PubMed Health⌕ Search

Biomedical subjects

R A Irving

Publications and source records attributed to R A Irving.

At least 19 recordsLinked to original sources
PubMed2001

Panning and selection of proteins using ribosome display.

Eukaryotic ribosome complexes can be used as a means to display a library of proteins, and isolate specific binding reagents by screening against target molecules. Here we present, as an example, a method for the display of a library of immunoglobulin variable-like domains (VLDs) for the production of stable mRNA/ribosome/protein complexes. These complexes are produced by the addition of specific in vitro transcriptional promoter elements and translation control sequences to the template DNA. Furthermore, an appropriate spacer (anchor) domain is included for efficient folding of the nascent translated protein, which remains attached to the ribosome complex. Ribosome complexes are panned against hen egg lysozyme-conjugated magnetic beads and genes encoding specific, binding, V-like domains are recovered by RT-PCR and cloned into an Escherichia coli expression vector.

Cloning, Molecular↗
PubMed2001

Protein affinity maturation in vivo using E. coli mutator cells.

This protocol provides a simple in vivo strategy for introducing random mutations to a target DNA sequences using E. coli mutator cells. The method has been used in our laboratory for affinity maturation of proteins encoded by target DNA sequences. Selection conditions can be modified for antibody fragments with increased production levels. Growth conditions in E. coli mutator cells can be adjusted to introduce a single random point mutation per kilobase of DNA, approximately equivalent to one codon change per scFv fragment.

Antibodies↗
PubMed2001

Ribosome display and affinity maturation: from antibodies to single V-domains and steps towards cancer therapeutics.

Protein affinity maturation using molecular evolution techniques to produce high-affinity binding proteins is an important step in the generation of reagents for cancer diagnosis and treatment. Currently, the most commonly used molecular evolution processes involve mutation of a single gene into complex gene repertoires followed by selection from a display library. Fd-bacteriophage are the most popular display vectors, but are limited in their capacity for library presentation, speed of processing and mutation frequency. Recently, the potential of ribosome display for directed molecular evolution was recognised and developed into a rapid and simple affinity selection strategy using ribosome complexes to display antibody fragments (scFv). Ribosome display and selection has the potential to generate and display large libraries more representative of the theoretical optima for naïve repertoires (10(14)). Even more important is the application of ribosome display for the affinity maturation of individual proteins by rapid mutation and selection cycles. These display strategies can apply to other members of the immunoglobulin superfamily; for example single V-domains which have an important application in providing specific targeting to either novel or refractory cancer markers. We discuss the application of ribosome display and selection in conjunction with variable domain (CTLA-4) libraries as the first step towards this objective and review affinity maturation strategies for in vitro ribosome display systems.

Amino Acid Sequence↗
PubMed2001

Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries.

The new antigen receptor (NAR) from nurse sharks consists of an immunoglobulin variable domain attached to five constant domains, and is hypothesised to function as an antigen-binding antibody-like molecule. To determine whether the NAR is present in other species we have isolated a number of new antigen receptor variable domains from the spotted wobbegong shark (Orectolobus maculatus) and compared their structure to that of the nurse shark protein. To determine whether these wNARs can function as antigen-binding proteins, we have used them as scaffolds for the construction of protein libraries in which the CDR3 loop was randomised, and displayed the resulting recombinant domains on the surface of fd bacteriophages. On selection against several protein antigens, the highest affinity wNAR proteins were generated against the Gingipain K protease from Porphyromonas gingivalis. One wNAR protein bound Gingipain K specifically by ELISA and BIAcore analysis and, when expressed in E. coli and purified by affinity chromatography, eluted from an FPLC column as a single peak consistent with folding into a monomeric protein. Naturally occurring nurse shark and wobbegong NAR variable domains exhibit conserved cysteine residues within the CDR1 and CDR3 loops which potentially form disulphide linkages and enhance protein stability; proteins isolated from the in vitro NAR wobbegong library showed similar selection for such paired cysteine residues. Thus, the New Antigen Receptor represents a protein scaffold with possible stability advantages over conventional antibodies when used in in vitro molecular libraries.

Adhesins, Bacterial↗
PubMed2001

A single chain Fv antibody displayed on phage surface recognises conformational group-specific epitope of bluetongue virus.

A single chain fragment variable (scFv) antibody gene was isolated from hybridoma cell line secreting monoclonal antibody (MAb) 20E9 that recognises bluetongue virus (BTV) VP7. DNA fragments encoding variable regions of heavy and light chains were amplified by RT-PCR and library of scFv was constructed in phage vector. Two scFv clones that were selected showed specific reactivity with conformational epitope VP7. The N-terminal 22 amino acid residues of 20E9 light chain were identical to that deduced from scFv DNA sequence. An in-frame TAG stop codon was found in the coding sequence and its potential role in regulating the expression and stability of scFv in phage is discussed.

Amino Acid Sequence↗
PubMed2000

The ShBle resistance determinant from Streptoalloteichus hindustanus is expressed in Haloferax volcanii and confers resistance to bleomycin.

We have designed a gene cassette for expression of the bleomycin-resistance protein from Streptoalloteichus hindustanus (ShBle) in the extremely halophilic archaeon Haloferax volcanii, and shown that transformed haloarchaea are resistant to bleomycin. Recombinant ShBle was purified by a one-step affinity-chromatography procedure as a correctly folded, dimeric protein. ShBle thus provides a useful haloarchaeal selectable marker and represents the first non-halophilic and soluble heterologous protein to be expressed in the Haloarchaea.

Antibiotics, Antineoplastic↗
PubMed2000

Immunoglobulin VH domains and beyond: design and selection of single-domain binding and targeting reagents.

Identification of the smallest antibody fragment still capable of binding to antigen has progressed from full antibody molecules to Fab and recombinant single chain Fv fragments. Now, a further reduction to single domain binding proteins based upon immunoglobulin VH and VH-like domains offers exciting prospects in the development of novel immunotherapeutics and immunodiagnostics. Minimisation of the antigen-binding fragment to such small single-domain proteins offers the advantages of enhanced stability, and possibly access to a class of antigenic epitopes not generally recognised by conventional antibodies.

Animals↗
PubMed1999

Design and expression of soluble CTLA-4 variable domain as a scaffold for the display of functional polypeptides.

We have designed and engineered the human cytotoxic T-lymphocyte associated protein-4 (CTLA-4) variable (V-like) domain to produce a human-based protein scaffold for peptide display. First, to test whether the CTLA-4 CDR-like loops were permissive to loop replacement/insertion we substituted either the CDR1 or CDR3 loop with somatostatin, a 14-residue intra-disulfide-linked neuropeptide. Upon expression as periplasmic-targeted proteins in Escherichia coli, molecules with superior solubility characteristics to the wild-type V-domain were produced. These mutations in CTLA-4 ablated binding to its natural ligands CD80 and CD86, whereas binding to a conformation-dependent anti-CTLA-4 monoclonal antibody showed that the V-domain framework remained correctly folded. Secondly, to develop a system for library selection, we displayed both wild-type and mutated CTLA-4 proteins on the surface of fd-bacteriophage as fusions with the geneIII protein. CTLA-4 displayed on phage bound specifically to immobilized CD80-Ig and CD86-Ig and in one-step panning enriched 5,000 to 2,600-fold respectively over wild-type phage. Bacteriophage displaying CTLA-4 with somatostatin in CDR3 (CTLA-4R-Som3) specifically bound somatostatin receptors on transfected CHO-K1 cells pre-incubated with 1 microg/ml tunicamycin to remove receptor glycosylation. Binding was specific, as 1 microM somatostatin successfully competed with CTLA-4R-Som3. CTLA-4R-Som3 also activated as well as binding preferentially to non-glycosylated receptor subtype Sst4. The ability to substitute CDR-like loops within CTLA-4 will enable design and construction of more complex libraries of single V-like domain binding molecules. Proteins 1999;36:217-227.

Abatacept↗
PubMed1997

Use of mutator cells as a means for increasing production levels of a recombinant antibody directed against Hepatitis B.

A mutation strategy which utilises phage display technology and the Escherichia coli mutator strains, mutD5-FIT and XL1-RED, was applied to a Hepatitis B (HepB) specific single-chain Fv (scFv) to incorporate random mutations throughout the gene. Messenger RNA from a hybridoma producing antibodies against HepB was isolated, reverse transcribed and used as template for the production of scFv. Following production of the scFv protein using an E. coli expression vector (pGC), the scFv gene was recloned into a phage display vector (pHFA). This gene construct was introduced into E. coli mutator cells and the transformed cells were used as an inoculum for liquid cultures. After five cycles of growth at 37 degrees C, each followed by dilution and re-inoculation of fresh media, recombinant phage were recovered. Nucleotide sequence analysis of the scFv gene in phage selected on HBsAg-coated magnetic beads identified amino acid substitutions which produced an increase of greater than 10-fold in apparent production levels. Competitive ELISA studies showed that the selected scFv mutants appeared to have similar affinity to HBsAg as the parent scFv. The apparent increase in production was not the result of improved surface characteristics of regions uniquely exposed in scFvs, as the sites did not correlate with the variable/constant interface of the scFv variable region normally masked in Fabs or IgGs.

Amino Acid Sequence↗
PubMed1996

Affinity maturation of recombinant antibodies using E. coli mutator cells.

BACKGROUND: Phage libraries can display repertoires of antibodies which are greater in number than the mammalian immune response. However, the selected antibodies often have low binding affinity to their target antigen or hapten (KD below 10(-6) M), which is characteristic of the primary immune repertoire. There is a need for procedures to mimic somatic hypermutation through antigen driven affinity maturation, thereby increasing the affinity of selected immunoglobulins. OBJECTIVE: To investigate the effectiveness of mutation and affinity selection of recombinant antibody genes with mutator E. coli cells, incorporating phage-display strategies. STUDY DESIGN: Unique human scFvs were selected from a naive Fd-phage library. These genes were mutated by propagation in mutD5 mutator E. coli cells (mutD5-FIT) which were competent for Fd (M13) based phagemid transfections and generated point mutations (transversions and transitions) in the scFv genes. Individual phage-displayed scFvs were affinity selected from the mutation library and were assayed as soluble scFvs by ELISA and BIAcore for binding to antigen. RESULTS: The in vivo mutation of phage-displayed scFvs in E. coli mutD5-FIT, combined with affinity selection against antigen, produced scFv molecules with improved binding activity. The point mutations which resulted in single amino acid substitutions frequently produced ten fold increases in apparent binding affinity. Structural comparisons revealed that these point mutations were in framework regions (adjacent to the CDRs) and within the CDRs. In one case the apparent affinity of an anti-glycophorin scFv after mutation in the VL framework region close to CDR3 increased by 10(3). However, this increase in apparent affinity was accompanied by an increased propensity to dimerise and form aggregates. CONCLUSIONS: A strategy for the rapid affinity maturation of scFv and Fab antibody fragments has been developed which utilises mutator strains of E. coli and incorporates phage display of antibody repertoires (libraries).

Antibody Affinity↗
PubMed1992

High-level temperature-induced synthesis of an antibody VH-domain in Escherichia coli using the PelB secretion signal.

We have constructed a temperature-inducible Escherichia coli expression vector (pPOW) for enhanced secretion of antibody (Ab) domains and other foreign proteins. The vector contains the lambda pRpL promoters in tandem, and the cI857 gene encoding the temperature-sensitive repressor which provide tight control over protein production. The PelB secretion signal directs the synthesized foreign protein through the cytoplasmic membrane. A mouse Ab fragment (the variable heavy (VH) domain of NC41) was synthesized efficiently by this vector and accumulated with the cell membranes (not as inclusion bodies) at levels of 30 mg/l. This represents the highest yields reported to date for Ab fragments with a native N terminus. An octapeptide (FLAG) tail was fused to the C terminus of the VH domain to aid in purification, and remained intact throughout the protein purification process. The optimum conditions for protein production were controlled by the type of culture medium used, the age of the bacterial population at the time of induction, and the period of synthesis of the protein product. The purified Ab VH fragment showed binding affinity (Ka less than 10(4)/M) to its target antigen (neuraminidase).

Animals↗
PubMed1991

Evaluation of renal function and fluid homeostasis during recovery from exercise-induced hyponatremia.

Renal function including fluid and electrolyte balance was studied during recovery in eight subjects who developed symptomatic hyponatremia (HN; plasma sodium concentration less than 130 mM) during an 88-km ultramarathon footrace and compared with results for normonatremic runners [NN; n = 18, mean postrace plasma sodium concentration, 138.2 +/- 1.2 (SE) mM]. Estimated fluid intake during the race for HN was 12.5 +/- 1.6 (SE) liters over 9 h 41 min (+/- 28 min). HN excreted a net fluid excess of 2.95 +/- 0.56 (range 1.2-5.9) liters compared with a fluid deficit of 2.7 +/- 0.3% body weight in NN. The sodium deficit was 153 +/- 35 mmol in HN and 187 +/- 37 mmol in NN. Despite the fluid overload, plasma volume was decreased by 24.1 +/- 5.0% in HN compared with 8.2 +/- 2.6% in NN. Serum renin activity (5.1 +/- 2.0 ng.ml-1.h-1), aldosterone concentrations (410 +/- 34 ng/l), creatinine clearances (174.8 +/- 28.2 ml/min), and urine output (6.4 +/- 1.0 ml/min) were markedly elevated in HN during recovery. Thus the hyponatremia of exercise results from fluid retention in subjects who ingest abnormally large fluid volumes during prolonged exercise.

Adult↗
PubMed1990

Expression and characterization of infectious bursal disease virus polyprotein in yeast.

Various expression vectors containing a cDNA fragment encoding all but the first five amino acids (aa) of the large polyprotein (N-VP2-VP4-VP3-C) of infectious bursal disease virus were transformed into yeasts. In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, co- or post-translational processing of the unfused large polyprotein occurred, generating a stable C-terminal product (VP3) or correct size, but without any detectable N-terminal product (VP2). Furthermore, when the processing of the polyprotein was interrupted, because of an engineered in-frame site-specific insertion of 4 aa, even VP3 (as part of the unprocessed polyprotein) was undetected. VP2 was detected in S. cerevisiae only when fused to yeast pre-sequences at the N terminus, suggesting that in yeast, VP2 or the unprocessed polyprotein, in the absence of its native N terminus or proper protection of its N-terminal aa residues is susceptible to proteolytic degradation. The first 8 aa of a modified pre-sequence of the CUP1 gene product and the pre-pro sequence of MF alpha 1 gene product have been used for stable intra- and extra-cellular production of VP2, respectively.

Amino Acid Sequence↗
PubMed1990

Plasma volume and renal function during and after ultramarathon running.

Plasma volume (PV) and renal function were studied in eight subjects for 3 d prior to and 6 d after a 56 km footrace. Immediately following the race, PV, creatinine clearance, and urine flow were unchanged from pre-race values. Over the subsequent 3 d, PV increased due initially to a 17 g influx of serum albumin and an associated increase in plasma sodium content, which persisted throughout the study period. A reduction in urine sodium secretion occurred during the race day. Creatinine clearance increased after the race and remained elevated for 48 h. Increases serum enzyme activities, C-reactive protein concentration, serum uric acid content, and plasma creatinine concentration and production suggest muscle damage. We suggested the following. First, the persistent post-exercise plasma volume expansion is initiated by an influx of albumin into the intravascular space with an associated increase in plasma sodium content. A decrease in urine sodium excretion during the race day would contribute to the latter. Second, the interpretation of post-race changes in serum constituents must take account of changes in plasma volume. Third, there is an increase in creatinine clearance, indicating an increase in glomerular filtration rate, after both standard and ultramarathon running. This may be caused by the products of muscle cell damage although the physiologic mechanism for this is unclear.

Adult↗
PubMed1990

Transient oliguria with renal tubular dysfunction after a 90 km running race.

In the course of a 19-d study of renal function in five ultramarathon runners, before, during and after a 90 km race, one runner developed transient oliguria with renal tubular dysfunction and anuria during and immediately after the race. Other features of the renal failure were an 84-fold increase in urine beta 2-microglobulin excretion (from 0.19 to 16.0 micrograms.min-1) and a much smaller increase in urine total protein excretion (from 0.07 to 0.18 mg.min-1) during the post-race period. Post-race creatinine clearance remained below pre-race levels throughout the study, varying between 42.8 and 72.9 ml.min-1, in contrast to the post-race 49% increase in the remaining runners (from 138.1 +/- 12.9 to 205.5 +/- 59.9 ml.min-1). Osmolal clearance also remained low (0.31 to 0.98 ml.min-1) compared with the pre-race values (1.46 +/- 0.02 ml.min-1), as did the urine flow rates (0.11 to 0.18 ml.min-1) compared with the pre-race values (0.34 +/- 0.02 ml.min-1). This renal dysfunction persisted despite the patient receiving 2 l of intravenous fluids immediately after the race and probably resulted from fluid restriction during the race. There was full recovery of renal function 1 yr later when the subject again ran the Comrades Marathon.

Adult↗
PubMed1989

Metabolic and renal changes in two athletes during a world 24 hour relay record performance.

Metabolic parameters and renal function were studied in two subjects before, during and after they established a world two-man 24 hour relay record. During the race, the athletes expended an estimated 37.747 and 42.880 kJ running at 54 and 61 per cent of maximum oxygen consumption (VO2max). Rectal temperatures reached maxima of 38.6 and 39.2 degrees C respectively during the race. Serum free fatty acid levels peaked at 2108 and 1875 mumol ml-1 after 24 hours; blood glucose levels varied from 4.3-6.5 and 4.9-8.5 mmol.l-1 respectively. Plasma insulin levels fell from 42.9 and 22.7 microU.ml-1 to 11.5 microU.ml-1. Plasma urea, creatinine, beta 2-microglobulin and C-reactive protein concentrations were elevated at the end of the race (to 9.0 and 8.0 mmol.l-1, 119 and 102 mumol.l-1, 3.508 and 3203 micrograms.l-1 and 2.7 and 3.9 mg per cent respectively). Plasma osmolality was altered from 293 and 304 to 302 and 280 mosmol.Kg-1 during the race but increased to 312 and 318 mosmol.Kg-1 the following day probably due to intercompartmental fluid shifts. Plasma creatinine concentration was increased by 38 and 26 per cent due to reduced urinary excretion. Urine flow rate increased 40 and 123 per cent respectively during the race, but creatinine clearance decreased by 38 and 40 per cent. Urine osmolality decreased by 38 and 65 per cent and osmolal clearance decreased by 15 and 16 per cent respectively. Urine sodium excretion was greatly reduced (85 and 90 per cent) on the post-race days (by 88 and 92 per cent on day 2). Both urine total protein and beta2-microglobulin excretion increased during the race (by 89 and 35 per cent and by 334 and 136 per cent respectively), but owing to the increased beta2-microglobulin production renal clearance was unaltered. The changes in renal function were temporary and some aspects of renal tubular function were enhanced during the post-race days. We conclude that, although C-reactive protein concentrations increased sooner and were higher than other shorter events and although creatinine, urine excretion and urine osmolality decreased markedly, the intermittent nature of the event, the mild environmental conditions, the moderate percentage of VO2 max maintained by the well conditioned subjects and a high fluid intake enabled a rapid return to normality and indeed to enhanced renal tubular function. The only moderate increases in body temperature would be due to the same factors.

Adult↗
PubMed1988

Crystalluria in marathon runners. II. Ultra-marathon--males and females.

Particle-volume size distribution curves for the urines of a group of male ultra-marathon runners have been recorded and show the same features as those reported in an earlier study involving standard marathon runners. It is again suggested that these features indicate risk of urinary stone formation although this does not appear to be increased by the more stressful ultra-marathon. Distribution curves obtained for female runners closely resemble those recorded for controls, thus rendering it impossible to assess their risk profile. A commonly observed feature of both the male and female urines was the presence of various urinary salts. Profuse calcium oxalate dihydrate crystals were detected in some samples 11 days after the race. It is suggested that entrapment sites within the urinary tract have a selective specificity for these crystals while urinary salts pass through unhindered.

Adult↗