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Proteins Used (protein + used)
Selected AbstractsInorganic Materials and Ionic Liquids: Large-scale Nanopatterning of Single Proteins used as Carriers of Magnetic Nanoparticles (Adv. Mater.ADVANCED MATERIALS, Issue 5 20105/2010) Ricardo Garcia, Eugenio Coronado, and co-workers demonstrate on p. 588 large-scale patterning of single ferritin molecules by sequential (atomic force microscopy local oxidation) and parallel approaches (lithographically controlled wetting). The nanopattern size matches the size of the protein (,10 nm). Electrostatic interactions, capillary forces, surface functionalization, and nanolithography are used to achieve the desired protein organization. [source] Large-scale Nanopatterning of Single Proteins used as Carriers of Magnetic NanoparticlesADVANCED MATERIALS, Issue 5 2010Ramsés V. Martínez Patterning of single ferritin molecules by sequential (atomic force microscopy local oxidation) and parallel approaches (lithographically controlled wetting). The nanopattern size matches the size of the protein (,10 nm). Electrostatic interactions, capillary forces, surface functionalization, and nanolithography are used to achieve the desired protein organization. [source] Assembly of Gold Nanoparticles in a Rod-Like Fashion Using Proteins as Templates,ADVANCED FUNCTIONAL MATERIALS, Issue 3 2006R. Bhattacharya Abstract An area of considerable current interest is the development of a practical approach for assembling inorganic nanoparticles into well-defined arrays because such a technique would offer immense opportunities leading to applications in microimaging, optoelectronics, therapeutics, etc. This paper illustrates a new, simple one-step process in which proteins act as templates to assemble gold nanoparticles in a shape-selective fashion. We show, for the first time, that antibodies to vascular endothelial growth factor 165 isoform, 2C3, and epidermal growth factor receptor can act as templates when present in solution during the synthesis of gold nanoparticles. These proteins direct the assembly of the gold nanoparticles into rod-like shapes when cooled to ,20,°C followed by thawing at room temperature. Immunoglobulin,G and bovine serum albumin can also direct the assembly process in a similar fashion; however, small molecules, such as poly(L -lysine) and lysine, cannot. The formation of a self-assembled structure in the form of a continuous rod, or the assembly of discrete nanoparticles in a rod-like fashion, can be tailored by controlling the ratio of the precursor gold salt, HAuCl4, to the antibody/protein used as the template. The nanoconjugates are characterized using UV-vis spectroscopy, transmission electron microscopy, and infrared spectroscopy. The nano-bioconjugates obtained via this process may find wide application in areas ranging from optoelectronics and biosensors to therapeutics in neoplastic disorders. [source] Aggregation kinetics of recombinant human FVIII (rFVIII)JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 9 2005Karthik Ramani Abstract The physical phenomenon of aggregation can have profound impact on the stability of therapeutic proteins. This study focuses on the aggregation behavior of recombinant human FVIII (rFVIII), a multi-domain protein used as the first line of therapy for hemophilia A, a bleeding disorder caused by the deficiency or dysfunction of factor VIII (FVIII). Thermal denaturation of rFVIII was investigated using circular dichroism (CD) spectroscopy and size exclusion chromatography (SEC). The dependence of unfolding on heating rate indicated that the thermal denaturation of the protein was at least partly under kinetic control. The data was interpreted in terms of a simple two-state kinetic model, , where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation. Analysis of the data in terms of the above scheme suggested that under the experimental conditions used in this study, the rate-controlling step in the aggregation of rFVIII may be a unimolecular reaction involving conformational changes. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:2023,2029, 2005 [source] Separation with zwitterionic hydrophilic interaction liquid chromatography improves protein identification by matrix-assisted laser desorption/ionization-based proteomic analysisBIOMEDICAL CHROMATOGRAPHY, Issue 6 2009Atsushi Intoh Abstract Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC-HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC-HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC-HILIC has better peptide fractionation ability. We further demonstrated that ZIC-HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC-HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses. Copyright © 2009 John Wiley & Sons, Ltd. [source] Familial dysautonomia: A diagnostic dilemma.PEDIATRIC PULMONOLOGY, Issue 6 2001Chronic lung disease with signs of an autoimmune disease Abstract We present an 11-year-old girl with sensory and autonomic neurological dysfunction, and respiratory insufficiency caused by recurrent aspiration. The diagnosis of familial dysautonomia (FD) was confirmed by a missing axonal flare to histamine, miosis in response to conjunctival methacholine and homozygous polymorphic linked markers DS58(18) and DS159(7) on chromosome 9. Ashkenazi Jewish descent could not be ascertained by history. A variety of positive tests for autoantibodies were initially interpreted as evidence for systemic lupus erythematosus vs. overlap syndrome with pulmonary, cerebral, skin, and ocular involvement. The diagnosis of FD was delayed because of the rarity of this disorder in Germany (second case reported). We discuss possible explanations for the misleading immunological findings, including interference by antibodies binding to milk proteins used as blocking reagents in enzyme-linked immunoassays and circulating immune-complexes due to chronic aspiration pneumonitis. Pediatr Pulmonol. 2001; 31:478,481. © 2001 Wiley-Liss, Inc. [source] Identification of neutrophil gelatinase-associated lipocalin (NGAL) as a discriminatory marker of the hepatocyte-secreted protein response to IL-1,: a proteomic analysisBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2005Arul Jayaraman Abstract The liver is the major source of proteins used throughout the body for various functions. Upon injury or infection, an acute phase response (APR) is initiated in the liver that is primarily mediated by inflammatory cytokines such as interleukin-1, (IL-1,) and interleukin-6. Among others, the APR is characterized by an altered protein synthetic profile. We used two-dimensional gel electrophoresis to study the dynamics of changes in protein synthesis in hepatocytes exposed to these inflammatory cytokines. Protein profiles were quantified using image analysis and further analyzed using multivariate statistical methods. Our results indicate that IL-1, and IL-6 each induces secreted protein responses with distinct dynamics and dose-dependence. Parallel stimulation by IL-1, and IL-6 results in a protein pattern indistinguishable from the IL-1, pattern, indicating a dominant effect of IL-1, over IL-6 at the doses tested. Multidimensional scaling (MDS) of correlation distances between protein secretion levels revealed two protein pairs that are robustly co-secreted across the various cytokine stimulation conditions, suggesting shared regulatory pathways. Finally, we also used multivariate alternating conditional expectation (MACE) to identify transformation functions that discriminated the cytokine-stimulated and untreated hepatocyte-secreted protein profiles. Our analysis indicates that the expression of neutrophil gelatinase-associated lipocalin (NGAL) was sufficient to discriminate between IL-1, and IL-6 stimulation. The combination of proteomics and multivariate analysis is expected to provide new information on the cellular regulatory networks involved in generating specific cellular responses. © 2005 Wiley Periodicals, Inc. [source] Rapid and selective isolation of ,-xylosidase through an activity-based chemical approachBIOTECHNOLOGY JOURNAL, Issue 2 2006Lee-Chiang Lo Dr. Abstract ,-Xylosidase is a key enzyme in the xylanolytic system with a great potential in many biotechnological applications, especially in the food as well as the pulp and paper industries. We have developed a chemical approach for the rapid screening and isolation of ,-xylosidase. Activity probe LCL-6X targeting ,-xylosidase was utilized in this study. It carries a ,-xylopyranosyl recognition head, a latent trapping device consisting of a 2-fluoromethylphenoxyl group, and a biotin reporter group. The biotin reporter group serves both as a readout device and as a tool for enriching the labeled proteins. LCL-6X could selectively label a model ,-xylosidase from Trichoderma koningii. All other bystander proteins used in this study, including phosphorylase b, BSA, ovalbumin, carbonic anhydrase, and trypsin inhibitor, gave negligible cross-labeling effect. With the assistance of streptavidin agarose beads and mass spectrophotometry for the recovery and identification of the biotinylated proteins, we demonstrated that LCL-6X could be successfully applied to identify a bi-functional enzyme with ,- L -arabinofuranosidase/,-xylosidase activity from the total protein extract of a Pichia expressing system and a prospective ,-xylosidase in the culture medium of Aspergillus fumigatus. The ,-xylosidase activities from numerous microbes were also screened using the LCL-6X probe. Preliminary results showed significant differences among these microbial sources and some distinct protein bands were observed. Thus, we have successfully developed a novel chemical probe that has potential applications in xylan-related research. [source] pH-insensitive glucose indicatorsBIOTECHNOLOGY PROGRESS, Issue 5 2008Jared R. Garrett Abstract There is an urgent need for developing a biosensor that can real-time and noninvasively determine glucose concentration within living cells. In our previous study, we have engineered a glucose indicator protein (GIP) that can provide continuous glucose monitoring through a conformation change-induced Förster resonance-energy transfer measurement. Because of the pH-sensitivity of the fluorescent proteins used in the GIP construction, the GIP made from these fluorescent proteins is less tolerant to a pH change, especially to the acidic environment. It has been well documented that intracellular pH does not always remain the same, and it fluctuates in metabolism and other cellular activities and also differs between cellular compartments. To address these issues, we developed a GIP that can tolerate to pH change. This GIP was constructed by flanking a glucose binding protein with a cyan fluorescent protein and a pH-insensitive yellow fluorescent protein. Our experimental results indicated that the new GIP is more tolerant to pH change. The glucose response of this new GIP kept almost unchanged from pH 7.3 to 5.3, suggesting its capability of tolerating to acidic environment. This capability is desirable for intracellular glucose measurement. [source] Disulfide Bond Substitution by Directed Evolution in an Engineered Binding ProteinCHEMBIOCHEM, Issue 8 2009Antoine Drevelle Dr. Abstract Breaking ties: The antitumour protein, neocarzinostatin (NCS), is one of the few drug-carrying proteins used in human therapeutics. However, the presence of disulfide bonds limits this protein's potential development for many applications. This study describes a generic directed-evolution approach starting from NCS-3.24 (shown in the figure complexed with two testosterone molecules) to engineer stable disulfide-free NCS variants suitable for a variety of purposes, including intracellular applications. The chromoprotein neocarzinostatin (NCS) has been intensively studied for its antitumour properties. It has recently been redesigned as a potential drug-carrying scaffold. A potential limit of this protein scaffold, especially for intracellular applications, is the presence of disulfide bonds. The objective of this work was to create a disulfide-free NCS-derived scaffold. A generic targeted approach was developed by using directed evolution methods. As a starting point we used a previously engineered NCS variant in which a hapten binding site had been created. A library was then generated in which cysteine Cys88 and Cys93 and neighbouring residues were randomly substituted. Variants that preserved the hapten binding function were selected by phage display and further screened by colony filtration methods. Several sequences with common features emerged from this process. The corresponding proteins were expressed, purified and their biophysical properties characterised. How these selected sequences rescued folding ability and stability of the disulfide-free protein was carefully examined by using calorimetry and the results were interpreted with molecular simulation techniques. [source] | ||||||||||||||||