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Biomedical subjects

K Ashman

Publications and source records attributed to K Ashman.

34 records · Page 2Linked to original sources
PubMed1996

Purification of hydrophobic integral membrane proteins from Mycoplasma hyopneumoniae by reversed-phase high-performance liquid chromatography.

A general and practical approach for isolating, fractionating and purifying large quantities of outer membrane hydrophobic proteins is described as applied to membrane proteins of Mycoplasma hyopneumoniae. Outer membrane proteins were extracted with Triton X-114 detergent and were precipitated from the detergent phase with 90% ethanol. Precipitated proteins were dissolved in 65% formic acid and separated by RP-HPLC using a formic acid-acetonitrile gradient. A M(r) 48 000 protein was obtained in high yield and at greater than 90% purity by optimisation of parameters for RP-HPLC. The combination of Triton X-114 extraction followed by high resolution RP-HPLC is a novel and rapid procedure for the isolation and purification of hydrophobic proteins. Proteins purified by this approach were suitable for subsequent characterisation by direct sequencing of the amino terminus as well as generation of peptides by digestion with cyanogen bromide.

Amino Acid Sequence↗
PubMed1995

Automation of micro-preparation and enzymatic cleavage of gel electrophoretically separated proteins.

To achieve high throughput, protein microcharacterization sample preparation must be automated. We describe a cartesian robot capable of processing 32 protein samples in parallel. The system is based on specially designed flow-through reactors for contamination-free reagent delivery and removal. Washing of excised gel pieces, reduction and alkylation, proteolytic cleavage and peptide extraction are performed in these reactors. Compatibility of the system with HPLC peptide separation and Edman degradation as well as with laser desorption mass spectrometry of the unseparated mixture is demonstrated. This is the first report describing automated preparation and processing of multiple protein samples.

Amino Acid Sequence↗
PubMed1995

Fasciola hepatica: rapid identification of newly excysted juvenile proteins.

The sensitivity of N-terminal sequencing has been used to identify proteins expressed by the newly excysted juvenile stage of the parasite Fasciola hepatica. Of the seven proteins identified, a number have significant sequence homology to the cysteine proteases: cathepsin B, cathepsin L and asparaginyl endoproteinase. Proteolytic activity was demonstrated using gelatin substrate sodium dodecyl sulphate polyacrylamide gel electrophoresis. In addition, a number of novel proteins were identified which shared no significant sequence homology to proteins in the databases. The availability of such N-terminal sequence information allows rapid identification of major proteins from scarce developmental stages and provides the basis for further molecular studies.

Amino Acid Sequence↗
PubMed1995

Humoral and cellular responses following local immunization with a surface antigen of the gastrointestinal parasite Haemonchus contortus.

A purified, larval specific antigen of the abomasal parasite Haemonchus contortus was used to immunize sheep. In an attempt to induce a local immune response in the abomasum, the antigen was injected twice into the abomasal wall after one peripheral immunization. Serum antibody responses were boosted by each intra-abomasal immunization but not by the challenge infection given 3.5 weeks after the last immunization. Examination of the specific antibody secreting cells (ASC) recirculating in the peripheral blood indicated that there was an increase in blood ASC 5 days after local stimulation. This increase was maintained only after immunization and not after infection, probably reflecting the different responses induced when antigen is presented by injection in an adjuvant or by the parasite during infection. High proliferative T cell responses in the abomasal lymph nodes were only observed in one of the five sheep immunized with antigen; this was also the only sheep in this group to maintain an adult parasite burden at postmortem corresponding with the lowest antibody response. Peak faecal egg counts after infection were reduced by 54% in the immunized group compared to control sheep. Egg counts in the control sheep were, however, variable and dropped quickly, probably as a consequence of the inflammatory response induced by the injection of aluminium hydroxide into the abomasal wall.

Abomasum↗
PubMed1993

Molecular cloning and characterization of a ruminant interleukin-6 cDNA.

By hybridization with a human interleukin-6 (IL-6) cDNA fragment a corresponding ruminant (ovine) cDNA was isolated from a lipopolysaccharide (LPS)-stimulated alveolar macrophage library. The nucleotide sequence of the cDNA and the predicted amino acid sequence of the protein showed significant homology to the human and murine molecules. Ovine IL-6 cDNA encodes a polypeptide of 208 amino acids that, based on analysis of human IL-6, is processed to a protein of 180 amino acids. Northern blot analysis and the 7TD1 bioassay were used to analyse regulatory aspects of IL-6 production by primary ovine fibroblasts. Both LPS and recombinant ovine IL-1 alpha were shown to induce IL-6 mRNA with peak levels occurring at 1 h post-stimulation and declining thereafter. When fibroblasts were pretreated with cyclohexamide prior to stimulation the level of induction by LPS and IL-1 alpha increased dramatically and peak levels were observed at 5 h post-stimulation. The level of secreted IL-6 increased rapidly over the first 24 h and continued to increase over the next 48 h.

Amino Acid Sequence↗
PubMed1993

Recombinant cytokines as immunological adjuvants.

This paper describes the bacterial expression and purification of bioactive recombinant ovine interleukin-2 (rovIL-2), interleukin-1 alpha (rovIL-1 alpha) and tumour necrosis factor alpha. These purified proteins had specific activities in appropriate bioassays of 1 x 10(7) 1 x 10(7) and 1 x 10(5) U/mg, respectively. Recombinant ovIL-1 alpha was assessed as an immunological adjuvant for the sheep response to the model protein avidin. When delivered either intradermally or intramuscularly in conjunction with avidin in aluminium hydroxide the rovIL-1 alpha significantly enhanced the secondary humoral response. Doses of 1, 10 or 100 micrograms per sheep enhanced the humoral response to a similar extent. Recombinant ovIL-1 beta had similar adjuvant activity in that it was demonstrated to significantly enhance the sheep humoral response to an experimental H. contortus antigen. This increase in specific antibody, however, did not correlate with enhanced protection against infection with third stage H. contortus larvae. In addition incorporation of rovIL-1 beta into the formulation was shown not to alter the isotype profile of H. contortus antigen specific antibody.

Adjuvants, Immunologic↗
PubMed1989

Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010.

We present the complete nucleotide sequence of RSF1010, a naturally occurring broad-host-range plasmid belonging to the Escherichia coli incompatibility group Q and encoding resistance to streptomycin and sulfonamides. A molecule of RSF1010 DNA consists of 8684 bp and has a G + C content of 61%. Analysis of the distribution of translation start and stop codons in the sequence has revealed the existence of more than 40 open reading frames potentially capable of encoding polypeptides of 60 or more amino acids. To date, products of eleven such potential RSF1010 genes have been identified through the application of controlled expression vector systems, and for eight of them, the reading frame has been confirmed by N- and/or C-terminal amino acid sequence determinations on the purified proteins. The sequencing results are discussed in relation to the systems of replication, host range, conjugal mobilization and antibiotic resistance determinants associated with the RSF1010 plasmid.

Amino Acid Sequence↗
PubMed1989

Chemical synthesis, expression and product assessment of a gene coding for biologically active human tumour necrosis factor alpha.

A gene encoding human tumour necrosis factor alpha (TNF-alpha) has been chemically synthesized, cloned and expressed to yield a biologically active protein in Escherichia coli. The 480-bp gene was assembled by enzymic ligation of 32 oligonucleotides, cloned directly into M13mp18 for sequence verification and expressed in the broad host range high-level expression vector pMMB66EHST. Expressed recombinant TNF-alpha was shown to have the correct molecular weight, processed N-terminal sequence, antibody cross-reactivity and tumour cell killing activity. The expression product of the synthetic gene has been purified to homogeneity by a two-step ion-exchange procedure and the purified material shown to be active.

Amino Acid Sequence↗
PubMed1987

High-speed preparative reversed-phase high-performance liquid chromatography of synthetic oligonucleotides.

A rapid method is described for the purification and analysis of synthetic oligonucleotides, based on reversed-phase high-performance liquid chromatography. Volatile buffers and a short column (40 mm X 4.6 mm) packed with Nucleosil 300-5 C4 were employed. Monitoring the column effluent with an UV detector provides an excellent means of controlling product quality. The method is suitable for the purification of crude synthesis products, as well as for desalting and removing gel contaminants from oligonucleotides eluted from polyacrylamide gels. The total time required per sample is less than 25 min.

Chromatography, High Pressure Liquid↗
PubMed1986

A new covalently modified support for gas-liquid phase sequencing.

A new method for activation of glass fiber supports for immobilisation of proteins and peptides in gas-liquid phase sequencing is described. The new support offers several advantages over the presently used carrier polybrene: no precycling is required, initial yields are improved and background contamination is lower. This leads to an overall increase in detection sensitivity. The derivatisation method includes acid activation and subsequent covalent coating of glass fibers with quaternary ammonium groups thereby giving the glass surface a high binding capacity for both proteins and peptides. The activated glass has been successfully used for sequencing proteins and peptides isolated by HPLC as well as by electroelution from polyacrylamide gels.

Chromatography, Gas↗
PubMed1985

The complete primary structure of ribosomal proteins L1, L14, L15, L23, L24 and L29 from Bacillus stearothermophilus.

The amino acid sequences of ribosomal proteins L1, L14, L15, L23, L24 and L29 from Bacillus stearothermophilus have been completely determined. This has been achieved by sequence analyses of peptides derived from enzymatic digestions of the proteins with trypsin, chymotrypsin, pepsin, Staphylococcus aureus protease, and Armillaria mellea protease as well as by chemical cleavage with hydroxylamine and cyanogen bromide. Based on the primary structures of the six proteins, their secondary structures were predicted using four different computer prediction programs. A comparison of the amino acid sequences of the studied proteins from B. stearothermophilus with the homologous proteins from Escherichia coli revealed that in four proteins (L1, L15, L24 and L29) between 40-50% of the residue in the sequences are identical, whereas this value is significantly higher (69%) for L14 and lower (28%) for L23. The distribution of those amino acid residues which are identical in the corresponding proteins from the two bacteria is not random along the protein chain: some regions are highly conserved whereas others are not. This finding indicates that the regions which are conserved during evolution are important for the spatial structure and/or function of the protein.

Amino Acid Sequence↗
PubMed1984

On the statistical significance of homologous structures among the Escherichia coli ribosomal proteins.

Completion of the sequence determination of all 52 Escherichia coli ribosomal proteins enabled a final comparison of their sequences. Similarities in amino acid compositions were compared to the relatedness of the sequences, which was analyzed statistically with the aid of the computer programs RELATE and ALIGN. Among the examined 52 X 52 possible protein pairs at least 40 pairs were found that can be regarded as distantly related (showing segment comparison score values slightly above 3.0 S.D. units). These protein pairs were further examined with the programs ALIGN and SEEK to locate homologous sequence stretches. In no case were two complete homologous sequences found (with the exception of the known identical pairs L7/L12 and S20/L26). However, short homologous sequence regions were observed. Beside those protein pairs that show significant although distant relatedness, other pairs were slightly below the threshold value of 3.0 S.D. units. Those pairs observed to be distantly related consisted either of two proteins from the same subunit or of one protein from each of the different subunits. A further analysis of these pairs revealed a correlation between their relatedness and their time of incorporation into the ribosome during assembly.

Amino Acid Sequence↗
PubMedDate not supplied

Use of mass spectrometric methods for protein identification in receptor research.

In recent years, mass spectrometry has become the method of choice for identifying small amounts of gel separated proteins. Using high mass accuracy peptide mass mapping followed if necessary by nanoelectrospray sequencing, most mammalian proteins can now be identified quickly and sensitively either in amino acid or in EST sequence databases. These methods are illustrated here using an ongoing project in the author's laboratory, a mass spectrometric screen for new mouse brain receptors and their interaction partners.

Animals↗
PubMedDate not supplied

Identification of cysteine-containing peptides during the peptide mapping of recombinant proteins.

Confirmation of a protein's cysteine content and of its location within the amino acid sequence is crucial in investigating the structural integrity of recombinant proteins. A combination of thiol-specific chemistry and peptide mapping by reversed-phase microbore HPLC was used to confirm the presence and map the location of cysteine residues in the primary sequences of recombinant porcine growth hormone and human tumor necrosis factor alpha. Recombinant proteins were conjugated with a hydrophobic iodoacetamide derivative, dimethylaminoazo-benzene iodoacetamide, and digested with trypsin. The peptide fragments were separated on a C8 microbore reversed-phase column using a linear acetonitrile gradient. The peptides containing the cysteine residues were selectively identified by monitoring with a diode-array detector at 215 nm with the reference wavelength set at 450 nm. Cysteine-containing peptides could be readily distinguished as inverted "negative" peaks relative to the baseline and noncysteine-containing peptides. Isolated peptide fragments were then sequenced in order to confirm the location of the cysteines in the proteins. This approach offers the benefits of selectively and rapidly identifying, from a single chromatrophic step, the cysteine-containing peptides of proteins. Furthermore, the use of the labeling reagent renders the cysteine-containing peptides more hydrophobic, thereby making them easier to separate from noncysteine-containing peptides.

Amino Acid Sequence↗