PubMed Health⌕ Search

Biomedical subjects

W Cockburn

Publications and source records attributed to W Cockburn.

At least 19 recordsLinked to original sources
PubMed2000

High affinity ScFvs from a single rabbit immunized with multiple haptens.

We report the generation of single-chain Fv (scFv) fragments with high affinities against four different hapten molecules from a single immunised rabbit. The rabbit was immunised with a mixture of protein conjugates of four different haptens, namely the herbicide mecoprop and derivatives of the herbicides atrazine, simazine, and isoproturon. An scFv phage display library was constructed, and several scFvs with high affinity against each hapten were isolated. For each hapten, a single binder was selected by k(off) ranking and used for affinity determination. The affinities were in sub-nanomolar range and the lowest K(d) value obtained was 6.75 x 10(-10) M. An unusual feature of one of the anti-isoproturon scFvs was its ability to retain binding activity at pH1.7. The utility and potential of using a single animal and immunisation with multiple antigens for the production of multiple, specific, high affinity scFvs by phage display is discussed.

Amino Acid Sequence↗
PubMed2000

Cytoplasmic expression of a soluble synthetic mammalian metallothionein-alpha domain in Escherichia coli. Enhanced tolerance and accumulation of cadmium.

Bacteria are commonly used for bioremediation of heavy metal pollution and strategies to improve their performance in this respect are desirable. In this study, an Escherichia coli strain was engineered to express a common metallothionein-alpha domain. The metallothionein-alpha domain was over-expressed in the cytoplasm of E. coli as a fusion to the carboxyl terminal of maltose binding protein. The fusion protein was highly soluble in the cytoplasm of E. coli. When grown in the presence of cadmium, cells expressing the metallothionein-alpha fusion protein showed increased viability compared with control cells. Cells expressing the metallothionein-alpha also demonstrated increased accumulation of cadmium.

ATP-Binding Cassette Transporters↗
PubMed1999

Rapid production of single-chain Fv fragments in plants using a potato virus X episomal vector.

We have used a plant virus episomal vector, based on potato virus X (PVX) to transiently express a single-chain Fv (scFv) and its diabody derivative in plants. The scFv was directed against a continuous epitope (cryptotope) on the coat protein of potato virus V. A cloned, full-length PVX vector sequence, containing the scFv gene, was used to direct in vitro transcription and the resulting RNA was used to inoculate Nicotiana clevelandii plants. Within a few days, plants developed characteristic symptoms and immunoblot analysis showed that accumulation of scFv protein coincided with accumulation of PVX. Targeting of the scFv to the apoplast greatly increased protein accumulation compared with cytosolic scFv and produced more severe symptoms on infected plants. ELISA demonstrated that the scFv and diabody extracted from infected plants showed the same antigen-binding specificity as that of the parental monoclonal antibody. The PVX vector is a convenient, rapid, low-cost in planta expression system that can also be used for assessment of scFv production and function prior to stable plant transformation.

Amino Acid Sequence↗
PubMed1999

Selection of phage antibodies to surface epitopes of Phytophthora infestans.

Antibodies specific for surface-exposed epitopes on germlings of the plant pathogen, Phytophthora infestans, were isolated from a diverse phage library displaying single-chain Fv (scFv) antibody fragments. The library was subpanned against external soluble components released from mycelia, sporangia and germlings and a discrete population of phage antibodies isolated. Binding of monoclonal phage antibodies was demonstrated by enzyme-linked immunosorbent assay (ELISA) and diversity was established by BstNI restriction enzyme digest patterns. Antibodies were subcloned as fusions at the C-terminus of maltose binding protein (MBP) and expressed as soluble proteins in Escherichia coli. These antibody fusion proteins bound to P. infestans germlings and to mycelial homogenates from various Phytophthora species. The binding activities to mycelial homogenates of fungal species not belonging to the order Peronosporales were substantially lower. Several phage-displayed scFvs were used in conjunction with fluorescently labelled antiphage antibody to visualise the distribution of their cognate epitopes on the surface of the germlings. The combination of procedures developed here with Phytophthora demonstrates the potential of phage antibody technology in isolating antibodies to cell surface and external soluble components of pathogens, some of which may play a role in host/pathogen interactions.

Animals↗
PubMed1999

Selection of phage-display peptides that bind to cucumber mosaic virus coat protein.

Several discrete peptides that bind specifically to the coat protein of cucumber mosaic virus (CMV) were isolated from a diverse phage library displaying random nonapeptides on the major coat protein VIII. Enrichment was shown by polyclonal phage enzyme linked immunosorbent assay (ELISA) after three rounds of selection. Sequencing of the genes encoding 10 of these peptides revealed an absence of any conserved motifs, although nine of them contained a high proportion of proline residues. Some of the selected peptides were displayed at the N-terminus of thioredoxin and expressed in the cytoplasm of Escherichia coli. Both the phage-displayed and thioredoxin-fusion versions of the peptides could detect purified CMV and CMV present in crude leaf extracts from infected plants. By dot blot analysis, a thioredoxin-peptide fusion could readily detect as little as 5 ng of CMV. The peptides did not bind to other plant viruses. These peptides have been shown to be specific and highly sensitive tools in the detection of CMV and, as well as their diagnostic potential, they could form the basis for a novel disease resistance strategy.

Amino Acid Sequence↗
PubMed1998

Filamentous bacteriophage display of a bifunctional protein A::scFv fusion.

Filamentous bacteriophage display is a powerful and widely used technology for the selection of affinity ligands. However, the commonly used phagemid systems result in the production of a population of phage of which those displaying the ligand of interest represent only a small proportion. Through simple dilution and nonspecific binding effects, the presence of large numbers of ligand-free phage reduces the likelihood that weak binders will be successfully selected from a ligand library. To provide a means of avoiding such problems, we have introduced an affinity handle into the phage that permits the purification of ligand-displaying phage. The IgG binding domains of Staphylococcus aureus protein A (SpA) were fused to a ligand (single chain Fv[scFv]) which is displayed as a fusion with the phage surface protein delta pIII. Phage-displaying SpA were separated by affinity chromatography using immobilized human IgG from non-displaying phage and the purified phage were shown to possess functional scFv. Comparisons of fusion proteins in which either the scFv or the affinity handle occupied the amino terminus of the fusion protein showed that, whereas SpA function was unaffected by position, scFv function was compromised when the scFv did not occupy the amino terminus.

Animals↗
PubMed1996

Study of antibody-antigen interaction through site-directed mutagenesis of the VH region of a hybrid phage-antibody fragment.

An important aspect of the study of antibody structure-function relationships involves analysis of natural or synthetic mutations of antigen-combining sites. The anti-hen egg lysozyme monoclonal antibody HyHEL-10 has been a focus for antibody structure-function studies. We have displayed on bacteriophage of a hybrid single chain Fv, containing the light chain variable region of HyHEL-10 and the heavy chain variable region of a structurally related but functionally distinct antibody, AS32. By using a combination of site-directed mutagenesis, complementary determining region grafting and molecular modeling, we have identified a number of contact and non-contact residues that are important in the affinity of HyHEL-10 for lysozyme. In particular, the heavy chain variable region framework residue at position 94 was shown to be an important determinant of high-affinity binding. The phage display approach eliminates the need for purification of antibodies and, when used in combination with polymerase chain reaction for variable region sequence mutagenesis, facilitates the rapid generation and characterization of mutant antibodies.

Animals↗
PubMed1994

Antigen detection using recombinant, bifunctional single-chain Fv fusion proteins synthesised in Escherichia coli.

A gene fusion approach has been used to produce antibody conjugates for use in immunoassays. Escherichia coli expression vectors encoding fusions between the outer membrane protein A signal peptide, an anti-phytochrome single-chain Fv protein, and either Escherichia coli alkaline phosphatase or Staphylococcal protein A downstream of the T7 O10 promoter were constructed. A crude lysate from cells expressing the single-chain Fv-alkaline phosphatase fusion protein could be used directly for the sensitive and specific staining of phytochrome on protein blots by a single-step immunoassay procedure. Following purification by immunoglobulin G affinity chromatography, the Staphylococcal protein A-single-chain Fv fusion protein was also used for selective immunostaining of phytochrome on protein blots by a two-step procedure in which a rabbit immunoglobulin-alkaline phosphatase conjugate was used to detect antigen-bound Staphylococcal protein A. Recombinant antibody conjugates of the types described here are simple and inexpensive to produce and are a realistic alternative to conventional antibody conjugates.

Alkaline Phosphatase↗
PubMed1990

A microtiter plate-based assay for phosphoenolpyruvate carboxylase.

A sensitive, quantitative assay for phosphenolpyruvate carboxylase which utilizes microtiter plates is described. The assay depends upon the production of a colored compound in the reaction between oxaloacetate, the product of the phosphoenolpyruvate reaction, and the dye Fast Violet B. The method is particularly appropriate for monitoring chromatographic eluates and its utility for this purpose is demonstrated by the detection of phosphoenolpyruvate carboxylase in fractions of crude maize extract separated by size-exclusion chromatography.

Diazonium Compounds↗
PubMed1985

Photosynthetic Carbon Assimilation in a Shootless Orchid, Chiloschista usneoides (DON) LDL: A Variant on Crassulacean Acid Metabolism.

Photosynthetic carbon assimilation in the roots of a shootless orchid Chiloschista usneoides (DON) LDL involves the synthesis and accumulation of malic acid from CO(2) in darkness. Malic acid is consumed in the light.The roots do not possess stomata or any means of diurnally regulating the diffusive conductance of the pathway between the internal gas phase of the plant and the atmosphere. Regulation of internal CO(2) concentration near to atmospheric levels avoids a large net loss of CO(2) to the atmosphere during malic acid consumption in the light.The water-absorbing function of the velamen conflicts with the photosynthetic function of the roots. Plants with water-saturated velamina do not acquire CO(2) from the atmosphere at night.

Journal Article↗
PubMed1982

Effects of light quantity and quality on the decarboxylation of malic Acid in crassulacean Acid metabolism photosynthesis.

The rate of malic acid consumption in the Crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perrier was found to be more rapid than the rate of photosynthetic oxygen evolution under all levels of irradiation by white light. This accounts for the accumulation of carbon dioxide in CAM tissues in the light.Action spectra of malate consumption and photosynthetic oxygen evolution in Kalanchoë were similar. Experiments using monochromatic photosynthetically active light in addition to a range of narrow waveband irradiations demonstrated that malic acid consumption in the experiments from which the action spectrum of acid consumption was constructed was not limited by the rate of photosynthesis. These data indicate that light involved in the promotion of malate consumption in CAM is absorbed by the same pigments that absorb the light which powers photosynthesis.

Journal Article↗
PubMed1979

Relationships between Stomatal Behavior and Internal Carbon Dioxide Concentration in Crassulacean Acid Metabolism Plants.

Measurements of internal gas phase CO(2) concentration, stomatal resistance, and acid content were made in Crassulacean acid metabolism plants growing under natural conditions. High CO(2) concentrations, sometimes in excess of 2%, were observed during the day in a range of taxonomically widely separated plants (Opuntia ficus-indica L., Opuntia basilaris Engelm. and Bigel., Agave desertii Engelm., Yucca schidigera Roezl. ex Ortiges, Ananas comosus [L.] Merr., Aloe vera L., Cattleya sp. and Phalanopsis sp.) and below ambient air concentrations were observed at night.Stomatal resistance was always high when CO(2) concentration was high and experiments in which attempts were made to manipulate internal CO(2) concentrations gave data consistent with stomatal behavior in Crassulacean acid metabolism being controlled by internal CO(2) concentration. Exogenous CO(2) applied in darkness at a concentration similar to those observed in the light caused stomatal resistance to increase.In pads of Opuntia basilaris Engelm. and Bigel. subjected to severe water stress internal gas phase CO(2) concentrations exhibited fluctuations opposite in phase to fluctuations in acid content. Stomatal resistance remained high and the opening response to low CO(2) concentration was almost entirely eliminated.

Journal Article↗
PubMed1977

Changes in Metabolite Levels in Kalanchoë daigremontiana and the Regulation of Malic Acid Accumulation in Crassulacean Acid Metabolism.

Changes in glucose-6-P, fructose-6-P, fructose-1,6-diP, 6-phospho-gluconate, phosphoenolpyruvate, 3-phosphoglycerate, and pyruvate levels in the leaves of the Crassulacean plant Kalanchoë daigremontiana Hammet et Perrier were measured enzymically during transitions from CO(2)-free air to air, air to CO(2)-free air, and throughout the course of acid accumulation in darkness. The data are discussed in terms of the involvement of phosphoenolpyruvate carboxylase in malic acid synthesis and in terms of the regulation of the commencement of malic acid synthesis and accumulation through the effects of CO(2) on storage carbohydrate mobilization and its termination through the effects of malic acid on phosphoenolpyruvate carboxylase activity.

Journal Article↗
PubMed1975

The pathway of carbon dioxide fixation in crassulacean plants.

Combined gas chromatography-mass spectrometry of malic acid derivatives has been used to show unequivocally that malic acid, synthesized during active acid accumulation in the dark by Kalanchoë daigremontiana Hammet et Perrier in the presence of (13)CO(2) is produced by a pathway involving a single carboxylation. The significance of the finding that crassulacean malate synthesized in the dark and in the presence of (14)CO(2) often contains 66% of the total carboxyl label in carbon atom 4, which has previously been taken to indicate the operation of a double carboxylation pathway or has been dismissed as an artefact, is discussed.

Journal Article↗
PubMed1968

Photosynthesis by isolated chloroplasts. Reversal of orthophosphate inhibition by Calvin-cycle intermediates.

1. The orthophosphate inhibition of photosynthesis by isolated spinach chloroplasts can be reversed by 3-phosphoglycerate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate. 2. Metabolically related compounds such as ribulose 1,5-diphosphate, glucose 6-phosphate, 6-phosphogluconate and phosphoenolpyruvate are ineffective. 3. The kinetics of reversal are characteristic of the intermediate used, but, in each instance, the onset of oxygen evolution is accompanied by a carbon dioxide fixation and except with 3-phosphoglycerate the stoicheiometry is close to unity. 4. The nature of orthophosphate inhibition and its reversal is discussed in relation to metabolic control of photosynthesis.

Carbon Dioxide↗
PubMed1968

The isolation of spinach chloroplasts in pyrophosphate media.

A simplified procedure (involving disruption in sorbitol-pyrophosphate mixtures) permits the separation of spinach chloroplasts which retain the ability to catalyze the photosynthetic assimilation of carbon dioxide and its associated oxygen evolution.

Journal Article↗