichteverfahren wurden 153 mg Ecotin-Proinsulin pro Liter Fermentationsmedium produziert. Die Reinigung des Fusionsproteins erfolgte durch Aufarbeitung der Zellen mittels einer neu etablierten Affinitätschromatographie mit Trypsinogen als immobilisierten Bindungsmolekül.The production of proteins containing native disulfide bonds in Escherichia coli is a challenging task of research. Secretion of such proteins into the oxidizing periplasm of E. coli gives a chance of proper folding. In this study, human pepsinogen and human proinsulin were used as model proteins. As a new approach, human pepsinogen was fused to the C-terminus of ecotin, E. coli trypsin inhibitor, which is a highly stable homodimeric periplasmic protein (16 kDa). After production of ecotin-pepsinogen under control of a trc promoter, ecotin-pepsinogen was purified to homogeneity. The yield of purified native pepsinogen was 7.6 mg per liter fermentation broth. For the quantification of pepsinogen, a fluorogenic assay was developed using EGFP, enhanced green fluorescent protein, as a substrate. To evaluate the applicability of ecotin as a periplasmic fusion tag, human proinsulin was chosen as second model protein because it differs from pepsinogen in size, fold, pattern of disulfide bonds, and function. Proinsulin was fused to the C-terminus of ecotin and was expressed under control of the trc promoter. In high cell density cultivation, 153 mg ecotin-proinsulin per liter broth was produced. Downstream processing of ecotin-proinsulin was done in one step using a newly established affinity purification method based on the strong binding affinity of ecotin for trypsinogen.von Ajamaluddin Mali

A unique group of self-splicing introns in bacteriophage T4

289-293We describe in this review, the salient splicing features of group I introns of bac teriophage T4 and propose, a hypothetical model to fit in the self-splicing of nrdB intron ofT4 phage. Occurrence of non-coding sequences in prokaryotic cells is a rare event while it is common in ellkaryotic cells, especially the higher eukaryotes. Therefore. T4 bacteriophage can serve as a good model system to study the evoluti onary aspects of splicing of introns. Three genes of T4 phage were found to have st retches of non-coding sequences which belonged to the group IA type introns of self-splicing nature

Sunset Yellow Dye Induces Amorphous Aggregation in β-Lactoglobulin at Acidic pH: A Multi-Techniques Approach

Protein aggregation is of two types: (i) amorphous and (ii) amyloid fibril. Several extrinsic factors (temperature, pH, and small ligands) stimulate protein aggregation in vitro. In this study, we have examined the role of sunset yellow (SY) on the β-lactoglobulin (BLG) aggregation at pH 2.0. We have used spectroscopic (turbidity, Rayleigh light scattering (RLS), far-UV CD) and microscopic (transmission electron microscopy [TEM]) techniques to describe the effects of SY on BLG aggregation. Our results showed that BLG aggregation is dependent on SY concentrations. Very low concentrations (0.0–0.07 mM) of SY were unable to induce aggregation, while SY in the concentrations range of 0.1–5.0 mM induces aggregation in BLG. The kinetics of SY-stimulated aggregation is very fast and monomeric form of BLG directly converted into polymeric aggregates. The kinetics results also showed SY-induced BLG aggregation disappeared in the presence of NaCl. The far-UV CD and TEM results indicated the amorphous nature of SY-induced BLG aggregates. We believe that our results clearly suggest that SY dye effectively stimulates BLG aggregation

Iron response elements (IREs)-mRNA of Alzheimer's amyloid precursor protein binding to iron regulatory protein (IRP1): a combined molecular docking and spectroscopic approach

Abstract The interaction between the stem-loop structure of the Alzheimer's amyloid precursor protein IRE mRNA and iron regulatory protein was examined by employing molecular docking and multi-spectroscopic techniques. A detailed molecular docking analysis of APP IRE mRNA∙IRP1 reveals that 11 residues are involved in hydrogen bonding as the main driving force for the interaction. Fluorescence binding results revealed a strong interaction between APP IRE mRNA and IRP1 with a binding affinity and an average binding sites of 31.3 × 106 M−1 and 1.0, respectively. Addition of Fe2+(anaerobic) showed a decreased (3.3-fold) binding affinity of APP mRNA∙IRP1. Further, thermodynamic parameters of APP mRNA∙IRP1 interactions were an enthalpy-driven and entropy-favored event, with a large negative ΔH (–25.7 ± 2.5 kJ/mol) and a positive ΔS (65.0 ± 3.7 J/mol·K). A negative ΔH value for the complex formation suggested the contribution of hydrogen bonds and van der Waals forces. The addition of iron increased the enthalpic contribution by 38% and decreased the entropic influence by 97%. Furthermore, the stopped-flow kinetics of APP IRE mRNA∙IRP1 also confirmed the complex formation, having the rate of association (k on) and the rate of dissociation (k off) as 341 μM−1 s−1, and 11 s−1, respectively. The addition of Fe2+ has decreased the rate of association (k on) by ~ three-fold, whereas the rate of dissociation (k off) has increased by ~ two-fold. The activation energy for APP mRNA∙IRP1 complex was 52.5 ± 2.1 kJ/mol. The addition of Fe2+ changed appreciably the activation energy for the binding of APP mRNA with IRP1. Moreover, circular dichroism spectroscopy has confirmed further the APP mRNA∙IRP1 complex formation and IRP1 secondary structure change with the addition of APP mRNA. In the interaction between APP mRNA and IRP1, iron promotes structural changes in the APP IRE mRNA∙IRP1 complexes by changing the number of hydrogen bonds and promoting a conformational change in the IRP1 structure when it is bound to the APP IRE mRNA. It further illustrates how IRE stem-loop structure influences selectively the thermodynamics and kinetics of these protein-RNA interactions

Rational design of Zeocin binding protein variants for antibiotic resistance studies

Antibiotic resistance marker was used in this investigation because they are selected;e markers and effective in different hosts. This study used Zeocin binding protein (ZBP) due to its known 3D structure and applicability in prokaryotic and eukaryotic hosts. A library of 22 mutations was developed through a rational design strategy. Subsequently, a selection strategy was used to identify destabilizing mutations in the ZBP. ZBP variants were expressed in E.coli using a leaky expression approach, and minimum inhibitory concentration (MIC) was calculated by cultivating different variants at various Zeocin concentrations. Zeocin resistance was drastically decreased in some ZBP variants. Positive controls (wild-type ZBP) exhibited high resistance, while negative controls (pET vector without ZBP) showed susceptibility. Two variants (ZBP P9E and ZBP R26F) displayed drastic resistance loss. The variants reported in this study may identify molecular chaperones and folding modulators affecting proteostasis. These variants can potentially discover chemical chaperones that improve the stability and solubility of destabilized ZBP variants

Liposome-Mediated Delivery of MERS Antigen Induces Potent Humoral and Cell-Mediated Immune Response in Mice

The advancements in the field of nanotechnology have provided a great platform for the development of effective antiviral vaccines. Liposome-mediated delivery of antigens has been shown to induce the antigen-specific stimulation of the humoral and cell-mediated immune responses. Here, we prepared dried, reconstituted vesicles (DRVs) from DPPC liposomes and used them as the vaccine carrier system for the Middle East respiratory syndrome coronavirus papain-like protease (DRVs-MERS-CoV PLpro). MERS-CoV PLpro emulsified in the Incomplete Freund’s Adjuvant (IFA-MERS-CoV PLpro) was used as a control. Immunization of mice with DRVs-MERS-CoV PLpro did not induce any notable toxicity, as revealed by the levels of the serum alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and lactate dehydrogenase (LDH) in the blood of immunized mice. Immunization with DRVs-MERS-CoV PLpro induced greater antigen-specific antibody titer and switching of IgG1 isotyping to IgG2a as compared to immunization with IFA-MERS-CoV PLpro. Moreover, splenocytes from mice immunized with DRVs-MERS-CoV PLpro exhibited greater proliferation in response to antigen stimulation. Moreover, splenocytes from DRVs-MERS-CoV PLpro-immunized mice secreted significantly higher IFN-γ as compared to splenocytes from IFA-MERS-CoV PLpro mice. In summary, DRVs-MERS-CoV PLpro may prove to be an effective prophylactic formulation to prevent MERS-CoV infection

<em>Loranthus regularis</em> Ameliorates Neurodegenerative Factors in the Diabetic Rat Retina

Diabetic retinopathy remains a primary source of blindness with the growing pandemic of diabetes. Numerous studies have shown that early neurodegeneration caused by elevated oxidative stress may initiate microvascular damage in the diabetic retina during the last few decades. A variety of preventive and treatment strategies using phytochemicals that possess high antioxidants have shown great promise in reducing diabetes-induced neurodegeneration retinal damage. In this investigation, we employed an extract of Loranthus regularis, a traditional medicinal herb which is found to improve diabetes and associated complications in experimental studies. We orally treated STZ-induced diabetic rats with L. regularis and analyzed the neurodegenerative factors in the retina. After treatments, we used Western blotting techniques to analyze the protein content of neurotrophic factors (NGF, BDNF, TrkB), apoptotic factors (cytochrome c, Bcl-2, Bax), and phosphorylation of AKT in the diabetic retina. Additionally, we used ELISA methods to measure the contents of BDNF and the activity of Caspase-3 and biochemical procedures to determine the levels of glutathione and lipid peroxidation (TBARS). Our findings show that L. regularis treatments resulted in a considerable increase in neurotrophic factors and a decrease in apoptotic factors in the diabetic retina. Furthermore, in diabetic retina treated with L. regularis, the level of Bcl-2 protein increased, while the phosphor-AKT signaling improved. As a result, L. regularis may protect against diabetic-induced retinal neuronal damage by increasing neurotrophic support and reducing oxidative stress and apoptosis. Therefore, this study suggests that in diabetic retinopathy, L. regularis could be a potential therapy option for preventing neuronal cell death