Home About us Contact
|
Home About us Contact | ||
|
|
||
Resulting Peptides (resulting + peptide)
Selected AbstractsThe chromatography-free release, isolation and purification of recombinant peptide for fibril self-assemblyBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2009B.M. Hartmann Abstract One of the major expenses associated with recombinant peptide production is the use of chromatography in the isolation and purification stages of a bioprocess. Here we report a chromatography-free isolation and purification process for recombinant peptide expressed in Escherichia coli (E. coli). Initial peptide release is by homogenization and then by enzymatic cleavage of the peptide-containing fusion protein, directly in the E. coli homogenate. Release is followed by selective solvent precipitation (SSP) to isolate and purify the peptide away from larger cell contaminants. Specifically, we expressed in E. coli the self-assembling ,-sheet forming peptide P11 -2 in fusion to thioredoxin. Homogenate was heat treated (55°C, 15,min) and then incubated with tobacco etch virus protease (TEVp) to release P11 -2 having a native N-terminus. SSP with ethanol at room temperature then removed contaminating proteins in an integrated isolation-purification step; it proved necessary to add 250,mM NaCl to homogenate to prevent P11 -2 from partitioning to the precipitate. This process structure gave recombinant P11 -2 peptide at 97% polypeptide purity and 40% overall yield, without a single chromatography step. Following buffer-exchange of the 97% pure product by bind-elute chromatography into defined chemical conditions, the resulting peptide was shown to be functionally active and able to form self-assembled fibrils. To the best of our knowledge, this manuscript reports the first published process for chromatography-free recombinant peptide release, isolation and purification. The process proved able to deliver functional recombinant peptide at high purity and potentially low cost, opening cost-sensitive materials applications for peptide-based materials. Biotechnol. Bioeng. 2009; 104: 973,985. © 2009 Wiley Periodicals, Inc. [source] Novel N -(2,2-Dimethyl-2H -azirin-3-yl)- L -prolinates as Aib-Pro SynthonsHELVETICA CHIMICA ACTA, Issue 9 2006Simon Stamm Abstract The syntheses of phenacyl N -(2,2-dimethyl-2H -azirin-3-yl)- L -prolinate and allyl N -(2,2-dimethyl-2H -azirin-3-yl)- L -prolinate are reported. Reactions of these 2H -azirin-3-amine derivatives with Z-protected amino acids have shown them to be suitable synthons for the Aib-Pro unit in peptide synthesis. After incorporation into the peptide by means of the ,azirine/oxazolone method', the C-termini of the resulting peptides were deprotected selectively with Zn in AcOH or by a mild Pd0 -promoted procedure, respectively. [source] WHEN POSITIVELY CHARGED MILK PROTEINS CAN BIND TO DNAJOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2002MAHMOUD SITOHY ABSTRACT The binding of three esterified milk proteins (,-lactoglobutin, ,-lactalbumin and ,-casein) to plasmid DNAs at pH 7.1 was followed by agarose-gel electrophoresis. Highly esterified ,-lactoglobulin and ,-lactalbumin samples showed DNA-binding capacities comparable to those exhibited by native basic proteins such as lysozymes and histones. All the studied esterified ,-casein samples failed to bind to DNA at the applied pH. Complete retardation of DNA migration on agarose gel was observed at a 1:1 ratio of protein basic groups (Lys + Arg) to DNA phosphate add groups in the case of highly esterified ,-lactoglobulin, esterified ,-lactalbumin and native basic proteins (lysozyme and histone). Binding capacity was dependent on the degree of esterification of the milk proteins. Hydrolysis of esterified milk proteins either suppressed or reduced their DNA-binding capacities according to the degree of hydrolysis and consequently to the average size of the resulting peptides. A prolonged peptic hydrolysis (25% degree of hydrolysis) completely suppressed DNA-binding capacity probably because of the small sizes of the resulting peptic peptides (< 1 kDa). Treatment with trypsin, which hydrolyzed the esterified proteins into relatively large peptide fragments, reduced the DNA-binding capacity to levels inversely proportional to the degree of hydrolysis. In the range of 2.7,12.3 kb, there was no influence of the DNA size on the binding of esterified milk proteins. The interactions DNA-esterified milk proteins did not depend on the DNA shape (circular or linear). Circular dichroism spectra of DNA in complex with methylated ,-lactoglobulin were markedly altered as compared to those obtained when DNA was in complex with native ,-lactoghbutin. [source] Proteomics in Alzheimer's disease: insights into potential mechanisms of neurodegenerationJOURNAL OF NEUROCHEMISTRY, Issue 6 2003D. Allan Butterfield Abstract Proteomics involves the identification of unknown proteins following their separation, often using two-dimensional electrophoresis, digestion of particular proteins of interest by trypsin, determination of the molecular weight of the resulting peptides, and database searching to make the identification of the proteins. Application of proteomics to Alzheimer's disease (AD), the major dementing disorder of the elderly, has just begun. Differences in protein expression and post-translational modification (mostly oxidative modification) of proteins from AD brain and peripheral tissue, as well as in brain from rodent models of AD, have yielded insights into potential molecular mechanisms of neurodegeneration in this dementing disorder. This review surveys the proteomics studies relevant to AD, from which new understandings of the pathology, biochemistry, and physiology of AD are beginning to emerge. [source] Rapid mass spectrometric analysis of 15N-Leu incorporation fidelity during preparation of specifically labeled NMR samplesPROTEIN SCIENCE, Issue 9 2008Stephanie M.E. Truhlar Abstract Advances in NMR spectroscopy have enabled the study of larger proteins that typically have significant overlap in their spectra. Specific 15N-amino acid incorporation is a powerful tool for reducing spectral overlap and attaining reliable sequential assignments. However, scrambling of the label during protein expression is a common problem. We describe a rapid method to evaluate the fidelity of specific 15N-amino acid incorporation. The selectively labeled protein is proteolyzed, and the resulting peptides are analyzed using MALDI mass spectrometry. The 15N incorporation is determined by analyzing the isotopic abundance of the peptides in the mass spectra using the program DEX. This analysis determined that expression with a 10-fold excess of unlabeled amino acids relative to the 15N-amino acid prevents the scrambling of the 15N label that is observed when equimolar amounts are used. MALDI TOF-TOF MS/MS data provide additional information that shows where the "extra" 15N labels are incorporated, which can be useful in confirming ambiguous assignments. The described procedure provides a rapid technique to monitor the fidelity of selective labeling that does not require a lot of protein. These advantages make it an ideal way of determining optimal expression conditions for selectively labeled NMR samples. [source] Elongation of the BH8 ,-hairpin peptide: Electrostatic interactions in ,-hairpin formation and stabilityPROTEIN SCIENCE, Issue 7 2001Marina Ramírez-Alvarado Abstract An elongated version of the de novo designed ,-hairpin peptide, BH8, has allowed us to gain insight into the role of electrostatic interactions in ,-hairpin stability. A Lys,Glu electrostatic pair has been introduced by adding a residue at the beginning and at the end of the N-terminal and C-terminal strands, respectively, of the ,-hairpin structure, in both orientations. The two resulting peptides and controls having Ala residues at these positions and different combinations of Ala with Lys, or Glu residues, have been analyzed by nuclear magnetic resonance (NMR), under different pH and ionic strength conditions. All of the NMR parameters, in particular the conformational shift analysis of C, protons and the coupling constants, 3JHN,, correlate well and the population estimates are in reasonable agreement among the different methods used. In the most structured peptides, we find an extension of the ,-hairpin structure comprising the two extra residues. Analysis of the pH and salt dependence shows that ionic pairs contribute to ,-hairpin stability. The interaction is electrostatic in nature and can be screened by salt. There is also an important salt-independent contribution of negatively charged groups to the stability of this family of ,-hairpin peptides. [source] High-resolution imaging and proteomics of peptide fragments by TOF-SIMSPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 8 2010Hĺkan Nygren Abstract Thyroglobulin is an iodinated glycoprotein (m.w. 660,kD) required for the storage and formation of thyroid hormone. Thyroglobulin was digested by trypsin in distilled water and the resulting peptides were identified by TOF-secondary ion mass spectrometry, using TFA as a matrix to catalyze the ionization of the peptides. Cryostate sections of pig thyroid glands were incubated with trypsin in distilled water, followed by deposition of TFA. The sections were analyzed with TOF-secondary ion mass spectrometry, and the peptides formed were identified through comparison with the peptides of the thyroglobulin reference sample. The thyroglobulin fragments were localized in the thyroid follicle cells with a spatial resolution of 3 microns, a mass resolution m/,m of >6000 and a mass accuracy of <60,ppm. The thyroglobulin was found localized heterogeneously in the follicle cells. The heterogeneity may be due to thyroglobulin synthesis, uptake and degradation or globules representing insoluble polymers of thyroglobulin considered to be a mechanism for storing hormone at high concentrations. [source] Large-scale analysis of the human ubiquitin-related proteomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2005Masaki Matsumoto Abstract Protein ubiquitylation contributes to the regulation of many cellular processes including protein degradation, receptor internalization, and repair of DNA damage. We now present a comprehensive characterization of ubiquitin-conjugated and ubiquitin-associated proteins in human cells. The proteins were purified by immunoaffinity chromatography under denaturing or native conditions. They were then digested with trypsin, and the resulting peptides were analyzed by 2-D LC and MS/MS. A total of 670 distinct proteins were identified; 345 proteins (51%) were classified as Urp-D (ubiquitin-related proteome under the denaturing condition) and comprised ubiquitin-conjugated molecules, whereas 325 proteins (49%) were classified as Urp-N (ubiquitin-related proteome only under the native condition) and included molecules that associated with ubiquitylated proteins. The proportions of proteins in various functional categories differed substantially between Urp-D and Urp-N. Many ribosomal subunits were detected in the Urp-D group of proteins and several of these subunits were directly shown to be ubiquitylated by mass spectrometric analysis, suggesting that ubiquitylation might play an important role in the regulation and/or quality control of ribosomal proteins. Our results demonstrate the potential of proteomics analysis of protein ubiquitylation to provide important insight into the regulation of protein stability and other ubiquitin-related cellular functions. [source] Secreted proteome of the murine multipotent hematopoietic progenitor cell line DKmixRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2010Nina Luecke Administration of the multipotent hematopoietic progenitor cell (HPC) line DKmix improved cardiac function after myocardial infarction and accelerated dermal wound healing due to paracrine mechanisms. The aim of this study was to analyse the secreted proteins of DKmix cells in order to identify the responsible paracrine factors and assess their relevance to the wide spectrum of therapeutic effects. A mass spectrometry (MS)-based approach was used to identify secreted proteins of DKmix cells. Serum free culture supernatants of DKmix-conditioned medium were collected and the proteins present were separated, digested by trypsin and the resulting peptides were then analyzed by matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) MS. Overall 95 different proteins were identified. Among them, secretory proteins galectin-3 and gelsolin were identified. These proteins are known to stimulate cell migration and influence wound healing and cardiac remodelling. The remaining proteins originate from intracellular compartments like cytoplasm (69%), nucleus (12%), mitochondria (4%), and cytoplasmic membrane (3%) indicating permeable or leaky DKmix cells in the conditioned medium. Additionally, a sandwich immunoassay was used to detect and quantify cytokines and chemokines. Interleukin-6 (IL-6), interleukin-13 (IL-13), monocyte-chemoattractant protein-1 (MCP-1), monocyte-chemoattractant protein-3 (MCP-3), monocyte-chemoattractant protein-1, (MIP-1,) and monocyte-chemoattractant protein-1, (MIP-1,) were detected in low concentrations. This study identified a subset of proteins present in the DKmix-conditioned medium that act as paracrine modulators of tissue repair. Moreover, it suggests that DKmix-derived conditioned medium might have therapeutic potency by promoting tissue regeneration. Copyright © 2010 John Wiley & Sons, Ltd. [source] Use of different proteases working in acidic conditions to improve sequence coverage and resolution in hydrogen/deuterium exchange of large proteinsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 21 2003Laetitia Cravello The combination of hydrogen exchange and mass spectrometry has been widely used in structural biology, providing views on protein structure and protein dynamics. One of the constraints is to use proteases working at low pH and low temperature to limit back-exchange during proteolysis. Although pepsin works in these conditions and is currently used in such experiments, sequence coverage is not always complete especially for large proteins, and the spatial resolution of the exchange rate is limited by the size of the resulting peptides. In this study we tried two other proteases, protease type XIII from Aspergillus saitoi and protease type XVIII from Rhizhopus species. The penicillin-binding protein X (PBP-2X*), a 77-kDa protein, was selected as a model. Like pepsin, neither of these proteases is really specific, but we found very good reproducibility in the digestion pattern. Compared with using pepsin alone, combining the results of the three independent proteolyses increased the coverage for the peptide mapping, thus avoiding missing some potentially interesting regions of the protein. Furthermore, we obtained a better spatial resolution for deuterium incorporation data, specifying accurately the deuterated regions. Copyright © 2003 John Wiley & Sons, Ltd. [source] Peptidylarginine deiminase from Porphyromonas gingivalis citrullinates human fibrinogen and ,-enolase: Implications for autoimmunity in rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 9 2010Natalia Wegner Objective To investigate protein citrullination by the periodontal pathogen Porphyromonas gingivalis as a potential mechanism for breaking tolerance to citrullinated proteins in rheumatoid arthritis (RA). Methods The expression of endogenous citrullinated proteins was analyzed by immunoblotting of cell extracts from P gingivalis and 10 other oral bacteria. P gingivalis,knockout strains lacking the bacterial peptidylarginine deiminases (PADs) or gingipains were created to assess the role of these enzymes in citrullination. Citrullination of human fibrinogen and ,-enolase by P gingivalis was studied by incubating live wild-type and knockout strains with the proteins and analyzing the products by immunoblotting and mass spectrometry. Results Endogenous protein citrullination was abundant in P gingivalis but lacking in the other oral bacteria. Deletion of the bacterial PAD gene resulted in complete abrogation of protein citrullination. Inactivation of arginine gingipains, but not lysine gingipains, led to decreased citrullination. Incubation of wild-type P gingivalis with fibrinogen or ,-enolase caused degradation of the proteins and citrullination of the resulting peptides at carboxy-terminal arginine residues, which were identified by mass spectrometry. Conclusion Our findings demonstrate that among the oral bacterial pathogens tested, P gingivalis is unique in its ability to citrullinate proteins. We further show that P gingivalis rapidly generates citrullinated host peptides by proteolytic cleavage at Arg-X peptide bonds by arginine gingipains, followed by citrullination of carboxy-terminal arginines by bacterial PAD. Our results suggest a novel model where P gingivalis,mediated citrullination of bacterial and host proteins provides a molecular mechanism for generating antigens that drive the autoimmune response in RA. [source] | |||||||||||||