From the data is possible to determine that the relation between the experimental assays and the automated results is the same for all the ligands (Figure 3.14). The study of interactions in silico presents several limitations, because neither the support (e.g. agarose resin) or the solvatation ofprotein-ligand are considered (Platis et al. 2006). These points are important, since when the ligand is attached to a support the conformation and the mobility of the ligand are limited, besides it can only interact with residues that are exposed in the protein surface and not in the core, although in these study was considered the spacer-arm - epoxide group activated in the resin, like suggested by other works (Haigh et al. 2009). Furthermore, the consideration of the presence of a solvent is important for the mobility of the structure and it is able of mediate some interactions, e.g. hydrogen bonds in the complex, because the angles of the complex are more flexible (Haigh et al. 2009). However, when the flexibility of the ligands is taken into account that would increase the number of possible interactions, to analyse all that binding possibilities, that would required even with computers more time and improved softwares (Thomsen and Christensen 2006). Furthermore, in this particular study was not considered the secondary structure of the AffinityTag that can interfere in the interaction between this molecule and the ligands, since the more or less rigidity of the molecule can restrict or improve the interactions.
Interleukin-24 (IL-24) is a novel tumor-suppressor gene that has different alternative splice isoforms. It has been shown that new smallest isoform of human IL-24 gene, lacking three exons, induces higher levels of cytotoxicity than all the isoforms, indicating shortest isoform of IL-24 may be a new promising anti-cancer agent. In this study, we aimed to provide a reproducible method for recombinant production of the smallest isoform of IL-24 (sIL-24). The Structure of sIL-24 was analyzed using bioinformatics tools (I-TASSER, Prosa, RAMPAGE and SPDBV version 4.1). The DNA sequence encoding sIL-24 was chemically synthesized and sub-cloned into the pET-32a (+) vector for further proteinexpression in Escherichia coli BL21 (DE3) strain. Upon IPTG induction, sIL-24 peptide was expressed as a thioredoxin fusion protein. The recombinant sIL-24 was released from the fusion by TEV protease cleavage followed by nickel affinity chromatography. The yield of the purified sIL-24 was estimated about 380 μg/ml. MTT assay showed that sIL-24 peptide inhibited the proliferation of PC-3 cancer cells more effectively than full length IL-24 protein, while none affect the survival of MRC-5 normal cells. These results indicate that the presented expression system is an efficient system for the production of small functional recombinant sIL-24 peptide.This functional peptide may have cancer therapeutic application.
Q-Sepharose (GE Healthcare Biosciences) was packed according to company guide-lines (20 mL of gel volume) into a C 16/20 [16 mm (diameter) x 200 mm (length)] glass column purchased from GE Healthcare Biosciences. Screening experiments were performed at different salt concentrations in order to assess the ideal sodium chloride concentration required for SCOMT_6His retention. The column was initially equilibrated with 10 mM Tris-HCl buffer pH 7.8. Aliquots ofrecombinant SCOMT_6His containing supernatant (3 mL) were injected into column at 1 mL/min with 10 mM Tris-HCl pH 7.8. The elution of unretained species occurred with an increasing sodium chloride gradient from 0 mM to 100 mM (3 CV). After elution, sodium chloride concentration was gradually increased from 100 mM to 310 mM (3.5 CV) to ensure that the protein was completely bound to the column. Subsequently, sodium chloride concentration in mobile phase was increased to 450 mM in a step mode (2.5 CV). Finally, a washing step was applied with 1 M of sodium chloride (2 CV) in a step mode. In all chromatographic runs, conductivity was continuously monitored, as well as absorbance at 280 nm. Fraction volumes of 3 mL were collected and pooled according to the obtained chromatographic profile. Finally, samples were concentrated and desalted with 1 mL 10 mM Tris-HCl pH 7.8 with Vivaspin concentrators (10.000 MWCO) and conserved at 4 ºC until further analysis.
was obtained from 1 L culture medium. The enzymatic activity of the fusion protein was reached 6.0×10 5 U/µM, which was seven times that of EKMax TM . It could be concluded that the specific activity reported in this work was the highest among the data currently available. The enzyme could be easily removed to prevent further degradation of the target protein after cleavage of fusion protein using amylose affinity chromatography. The fusion protein sequence also contained a human rhinovirus 3C protease cleavage recognition sequence between MBP-tagand hEK L for removal of the fusion handle. The
We report here the construction of a vector derived from pET3-His and pRSET plasmids for the expressionandpurificationofrecombinant proteins in Escherichia coli based on T7 phage RNA polymerase. The resulting pAE plasmid combined the advantages of both vectors: small size (pRSET), expressionof a short 6XHis tag at N-terminus (pET3- His) and a high copy number of plasmid (pRSET). The small size of the vector (2.8 kb) and the high copy number/cell (200-250 copies) facilitate the subcloning and sequencing procedures when compared to the pET system (pET3-His, 4.6 kb and 40-50 copies) and also result in high level expressionofrecombinant proteins (20 mg purified protein/liter of culture). In addition, the vector pAE enables the expressionof a fusion protein with a minimal amino-terminal hexa- histidine affinitytag (a tagof 9 amino acids using XhoI restriction enzyme for the 5’cloning site) as in the case of pET3-His plasmid and in contrast to proteins expressed by pRSET plasmids (a tagof 36 amino acids using BamHI restriction enzyme for the 5’cloning site). Thus, although proteins expressed by pRSET plasmids also have a hexa-histidine tag, the fusion peptide is much longer and may repre- sent a problem for some recombinant proteins.
Full length Ezrin is normally in a dormant confirmation masking the C-terminal actin bind- ing domain with its own N-terminal FERM domain [9, 10] resulting in the absence of interac- tion with Actin . Conformational change in full length Ezrin occurs after addition of Phosphatidylinositol bisphosphate (PIP2) [4, 5] which binds to the N terminal of the full length Ezrin and thus promotes binding to actin. In an alternative approach, we chose to employ the Ezrin C-terminal domain alone in combination with a lipid anchor 10xHis and a fluorescent tag in order to facilitate in vitro studies with Total Internal Reflection Fluorescence (TIRF) mi- croscopy. In particular, we present a design comprising a combination ofaffinity tags that allow high level expressionandaffinitypurificationof the Ezrin actin binding domain (ABD) without losing its functionality. We tested two constructs, viz., 1) 10xHis-Yellow Fluorescent Protein (YFP)-ezrinABD with Glutathione S-transferase (GST) and 2) 10xHis-Lysine-Cystine- Lysine (KCK)-ezrinABD with Maltose Binding Protein (MBP) for expression, solubility, purity and subsequent functionality test by TIRF microscopy (S1 and S2 Figs). N-terminus membrane binding FERM domain was replaced by 10xHis in above mentioned constructs. While con- struct1 was fluorescently labeled with YFP and called bright ezrinABD, construct 2 has a non- fluorescent KCK sequence instead, which we refer to as dark ezrinABD (Fig 1). By demonstrat- ing this successfully, we show how a strategic combination ofaffinity tags can be instrumental
Many diseases, such as coronary artery disease, coronary heart disease and stroke, have proven to be closely related with thrombosis. These diseases cause about 15 million death cases per year in the world and badly hurt the health of human beings  . Up to now, many anti-thrombus drugs have been developed, in which anti-platelet agents show great promise  . Decorsin, a protein isolated from the North American leech Macrobdella decora, acts as an antagonist of platelet glycoprotein IIb-IIIa (GPIIb-IIIa)------the fibrinogen receptorand is a potent inhibitor of platelet aggregation. The primary sequence of decorsin indicates that the protein is 39 amino acids long and contains 6 cysteine and 6 proline residues, as well as the sequence Arg-Gly-Asp, (RGD), a proposed recognition site of many adhesion proteins  . Annexin V, belonging to a family of proteins which was first discovered in 1990, the annexins, with anticoagulant properties has proven to preferentially bind to negatively charged phospholipids like PS (phosphatidylserine), which is greatly exposed on their membranes when platelets are activated, in the presence of Ca 2+[4-6] .
concentrações crescentes de insulina regular humana não marcada para saturação fria. O IVGTT não mostrou dife- renças entre as pacientes em diferentes fases do ciclo es- tral com relação a glicemia basal, ou na resposta glicêmica após infusão de glicose nos tempos estudados. Dois sítios de ligação da insulina, um de alta-afinidade, e outro de bai- xa afinidade, foram detectados pela análise de Scatchard, e diferenças significativas foram detectadas na constan- te de dissociação (Kd1) e capacidade de ligação máxima (Bmax1) dos sítios de ligação de alta-afinidade. O Kd1 para o grupo anestro (6,54±2,77nM/mg de proteína) foi menor (P<0,001) que os Kd1 dos grupos estro (28,54±6,94 nM/ mg de proteína) e diestro (15,56±3,88nM/mg de proteí- na). Os Bmax1 dos grupos estro (0,83±0,42nM/mg de proteína) e diestro (1,24±0,24nM/mg de proteína) tam- bém foram maiores que os valores encontrados no grupo anestro (0,35±0,06nM/mg de proteína). Estes resultados demonstram uma modulação das características de liga- ção da insulina nas diferentes fases do ciclo estral em cães, evidenciando uma menor afinidade de ligação da insulina ao seu receptor no tecido muscular durante o estro e dies- tro. Contudo, esta menor afinidade de ligação hormônio- -receptor é compensada por uma maior capacidade de ligação, o que fica também evidenciado pela ausência de diferenças na resposta glicêmica das pacientes após um desafio com glicose por via endovenosa.
Organisms have evolved proteins capable of reversibly storing iron, and many of these iron-storing proteins form a high order oligomer . Ferritins in mammals and bacteria are all composed of 24 subunits assembled to form a spherical protein. The frataxin is an oligomer of 48 subunits. Of particular note, the E. coli bacterioferritin (EcBFR), which is an iron storage and detoxification protein, forms a 24mer with 12 hemes bound [61,62]. In this study, we found that a supermolecule LipL41 made up of 36 subunits arranged in a Figure 3. Electron micrographs of LipL41. (A) TEM image of LipL41. The LipL41 with uranyl acetate negative staining existed as uniform and homogeneous particles. Class averages of LipL41 supermolecules are shown in top (B) and side views (C) with the scale bar 10 nm. The repeat of eighteen molecules was estimated from the top view of the particle (B), and each particle forms a two-layered structure from the side view (C). (D) Cryo-EM field of ice-embedded particles of LipL41. The shape of the particles is identical to LipL41 with negative staining (A), showing that negative staining did not interfere with particle formation.
Periodontal disease is an inflammatory disease of multifactorial etiology that develops in response to the presence of specific bacteria. It has the potential to compromise protective structures and the retention of teeth, resulting in attachment loss, irreversible bone loss, and eventually, tooth loss. The prevalence of periodontal disease is high; it is considered one of the most common infections observed in all populations and represents a significant cause for concern in public health worldwide. Conventional periodontal treatments typically involve radicular mechanical treatments that aim to remove the causative disease agents through the use of manual and ultrasonic techniques. More recently, photodynamic therapy (PDT) has begun to be incorporated as a periodontal treatment with specific antimicrobial characteristics. However, neither the effectiveness nor the mechanism of action of PDT at human periodontal sites is as yet completely clear, especially with regard to the possible molecular alterations resulting from this treatment. The current study was devised with the objective of using a split-mouth, controlled clinical trial to compare conventional mechanical debridement (scaling and root planing) treatment (T1) with conventional mechanical
Although we did not observe any changes in the glutamate transporters in the ACC, we observed that transcripts of various glutamate receptors andreceptor subunits were elevated in the ACC of mice with PINP. The increased expressionof some of the glutamate receptors andreceptor subunits could have been linked to astrocyte activation since all of the up-regulated receptors are expressed on astrocytes (Geurts et al., 2005; Mart´ınez-Lozada & Ortega, 2015 ). Several receptors have been reported to be differentially expressed in the ACC in rodent models of PINP. We observed an increase in the expressionof various GABA receptors in the ACC during PINP (Masocha, 2015). Ortega-Legaspi et al. (2011) and Ortega-Legaspi et al. (2010) . reported a differential expressionof muscarinic-1 and − 2 receptors and dopamine D1 and D2 receptors in the ACC of rodents with PINP. The increased expression glutamate receptors in the ACC also suggest a role of the glutamatergic system in the pathogenesis of PINP.
The growing interest and applications of biotechnology products have increased the developmentof new processes for recovery andpurificationof proteins. The expanded bed adsorption (EBA) has emerged as a promising technique for this purpose. It combines into one operation the steps of clarification, concentration andpurificationof the target molecule. Hence, the method reduces the time and the cost of operation. In this context, this thesis aim was to evaluate the recovery andpurificationof 503 antigen of Leishmania i. chagasi expressed in E. coli M15 and endotoxin removal by EBA. In the first step of this study, batch experiments were carried out using two experimental designs to define the optimal adsorption and elution conditions of 503 antigen onto Streamline chelating resin. For adsorption assays, using expanded bed, it was used a column of 2.6 cm in diameter by 30.0 cm in height coupled to a peristaltic pump. In the second step of study, the removal of endotoxin during antigen recovery process was evaluated employing the non-ionic surfactant Triton X-114 in the washing step ALE. In the third step, we sought developing a mathematical model able to predict the 503 antigen breakthrough curves in expanded mode. The experimental design results to adsorption showed the pH 8.0 and the NaCl concentration of 2.4 M as the optimum adsorption condition. In the second design, the only significant factor for elution was the concentration of imidazole, which was taken at 600 mM. The adsorption isotherm of the 503 antigen showed a good fit to the Langmuir model (R = 0.98) and values for q max (maximum
ability to detect subtle differences. We did not enroll infants younger than 12 months of age, and we were unable to determine if viral infection relates to induction of FceRI. Finally, defining sensitization status by selected ImmunoCAP may not be ideal— especially from a clinical perspective. However, given that our results were mostly independent of sensitization status, we believe this is not a major defect. Indeed, most of the limitations of our study would only lessen our ability to define statistically significant differences. Since we were able to document clear relationships between IgE (serum and cell bound) and cDC and pDC expressionof FceRI despite these limitations, their presence only strengthen the importance of our findings.
The most common type of complaint from students was un- questionably related to excessive lecturing on the part of the native TA (about 70% of negative comments related to excessive lecturing, not enough time to talk.) Students often felt shorthanded when not given opportunities to discuss topics in class. (Although this contra- dicts the statements above related to difficult discussion topics, we should assume that students who requested more discussion in class are expecting discussion at an appropriate language level.) One stu- dent said of a native male TA, “I think if there were more opportuni- ties to speak instead of hearing him speak for most of the class, it would have been more beneficial.” Another complained about a na- tive female TA, “She was very enthusiastic about teaching, but activi- ties that actually involved speaking were sparse.” More than anything else, students mentioned discussion in the classroom, and the majori- ty of these complaints and requests were aimed towards native female TAs: “I did not like how little we got to speak as a class. My under- standing increased but I feel my speaking skills went down”; “More class participation would have been good”; “I wish we would have had more opportunities to have class debates and class discussions”; “mostly a lecture [with] little interaction”; “I think the only thing that could be improved is if she would have us speak more in class.” Fi- nally, one student wrote an extensive comment for a female TA that depicted her as an outstanding TA, but then ended with the sugges- tion that “more emphasis on free class discussion might help.”
themselves must presumably undergo slow changes via additional layers of extrinsic regulation , perhaps involving random low frequency modulation of the underlying burst size or frequency. As others have suggested, chromatin is one logical candidate to mediate such regulation [30,32,38]. In fact, random fluctuations in chromatin folding have previously been linked to fluctuations in expression , and chromatin inheritance timescales are consistent with those prevailing in our system . It is also possible, though we believe less likely, that gains and losses of transgene copies or other factors such as genomic instability may be involved in the dynamic and reversible shifts in expression level we have observed. Moreover, although we have focused here on graded fluctuations, binary switching between expressing and non- expressing states are also possible [37,84]. The putative connection with chromatin raises the fascinating prospect of a functional link between expression noise and epigenetic gene silencing.
DNA replication occurs together with the expressionof viral components necessary for the assembly of new nucleocapsids. The newly assembled nucleocapsids are transported from the nucleus to the plasma membrane for budding through GP64- enriched areas, originating the budded viruses (BdVs; Passarelli, 2012). BdVs are non- occluded virions surrounded by a plasma membrane-derived envelope containing GP64 as a major structural protein (Washburn et al., 2003). BdVs are required for secondary infection: once released, they are transported through the hemolymph to infect new cells, a process which, in contrast to what is described for ODVs, is undertaken by GP64 via clathrin-mediated endocytosis (Blissard and Wenz, 1992; Long et al., 2006). In this regard, BdVs are the virus form responsible for viral dissemination through the host, culminating in a systemic infection. A secondary infection cycle begins with the entrance of BdVs into another cell of the insect. After entering the cell, the infection process is similar to what happens in a primary infection, with the nucleocapsids traveling to the nucleus for DNA replication and subsequent viral proteinexpression. The very late phase of infection (18 hpi) initiates with the expressionof proteins that constitute the crystalline matrix of the ODVs, namely polyhedrin. After nucleocapsid assembly, they are enveloped by the polyhedrin matrix to constitute the ODVs. The secondary cycle ends with the extensive infection of larvae tissues and cell lysis, culminating in insect larvae death, and dissemination of ODVs to the environment, where they can remain viable for several years until being ingested by other larvae (Volkman, 1997). Summarizing, BdVs and ODVs are genetically identical but differ in their envelope compositions and tissue tropisms, and are produced at different times during infection.
The polh-6xhis gene was removed from the cloning vec- tor by BglII and NotI restriction digestion following the manufacturer’s instructions (Promega) and analyzed by electrophoresis in a 0.8% agarose gel . The DNA fragment was then purified using the GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare) and cloned into the commercial donor vector pFastBac1® (pFB1 – Invitrogen) previously digested with BamHI and NotI (Promega) restriction enzymes in order to gen- erate the recombinant plasmid pFB1-polh-6xhis. The NcoI-flanked GarMbFV-cp gene, was removed from the pGem-T easy vector by NcoI restriction digestion, the fragment was analyzed by electrophoresis and purified from the gel as described above. The purified fragment was then introduced into the unique NcoI site present in the pFB1-polh-6xhis plasmid in order to construct the pFB1-polh-GarMbFV-cp-6xhis plasmid. Both vectors (pFB1-polh-6xhis and pFB1-polh-GarMbFV-cp-6xhis) were transformed into DH10-Bac cells (Invitrogen) by electroporation  andrecombinant bacmids were se- lected following the manufacturer’s instructions (Bac-to- Bac®, Baculovirus expression systems, Invitrogen). DNA from the bacmids were purified and the presence of the recombinant gene was checked by PCR using specific oligonucleotides as described by the manufacturer’s protocol (Invitrogen). One microgram of each recombin- ant bacmid was transfected into Tn5B cells (10 6 ) using liposomes (Cellfectin®) according to manufacturer’s in- structions (Invitrogen). Since naked baculovirus DNA is infectious, the supernatant of 7 days post-transfection Tn5B cells containing the recombinant viruses were col- lected, tittered, and the virus amplified by infection of 1,5 × 10 7 cells with a MOI of 1 in 75 cm 2 flasks (TPP) as described in O’Reilly et al. .
Agri-environment topics cover land uses, green house gases emissions, fertilizers and pesticides uses, etc. Expansion of agricultural land expansion is one of the most significant human alterations to the global environment (Zhang, 2016). In the past several hundred years, agricultural land area worldwide increased at the annual rate of 1.66% (Grigg, 1993; Matson et al., 1997). Excessive land use and agricultural intensification has been deteriorating global climate and environmental quality (Matson et al., 1997; Tilman et al., 2001; Tahir et al., 2013).
The biochemical pathways involved in nicotinamide adenine dinucleotide (NAD) biosynthesis converge at the enzymatic step catalysed by nicotinamide mononucleotide adenylyltransferase (NMNAT, EC: 220.127.116.11). The majority of NMNATs are assembled into homo-oligomeric states that comprise 2-6 subunits. Recently, the NMNAT of Plas- modium falciparum (Pf NMNAT) has been identified as a pharmacological target. The enzymatic characterisation, cellular location, and tertiary structure of the Pf NMNAT protein have been reported. Nonetheless, its quaternary structure remains to be explored. The present study describes the oligomeric assembly of the 6 x His-Pf NMNAT recombinantprotein using immobilised metal affinity chromatography coupled with size exclusion chromatography (SEC) and native protein electrophoresis combined with Ferguson plot graphing. These chromatographic approaches resulted in the elution ofan active monomer from the SEC column, whereas the Ferguson plot indicated a dimeric assembly of the 6 x His-Pf NMNAT protein.