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R J Fido

Publications and source records attributed to R J Fido.

8 recordsLinked to original sources
PubMed2001

Synthesis, expression and characterisation of peptides comprised of perfect repeat motifs based on a wheat seed storage protein.

We have developed a novel method for constructing synthetic genes that encode a series of peptides comprising perfect repeat motifs based on a high molecular weight subunit (HMW glutenin subunit), a highly repetitive storage protein from wheat seed. A series of these genes of sequentially increasing size was produced, four of which (called R3, 4, 5, 6) were expressed in Escherichia coli. Activity of the synthetic genes in E. coli was confirmed by Northern blot analysis but SDS-PAGE of crude protein extracts failed to show any expressed peptides when stained using Coomassie brilliant blue R250. However, Western blots probed with a HMW glutenin subunit-specific polyclonal antibody showed the presence of the R6 peptide (M(r) 22005) in the crude cell extracts and both this and the R3 peptide (M(r) 12005) were subsequently purified by extraction with hot aqueous ethanol followed by precipitation with acetone and separated by RP-HPLC. The R4 and R5 peptides were not purified. The purified R3 and R6 peptides absorbed Coomassie brilliant blue R250 or other protein stains only weakly and this was considered to account for their failure to be revealed by staining of separations of the crude protein extracts. Circular dichroism spectroscopy showed that both peptides had similar beta-turn rich structures similar to the repetitive sequences present in the whole HMW glutenin subunits. We conclude that expression of perfect repeat peptides in E. coli is a suitable system for the study of structure-function relationships in wheat gluten proteins and other highly repetitive proteins.

Amino Acid Motifs↗
PubMed2000

Overexpression, purification, and in vitro refolding of the 11S globulin from amaranth seed in Escherichia coli.

An amarantin 11S globulin cDNA encoding one of the most important storage proteins of amaranth seeds, with a high content of essential amino acids, was expressed in Escherichia coli. A good level of expression of recombinant amarantin with a molecular weight of 59 kDa was obtained. The recombinant protein was extracted by ammonium sulfate precipitation and purified to homogeneity using ion-exchange chromatography and reversed phase high-performance liquid chromatography. The expressed protein exhibited electrophoretic, immunochemical, and surface hydrophobicity properties similar to those of native amarantin from amaranth seed. Also, the recombinant protein was refolded in vitro using two different methods.

Chromatography, High Pressure Liquid↗
PubMed1998

Studies in vivo of omega-gliadins in gluten sensitivity (coeliac sprue disease).

1. Highly purified omega-gliadins from wheat were used to challenge gluten-sensitized individuals. Characteristic responses by mucosal CD3(+) and gamma delta+ lymphocytes were demonstrated. Each lymphocyte subset showed an increase within 8-12 h post-challenge, indicating a specific response by the rectal mucosa to this gliadin species.2. Available sequence data for the omega-gliadins and homologous proteins from barley and rye indicate a common repeating octapeptide motif (consensus PQQPFPQQ). The results indicate, therefore, that the octapeptide repeat, or a contained sequence such as PQQP, plays an important role in the mucosal immunopathology of gluten sensitivity.

CD3 Complex↗
PubMed1991

Isolation and partial amino acid sequence of domains of nitrate reductase from spinach.

Fragments of spinach nitrate reductase (NR) were prepared by limited proteolysis of immunopurified enzyme using both Staphylococcus aureus V8 protease and trypsin. Incubation of NR with V8 protease yielded two enzymically active fragments which could be size separated by FPLC on a Superose 12 column or subjected to further proteolysis while bound to a blue Sepharose affinity column. An NADH-ferricyanide (NADH-FR) active fragment bound to, and was eluted from, a blue Sepharose column by micromolar concentrations of NADH. A fragment with methyl viologen-NR activity was either eluted from the same column using 1 M KNO3 or on further treatment in situ on the blue Sepharose column with trypsin. Incubation of holo-NR with trypsin resulted in the loss of all terminal nitrate reducing activities but no loss in either NADH-FR activity or NADH-cytochrome c reductase activity. Two protease-sensitive regions of NR are shown which connect essentially between the flavin (FAD) and haem domains, and between the haem and molybdenum domains of NR. Amino acid analysis of the FAD- and FAD/haem-containing domains yielded two partial sequences which are compared with sequences deduced from complementary DNA (cDNA) of NR from Arabidopsis, tobacco and spinach. The deduced sequences from Arabidopsis and tobacco are found to be ca 80% and the spinach 100% homologous to the sequence obtained for spinach NR fragments.

Amino Acid Sequence↗
PubMed1983

Studies by electron-paramagnetic-resonance spectroscopy of the molybdenum centre of spinach (Spinacia oleracea) nitrate reductase.

The molybdenum centre of spinach (Spinacia oleracea) nitrate reductase has been investigated by e.p.r. spectroscopy of molybdenum(V) in reduced forms of the enzyme. The resting enzyme gives no signals attributable to Mo(V). However, on reduction with NADH, Mo(V) signals appeared at relatively short reaction times but decreased again on prolonged exposure to excess of the substrate as the enzyme was further reduced. On brief treatment of such samples with nitrate, Mo(V) signals reappeared but disappeared again on longer exposure to excess nitrate as the enzyme became fully reoxidized. Detailed investigation of the signals carried out in both 1H2O and 2H2O revealed the presence of two signal-giving species, referred to as 'signal A' and 'signal B', analogous to corresponding signals from nitrate reductase from Escherichia coli and from liver sulphite oxidase. Signal A has gav. 1.9767 and shows coupling to a single proton, exchangeable with the solvent, with A(1H)av. 1.3mT, whereas signal B shows no more than weak coupling to protons. Investigation of interconversion between the two species indicated that decreasing the pH from 8.0 to 6.7 had little effect, but that signal A was favoured by the presence of Cl-. This suggests, by analogy with recent work on sulphite oxidase by Bray, Gutteridge, Lamy & Wilkinson [Biochem. J. (1983) 211, 227-236] that Cl- is a ligand of molybdenum in the species giving signal A.

Chlorides↗