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Insoluble Proteins (insoluble + protein)
Selected AbstractsAmino Acid, Fatty Acid, and Mineral Profiles of Materials Recovered from Rainbow Trout (Oncorhynchus mykiss) Processing By-Products Using Isoelectric Solubilization/PrecipitationJOURNAL OF FOOD SCIENCE, Issue 9 2007Y.-C. Chen ABSTRACT:, Protein, lipid, and insolubles (bones, skin, scales, fins, insoluble protein, and more) were recovered from rainbow trout processing by-products by means of isoelectric solubilization/precipitation at basic pH and acidic pH. Isoelectric solubilization/precipitation of the trout processing by-products resulted in the recovery of protein that was higher (P < 0.05) in essential amino acids (EAAs), non-EAAs, and total EAA/total AA ratio when compared to the processing by-products. Basic pH treatments yielded a higher (P < 0.05) content of EAAs than the acidic pH treatments. Nutritional quality of the recovered protein was high based on EAAs meeting the FAO/WHO/UNU recommendations for adults. The presence of omega-3 and omega-6 fatty acids (,-3, ,-6 FAs) and the ,-3/,-6 ratio in the recovered lipids were similar to the trout processing by-products, indicating that the pH treatments had no effect on these FAs. Ca and P contents of the processing by-products exceeded the recommended dietary allowances (RDA), but Fe and Mg did not. Basic pH treatments yielded protein with the lowest (P < 0.05) amount of minerals and the highest (P < 0.05) amount of Ca, P, and Mg in the insolubles when compared to acidic pH. The isoelectric solubilization/precipitation of the processing by-products effectively removed minerals from the recovered protein without removal of the bones, skin, scales, fins, and so on, prior to processing. The results indicated that isoelectric solubilization/precipitation, particularly at basic pH, permitted recovery of high-quality protein and lipids from fish processing by-products for human food uses; also, the recovered insolubles may be used in animal feeds as a source of minerals. [source] NepA is a structural cell wall protein involved in maintenance of spore dormancy in Streptomyces coelicolorMOLECULAR MICROBIOLOGY, Issue 6 2009Wouter De Jong Summary Streptomycetes have a complex morphogenetic programme culminating in the formation of aerial hyphae that develop into chains of spores. After spore dispersal, environmental signals trigger dormant spores to germinate to establish a new colony. We here compared whole genome expression of a wild-type colony of Streptomyces coelicolor forming aerial hyphae and spores with that of the chp null mutant that forms few aerial structures. This revealed that expression of 244 genes was significantly altered, among which genes known to be involved in development. One of the genes that was no longer expressed in the ,chpABCDEFGH mutant was nepA, which was previously shown to be expressed in a compartment connecting the substrate mycelium with the sporulating parts of the aerial mycelium. We here show that expression is also detected in developing spore chains, where NepA is secreted to end up as a highly insoluble protein in the cell wall. Germination of spores of a nepA deletion mutant was faster and more synchronous, resulting in colonies with an accelerated morphogenetic programme. Crucially, spores of the ,nepA mutant also germinated in water, unlike those of the wild-type strain. Taken together, NepA is the first bacterial structural cell wall protein that is important for maintenance of spore dormancy under unfavourable environmental conditions. [source] Rapid Matrix-Assisted Refolding of Histidine-Tagged ProteinsCHEMBIOCHEM, Issue 5 2009Tetyana Dashivets Abstract Matrix refolded: The formation of inclusion bodies, which are amorphous aggregates of misfolded insoluble protein, during recombinant protein expression, is one of the biggest bottlenecks in protein science. We report a stepwise, rational optimization procedure for refolding of insoluble proteins (see scheme). In comparison to refolding in-solution, this parallelized, matrix-assisted approach allows the refolding of various proteins in a fast and efficient manner. The formation of inclusion bodies (IBs),amorphous aggregates of misfolded insoluble protein,during recombinant protein expression, is still one of the biggest bottlenecks in protein science. We have developed and analyzed a rapid parallel approach for matrix-assisted refolding of recombinant His6 -tagged proteins. Efficiencies of matrix-assisted refolding were screened in a 96-well format. The developed methodology allowed the efficient refolding of five different test proteins, including monomeric and oligomeric proteins. Compared to refolding in-solution, the matrix-assisted refolding strategy proved equal or better for all five proteins tested. Interestingly, specifically oligomeric proteins displayed significantly higher levels of refolding compared to refolding in-solution. Mechanistically, matrix-assisted folding seems to differ from folding in-solution, as the reaction proceeds more rapidly and shows a remarkably different concentration dependence,it allows refolding at up to 1000-fold higher protein concentration than folding in-solution. [source] Insoluble eggshell matrix proteins , their peptide mapping and partial characterization by capillary electrophoresis and high-performance liquid chromatographyELECTROPHORESIS, Issue 5 2003Ivan Mik Abstract Avian eggshell matrix proteins were studied by two analytical approaches. Peptide mapping was done by trypsin and pepsin followed by collagenase cleavage; analyses were carried out by capillary electrophoresis and reversed-phase high-performance liquid chromatography (HPLC). Comparison of peptide maps obtained by both methods revealed a complex mixture of peptides in the insoluble layers of the eggshell; it was concluded that there are at least three different insoluble protein/peptide layers in the avian eggshell (cuticle, palisade, and mammillary layer). Partial characterization of peptides in each layer was made by HPLC-mass spectrometry analysis. There is an evidence that the eggshell insoluble proteins contain species susceptible to collagenase cleavage, however, the sequences split by this enzyme probably are not those typical for the main triple-helical core of collagenous proteins. It is proposed that the action of collagenase upon eggshell proteins is caused by the side effect of collagenase described previously with synthetic peptides. Some of the proteins present are probably glycosylated. Fatty acid content in the insoluble eggshell layers (after decalcification) was in the range of 2,4% (which reflected both lipid and lipoproteins bound fatty acids). Porphyrin pigments are dominant in the cuticle layer. [source] Proteome analysis of human nuclear insoluble fractionsGENES TO CELLS, Issue 8 2009Hideaki Takata The interphase nucleus is a highly ordered and compartmentalized organelle. Little is known regarding what elaborate mechanisms might exist to explain these properties of the nucleus. Also unresolved is whether some architectural components might facilitate the formation of functional intranuclear compartments or higher order chromatin structure. As the first step to address these questions, we performed an in-depth proteome analysis of nuclear insoluble fractions of human HeLa-S3 cells prepared by two different approaches: a high-salt/detergent/nuclease-resistant fraction and a lithium 3,5-diiodosalicylate/nuclease-resistant fraction. Proteins of the fractions were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry, identifying 333 and 330 proteins from each fraction respectively. Among the insoluble nuclear proteins, we identified 37 hitherto unknown or functionally uncharacterized proteins. The RNA recognition motif, WD40 repeats, HEAT repeats and the SAP domain were often found in these identified proteins. The subcellular distribution of selected proteins, including DEK protein and SON protein, demonstrated their novel associations with nuclear insoluble materials, corroborating our MS-based analysis. This study establishes a comprehensive catalog of the nuclear insoluble proteins in human cells. Further functional analysis of the proteins identified in our study will significantly improve our understanding of the dynamic organization of the interphase nucleus. [source] Rheological and Functional Properties of Catfish Skin Protein HydrolysatesJOURNAL OF FOOD SCIENCE, Issue 1 2010Huaixia Yin ABSTRACT:, Catfish skin is an abundant and underutilized resource that can be used as a unique protein source to make fish skin protein hydrolysates. The objectives of this study were to isolate soluble and insoluble proteins from hydrolyzed catfish skin, study the rheological and functional properties of the protein hydrolysates, and evaluate the properties of emulsions made from the protein powders. Freeze-dried catfish skin soluble (CSSH) and insoluble hydrolysate (CSISH) powders were analyzed for proximate analysis, emulsion stability, fat absorption, amino acids, color, and rheological properties. CSSH had significantly (P,< 0.05) higher protein, ash, and moisture content but lower fat content than that of CSISH. The yield of CSSH (21.5%± 2.2%) was higher than that of CSISH (3%± 0.3%). CSISH had higher emulsion stability than CSSH. CSSH was light yellow in color and CSISH was darker. The mean flow index values for emulsion containing CSSH (ECSSH) and CSISH (ECSISH) were both less than 1, indicating that they were both pseudoplastic fluid. The G, and G, values for the ECSISH were higher than that of ECSSH, indicating that the viscoelastic characteristic of the emulsion containing CSISH was greater than that of the emulsion containing CSSH. The study demonstrated the CSSH and CSISH had good functional and rheological properties. They have potential uses as functional food ingredients. [source] Deamidation destabilizes and triggers aggregation of a lens protein, ,A3-crystallinPROTEIN SCIENCE, Issue 9 2008Takumi Takata Abstract Protein aggregation is a hallmark of several neurodegenerative diseases and also of cataracts. The major proteins in the lens of the eye are crystallins, which accumulate throughout life and are extensively modified. Deamidation is the major modification in the lens during aging and cataracts. Among the crystallins, the ,A3-subunit has been found to have multiple sites of deamidation associated with the insoluble proteins in vivo. Several sites were predicted to be exposed on the surface of ,A3 and were investigated in this study. Deamidation was mimicked by site-directed mutagenesis at Q42 and N54 on the N-terminal domain, N133 and N155 on the C-terminal domain, and N120 in the peptide connecting the domains. Deamidation altered the tertiary structure without disrupting the secondary structure or the dimer formation of ,A3. Deamidations in the C-terminal domain and in the connecting peptide decreased stability to a greater extent than deamidations in the N-terminal domain. Deamidation at N54 and N155 also disrupted the association with the ,B1-subunit. Sedimentation velocity experiments integrated with high-resolution analysis detected soluble aggregates at 15%,20% in all deamidated proteins, but not in wild-type ,A3. These aggregates had elevated frictional ratios, suggesting that they were elongated. The detection of aggregates in vitro strongly suggests that deamidation may contribute to protein aggregation in the lens. A potential mechanism may include decreased stability and/or altered interactions with other ,-subunits. Understanding the role of deamidation in the long-lived crystallins has important implications in other aggregation diseases. [source] An automatable screen for the rapid identification of proteins amenable to refoldingPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2006Nathan P. Cowieson Dr. Abstract Insoluble expression of heterologous proteins in Escherichia coli is a major bottleneck of many structural genomics and high-throughput protein biochemistry projects. Many of these proteins may be amenable to refolding, but their identification is hampered by a lack of high-throughput methods. We have developed a matrix-assisted refolding approach in which correctly folded proteins are distinguished from misfolded proteins by their elution from affinity resin under non-denaturing conditions. Misfolded proteins remain adhered to the resin, presumably via hydrophobic interactions. The assay can be applied to insoluble proteins on an individual basis but is particularly well suited for high-throughput applications because it is rapid, automatable and has no rigorous sample preparation requirements. The efficacy of the screen is demonstrated on small-scale expression samples for 15,proteins. Refolding is then validated by large-scale expressions using SEC and circular dichroism. [source] The effect of sodium dodecyl sulfate and anion-exchange silica gel on matrix-assisted laser desorption/ionization mass spectrometric analysis of proteinsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 11 2009Miwako Asanuma Sodium dodecyl sulfate (SDS), an anionic surfactant, is widely used in peptide and protein sample preparation. When the sample is analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), this surfactant can often cause signal suppression. We have previously reported an on-probe sample preparation method using a suspension of anion-exchange silica gel and sinapinic acid (i.e., gel-SA suspension) as a matrix, thereby greatly improving the MALDI signal detection of the protein solutions containing SDS. In this study, we found that a certain amount of SDS enhanced the MALDI signal intensity for protein samples. This effect was also observed when using sodium decyl sulfate and sodium tetradecyl sulfate instead of SDS. Furthermore, this on-probe sample preparation method using both SDS and the gel-SA suspension improved the detection limit of protein samples in the MALDI-MS analysis by about ten-fold as compared to that of protein samples without SDS and the gel-SA suspension. This method can be applied not only to the MALDI-MS analysis of samples containing SDS, but also to the examination of proteins at femtomole levels or insoluble proteins such as membrane proteins. Copyright © 2009 John Wiley & Sons, Ltd. [source] The effect of plant cytokinin hormones on the production of ethylene, nitric oxide, and protein nitrotyrosine in ageing tobacco leavesBIOFACTORS, Issue 1-4 2006N. Wilhelmová Abstract Transgenic plants with genetically increased or decreased levels of cytokinins were used to investigate the effect of cytokinin level on the production of ethylene, a plant hormone with suggested role in senescence, and the production of nitric oxide, potentially important signalling and regulatory molecule. The production of these gases was followed during the course of leaf development and senescence. The production of ethylene and nitric oxide is under genetic control of genes other than those involved in regulation of senescence. The difference in basic ethylene and NO levels in different tobacco cultivars was higher than their changes in senescence. The results of this study did not indicate a direct link between ethylene production and cytokinin levels. However, there was a decreased production of NO in senescent leaves. Low cytokinins level was associated with increased NO production during leaf development. Protein nitrotyrosine proved to be a better indicator of the reactive nitrogen species than measuring of the NO production. Higher nitrotyrosine concentrations were found in insoluble proteins than in the soluble ones, pointing to membrane proteins as the primary targets of the reactive nitrogen species. In plants with elevated cytokinin levels the content of nitrated proteins decreased both in soluble and insoluble fractions. This finding indicates an antioxidative function of cytokinins against reactive nitrogen species. [source] The influence of N-glycosylation and C-terminal sequence on secretion of HBV large surface antigen from S. cerevisiaeBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2005Jin-Seung Park Abstract In Saccharomyces cerevisiae, we synthesized and secreted L-HBVsAg (named as pre-S(Met1 to Asn174)::S(Met175 to Ile400)) and three mutants, i.e., pre-S°°::S (Asn15Gln and Asn123Gln), pre-S°°::S° (Asn15Gln, Asn123Gln, and Asn320Gln), and pre-S°°::S°° (Asn15Gln, Asn123Gln, Asn233Gln, and Asn320Gln). All of the secreted pre-S::S was N-glycosylated, i.e., hyper-mannosylated. In the secretion of pre-S°°::S and pre-S°°::S°, besides the hyper-mannosylated form, another immunoreactive protein with much lower molecular mass was observed, which seems to be unglycosylated form of pre-S°°::S and pre-S°°::S°. Only a part of the secreted pre-S°°::S or pre-S°°::S° molecules was N-glycosylated, and the site for the partial N-glycosylation seems to be Asn233 in S-antigen region. Compared to the N-glycosylated pre-S°°::S and pre-S°°::S°, pre-S°°::S°° (non-N-glycosylated mutant) was secreted with lower secretion efficiency but showed apparent immunoreactivity to anti-S antigen monoclonal Ab. Interestingly, unlike pre-S°°::S°° with authentic C-terminus, the recombinant pre-S°°::S°° with C-terminal myc or poly-histidine tag (pre-S°°::S°°::tag) was almost all aggregated into insoluble proteins in the intracellular region. Conclusively, the C-terminal sequence and glycosylation in S-antigen region seem to be of crucial importance in determining the secretion efficiency of L-HBVsAg in S. cerevisiae. © 2005 Wiley Periodicals, Inc. [source] Rapid Matrix-Assisted Refolding of Histidine-Tagged ProteinsCHEMBIOCHEM, Issue 5 2009Tetyana Dashivets Abstract Matrix refolded: The formation of inclusion bodies, which are amorphous aggregates of misfolded insoluble protein, during recombinant protein expression, is one of the biggest bottlenecks in protein science. We report a stepwise, rational optimization procedure for refolding of insoluble proteins (see scheme). In comparison to refolding in-solution, this parallelized, matrix-assisted approach allows the refolding of various proteins in a fast and efficient manner. The formation of inclusion bodies (IBs),amorphous aggregates of misfolded insoluble protein,during recombinant protein expression, is still one of the biggest bottlenecks in protein science. We have developed and analyzed a rapid parallel approach for matrix-assisted refolding of recombinant His6 -tagged proteins. Efficiencies of matrix-assisted refolding were screened in a 96-well format. The developed methodology allowed the efficient refolding of five different test proteins, including monomeric and oligomeric proteins. Compared to refolding in-solution, the matrix-assisted refolding strategy proved equal or better for all five proteins tested. Interestingly, specifically oligomeric proteins displayed significantly higher levels of refolding compared to refolding in-solution. Mechanistically, matrix-assisted folding seems to differ from folding in-solution, as the reaction proceeds more rapidly and shows a remarkably different concentration dependence,it allows refolding at up to 1000-fold higher protein concentration than folding in-solution. [source] Dynamics, stability and iron-binding activity of frataxin clinical mutantsFEBS JOURNAL, Issue 14 2008Ana R. Correia Friedreich's ataxia results from a deficiency in the mitochondrial protein frataxin, which carries single point mutations in some patients. In the present study, we analysed the consequences of different disease-related mutations in vitro on the stability and dynamics of human frataxin. Two of the mutations, G130V and D122Y, were investigated for the first time. Analysis by CD spectroscopy demonstrated a substantial decrease in the thermodynamic stability of the variants during chemical and thermal unfolding (wild-type > W155R > I154F > D122Y > G130V), which was reversible in all cases. Protein dynamics was studied in detail and revealed that the mutants have distinct propensities towards aggregation. It was observed that the mutants have increased correlation times and different relative ratios between soluble and insoluble/aggregated protein. NMR showed that the clinical mutants retained a compact and relatively rigid globular core despite their decreased stabilities. Limited proteolysis assays coupled with LC-MS allowed the identification of particularly flexible regions in the mutants; interestingly, these regions included those involved in iron-binding. In agreement, the iron metallochaperone activity of the Friedreich's ataxia mutants was affected: some mutants precipitate upon iron binding (I154F and W155R) and others have a lower binding stoichiometry (G130V and D122Y). Our results suggest that, in heterozygous patients, the development of Friedreich's ataxia may result from a combination of reduced efficiency of protein folding and accelerated degradation in vivo, leading to lower than normal concentrations of frataxin. This hypothesis also suggests that, although quite different from other neurodegenerative diseases involving toxic aggregation, Friedreich's ataxia could also be linked to a process of protein misfolding due to specific destabilization of frataxin. [source] | |||||||||||||