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At least 37 records · Page 2Linked to original sources
PubMed1999

Protein sequence analysis using Hewlett-Packard biphasic sequencing cartridges in an applied biosystems 473A protein sequencer.

Protein sequence analysis using an adsorptive biphasic sequencing cartridge, a set of two coupled columns introduced by Hewlett-Packard for protein sequencing by Edman degradation, in an Applied Biosystems 473A protein sequencer has been demonstrated. Samples containing salts, detergents, excipients, etc. (e.g., formulated protein drugs) can be easily analyzed using the ABI sequencer. Simple modifications to the ABI sequencer to accommodate the cartridge extend its utility in the analysis of difficult samples. The ABI sequencer solvents and reagents were compatible with the HP cartridge for sequencing. Sequence information up to ten residues can be easily generated by this nonoptimized procedure, and it is sufficient for identifying proteins by database search and for preparing a DNA probe for cloning novel proteins.

Immunoglobulin G↗
PubMed2004

Protein sequence analysis of a novel 103-kDa Dermatophagoides pteronyssinus mite allergen and prevalence of serum immunoglobulin E reactivity to rDer p 11 in allergic adult patients.

BACKGROUND: House dust mites are regarded as important indoor allergens. While the most studies mite allergens are low molecular weight (mw), a high mw Dermatophagoides farinae mite paramyosin (Der f 11) has recently been cloned. We have also cloned a novel high mw Dermatophagoides pteronyssinus (Dp) mite allergen, Der p 11. OBJECTIVE: The aim of this study was to isolate and express a cDNA gene coding for a Der p 11 allergen, to compare the sequence of Der p 11 with other antigens and to evaluate the presence of IgE reactivity to the recombinant protein (rDer p 11) in the sera of allergic adult patients. METHODS: The full-length Der p 11 gene was isolated by cDNA library screening, 5'-3' rapid amplification of cDNA ends and PCR. The cDNA gene was expressed as a glutathione-S-transferase fusion protein in Escherichia coli. The allergenicity of rDer p 11 was tested by human IgE immunodot or immunoblot assay in a large panel of 100 allergic patients with bronchial asthma, allergic rhinitis or eczema. RESULTS: Der p 11 is a 2965 bp cDNA gene with a 2625 bp open reading frame coding for a 875 amino acid protein. The deduced amino acid sequence of the Der p 11 showed significant homology with various invertebrate paramyosins. The prevalence of serum IgE reactivity to rDer p 11 on immunodot assay ranged from 41.7% to 66.7% in different allergic patient groups, whereas it was rare in non-atopic patients with urticaria (18.8%) and in normal individuals (8%). A high frequency (five out of eight) of MAST(Dp)- allergic serum samples had specific IgE-binding activity to rDer p 11 or its fragments on immunoblot assay, even though their IgE-binding activity to Dp extract was either weak or negative. CONCLUSION: The 103-kDa Der p 11 appears to be major Dp mite allergen with a high frequency of IgE reactivity in sera of patients allergic to mites.

Adolescent↗
PubMed1995

ANTHEPROT 2.0: a three-dimensional module fully coupled with protein sequence analysis methods.

ANTHEPROT is a fully interactive graphics program devoted to the analysis of the sequences and structures of proteins. This program, originally developed to facilitate the protein sequence analysis coupled with multiple alignments and predicted secondary structures of proteins, now comprises a powerful 3D module to display and handle macromolecular structures. All the methods that were previously integrated into ANTHEPROT are now directly coupled with a 3D window that provides the user all the classic features of a molecular modeling package. Indeed, it allows real-time rotation and translation of 3D structures with many kinds of models in depth-cueing mode (space filling, backbone, wire models, main chain, and ribbons), selections (atom type, residue type, segments, and chain), color-coding systems (amino acid properties, predicted or observed secondary structures, temperature B factor, and subunits), geometric calculations (Ramachandran plot, distances, and angles), and fitting molecules. Stereo views are possible as well as HPGL standard files. A module specifically devoted to the determination of 3D structures using nuclear magnetic resonance is also available. This major release of our program for IBM rs6000 workstations is available by anonymous ftp to ibcp.fr for academic institutions.

Antigens↗
PubMed2004

Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry.

Peptide sequence analysis using a combination of gas-phase ion/ion chemistry and tandem mass spectrometry (MS/MS) is demonstrated. Singly charged anthracene anions transfer an electron to multiply protonated peptides in a radio frequency quadrupole linear ion trap (QLT) and induce fragmentation of the peptide backbone along pathways that are analogous to those observed in electron capture dissociation. Modifications to the QLT that enable this ion/ion chemistry are presented, and automated acquisition of high-quality, single-scan electron transfer dissociation MS/MS spectra of phosphopeptides separated by nanoflow HPLC is described.

Amino Acid Sequence↗
PubMed1998

Expression in Escherichia coli of the lpdA gene, protein sequence analysis and immunological characterization of the P64k protein from Neisseria meningitidis.

By making use of recombinant DNA technology it is possible to characterize meningococcal outer membrane proteins (OMPs) capable of stimulating a host immune response. The lpdA gene, which codes for an OMP (P64k) from Neisseria meningitidis, was cloned in Escherichia coli. The recombinant protein was recognized by sera from patients convalescing from meningococcal disease. The monoclonal antibodies obtained against the recombinant protein recognized the natural protein on a Western blot, and monoclonal antibody 114 was assayed in ELISA with a panel of 85 N. meningitidis strains. The protein was recognized in 81 strains (95.3%); the strains that were not recognized were neither epidemic nor isolated from systemic disease. The complete amino acid sequence of P64k was obtained by automatic sequencing and MS.

Amino Acid Sequence↗
PubMed1987

Protein sequence analysis studies on the low molecular weight hydrophobic proteins associated with bovine pulmonary surfactant.

Lipid extracts of bovine pulmonary surfactant, which exhibit biophysical and biological activity, contain two hydrophobic proteins which have been designated surfactant protein-B (SP-B) and SP-C. Amino terminal amino acid sequence analysis of whole lipid extracts and partially purified protein fractions gave rise to three sequences, two major and one minor. The first sequence, identified as a member of the SP-B family, extended for 60 amino acids beginning with an amino terminal phe. The second polypeptide, identified as a member of the SP-C family, sequenced for 35 amino acids and had a leu amino terminus. The third minor sequence corresponded to amino acids 2-9 of SP-C (N-leu) and was designated SP-C (N-ile). Sequence analysis of cyanogen bromide peptides derived from methyl isocyanate-blocked lipid extract material produced two peptides which extended the amino acid sequence of SP-B to residue 79, which appears to be a glycine.

Amino Acid Sequence↗
PubMed1991

Amino acid sequence of Fel dI, the major allergen of the domestic cat: protein sequence analysis and cDNA cloning.

The complete primary structure of Fel dI (International Union of Immunological Societies nomenclature), the major allergen produced by the domestic cat, Felis domesticus, was determined by protein sequence analysis and cDNA cloning. Protein sequencing of Fel dI from an immunoaffinity-purified extract of house dust revealed that the allergen is composed of two polypeptide chains. Degenerate oligonucleotides derived from the protein sequence were used in polymerase chain reaction amplification of cat salivary gland cDNA to demonstrate that the two chains are encoded by different genes. Chain 1 of Fel dI shares amino acid homology with rabbit uteroglobin, while chain 2 is a glycoprotein with N-linked oligosaccharides.

Allergens↗
PubMed1990

Solid-phase protein sequence analysis.

The solutions to many protein analytical problems require flexible chemistries at a high sensitivity. Solid-phase sequence analysis is one way to eliminate limitations inherent in current methods.

Amino Acid Sequence↗
PubMed2004

Protein sequence analysis, cloning, and expression of flammutoxin, a pore-forming cytolysin from Flammulina velutipes. Maturation of dimeric precursor to monomeric active form by carboxyl-terminal truncation.

Flammutoxin (FTX), a 31-kDa pore-forming cytolysin from Flammulina velutipes, is specifically expressed during the fruiting body formation. We cloned and expressed the cDNA encoding a 272-residue protein with an identical N-terminal sequence with that of FTX but failed to obtain hemolytically active protein. This, together with the presence of multiple FTX family proteins in the mushroom, prompted us to determine the complete primary structure of FTX by protein sequence analysis. The N-terminal 72 and C-terminal 107 residues were sequenced by Edman degradation of the fragments generated from the alkylated FTX by enzymatic digestions with Achromobacter protease I or Staphylococcus aureus V8 protease and by chemical cleavages with CNBr, hydroxylamine, or 1% formic acid. The central part of FTX was sequenced with a surface-adhesive 7-kDa fragment, which was generated by a tryptic digestion of FTX and recovered by rinsing the wall of a test tube with 6 M guanidine HCl. The 7-kDa peptide was cleaved with 12 M HCl, thermolysin, or S. aureus V8 protease to produce smaller peptides for sequence analysis. As a result, FTX consisted of 251 residues, and protein and nucleotide sequences were in accord except for the lack of the initial Met and the C-terminal 20 residues in protein. Recombinant FTX (rFTX) with or without the C-terminal 20 residues (rFTX271 or rFTX251, respectively) was prepared to study the maturation process of FTX. Like natural FTX, rFTX251 existed as a monomer in solution and assembled into an SDS-stable, ring-shaped pore complex on human erythrocytes, causing hemolysis. In contrast, rFTX271, existing as a dimer in solution, bound to the cells but failed to form pore complex. The dimeric rFTX271 was converted to hemolytically active monomers upon the cleavage between Lys(251) and Met(252) by trypsin.

Amino Acid Sequence↗
PubMed1985

Purification and amino-terminal protein sequence analysis of the mumps virus fusion protein.

The fusion (F) protein of mumps virus was purified by immunoaffinity chromatography using an anti-F monoclonal antibody. The F protein was reduced and alkylated, and the F1 and F2 chains were isolated by high-pressure size exclusion chromatography. Twenty-three amino acid residues from the amino terminus of each chain were identified following automated Edman degradation. The amino-terminal sequence of the F1 chain was homologous to previously reported F1 sequences from three other paramyxoviruses (simian virus 5, Newcastle disease virus, and Sendai virus). Secondary structure predictions suggest an alpha-helical conformation for the mumps virus F1 amino-terminal sequence. A helical wheel model of the paramyxovirus F1 NH2 terminus is presented which defines conserved and variable arcs of the helix and provides a spatial representation of this critical functional domain of the paramyxovirus fusion protein.

Amino Acid Sequence↗
PubMed1986

GENEUS, a computer system for DNA and protein sequence analysis containing an information retrieval system for the EMBL data library.

We describe a comprehensive computer system, GENEUS, for extensive DNA, RNA and protein sequence analysis. The analysis system is developed for the DEC VAX/VMS computer and uses the EMBL Nucleic Acid Sequence Data Library. Help information is available on-line on terminal screen. To speed up system handling, a qualifier oriented user communication is employed. All results are stored on files making them accessible to the computer editor. An information retrieval system for the EMBL Nucleotide Sequence Data Library is also described. A defined data-base interface allows connection to other analysis programs.+

Amino Acid Sequence↗
PubMed2006

The MPI Bioinformatics Toolkit for protein sequence analysis.

The MPI Bioinformatics Toolkit is an interactive web service which offers access to a great variety of public and in-house bioinformatics tools. They are grouped into different sections that support sequence searches, multiple alignment, secondary and tertiary structure prediction and classification. Several public tools are offered in customized versions that extend their functionality. For example, PSI-BLAST can be run against regularly updated standard databases, customized user databases or selectable sets of genomes. Another tool, Quick2D, integrates the results of various secondary structure, transmembrane and disorder prediction programs into one view. The Toolkit provides a friendly and intuitive user interface with an online help facility. As a key feature, various tools are interconnected so that the results of one tool can be forwarded to other tools. One could run PSI-BLAST, parse out a multiple alignment of selected hits and send the results to a cluster analysis tool. The Toolkit framework and the tools developed in-house will be packaged and freely available under the GNU Lesser General Public Licence (LGPL). The Toolkit can be accessed at http://toolkit.tuebingen.mpg.de.

Computational Biology↗
PubMed2001

Integrated graphical analysis of protein sequence features predicted from sequence composition.

Several protein sequence analysis algorithms are based on properties of amino acid composition and repetitiveness. These include methods for prediction of secondary structure elements, coiled-coils, transmembrane segments or signal peptides, and for assignment of low-complexity, nonglobular, or intrinsically unstructured regions. The quality of such analyses can be greatly enhanced by graphical software tools that present predicted sequence features together in context and allow judgment to be focused simultaneously on several different types of supporting information. For these purposes, we describe the SFINX package, which allows many different sets of segmental or continuous-curve sequence feature data, generated by individual external programs, to be viewed in combination alongside a sequence dot-plot or a multiple alignment of database matches. The implementation is currently based on extensions to the graphical viewers Dotter and Blixem and scripts that convert data from external programs to a simple generic data definition format called SFS. We describe applications in which dot-plots and flanking database matches provide valuable contextual information for analyses based on compositional and repetitive sequence features. The system is also useful for comparing results from algorithms run with a range of parameters to determine appropriate values for defaults or cutoffs for large-scale genomic analyses.

Amino Acid Motifs↗
PubMed1993

Identification of cysteine residues alkylated with 3-bromopropylamine by protein sequence analysis.

A new reagent for the routine identification of cysteine residues during protein sequencing is described. This method employs 3-bromopropylamine to alkylate cysteines in proteins after reduction with dithiothreitol. Upon sequencing of the protein on an Applied Biosystems 477A protein sequencer, the aminopropylcysteine residue yields a phenylthiohydantoin (PTH) derivative which elutes reproducibly at a unique position immediately after PTH-leucine; baseline resolution is achieved without modification of the PTH analyzer gradient. Unlike PTH-pyridylethylcysteine, the relative elution position of the aminopropylcysteine PTH derivative is not affected by changes in the ionic strength of the analyzer solvents. In addition, the previewing of the next amino acid which is observed in proteins modified with 4-vinylpyridine does not occur in aminopropylated proteins. Preparation of alkylated proteins for electroblotting onto polyvinylidene difluoride (PVDF) membranes and methods for desalting alkylated proteins immobilized on precoated glass fiber filters or PVDF membranes are also described.

Alkylating Agents↗
PubMed1994

Maltose transport in Aeromonas hydrophila: purification, biochemical characterization and partial protein sequence analysis of a periplasmic maltose-binding protein.

A clinical isolate of Aeromonas hydrophila was demonstrated to transport [14C]maltose with similar kinetics to enteric bacteria (Km: 0.3 microM; Vmax: 22 nmol min-1 per 10(9) cells). The uptake of [14C]maltose was completely inhibited in the presence of unlabelled maltose or maltodextrins, whereas other mono- and disaccharides, such as glucose, galactose, sucrose, lactose or melibiose, had no effect. A protein with an apparent molecular mass of 39 kDa (maltose-binding protein; MBP) was identified in osmotic-shock fluid of maltose-grown cells by SDS-gel electrophoresis, and was purified to homogeneity by either amylose affinity chromatography or ion-exchange chromatography. Equilibrium dialysis experiments revealed the ability of the purified protein to bind [14C]maltose with high affinity (KD = 1.6 microM). Unlabelled maltose and maltodextrins competed for the binding site. In a reconstitution experiment, A. hydrophila MBP poorly restored the transport activity of a binding-protein-deficient Escherichia coli (delta malE) mutant. N-terminal sequence analyses of the purified native protein and of peptides generated by cleavage with CNBr and subsequently separated by HPLC revealed about 56% identical amino acid residues, as compared to enterobacterial MBPs. We conclude that maltose is transported into A. hydrophila via a binding-protein-dependent transport system.

ATP-Binding Cassette Transporters↗