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S Roberts

Publications and source records attributed to S Roberts.

396 records · Page 22Linked to original sources
PubMedDate not supplied

Biological activity and in vivo clearance of antitumor antibody/cytokine fusion proteins.

Several human cytokines including IL-2, GM-CSF, and tumor necrosis factors alpha and beta were engineered as fusion proteins to the carboxyl terminus of a chimeric anti-ganglioside antibody, ch14.18, and expressed in transfected hybridoma cells. All of the fusion proteins were expressed at high levels and were easily purified by affinity or ion-exchange chromatography from culture supernatants. The effect of fusion on antigen binding activity was tested and found to vary with the particular cytokine. No significant decreases in antigen binding were observed, and fusion of IL-2 had the greatest positive effect in a direct antigen binding assay. All fusion proteins maintained normal levels of biological activity except for GM-CSF, which was approximately 20% active, compared to recombinant GM-CSF produced in bacteria. The clearance of the fusion proteins was examined in normal Balb/c mice after intraperitoneal injection or in athymic (nu/nu) mice after intravenous injection and was generally quite rapid, relative to ch14.18. This was mainly due to a very rapid initial clearance rate (alpha phase) since the half-lives of the beta phase of the fusion proteins (about 30 h) were comparable to that of the free antibody (about 58 h). These results demonstrate that biologically active antibody/cytokine fusion proteins can be constructed by genetic engineering. Their relatively rapid clearance may require constant infusion rather than bolus injection in order to achieve clinical efficacy.

Animals↗
PubMedDate not supplied

Ubiquitin fusion technology: bioprocessing of peptides.

Ubiquitin fusion technology represents an emerging method for economically producing peptides and small proteins in the bacterium Escherichia coli. Our focus is on peptide production where the need for cost-effective, scaleable processes has recently been highlighted by Kelley (1996). There are two principal features: (1) the expression system consists of a suitable E. coli host strain paired with a plasmid that encodes the ubiquitin fusion and (2) an ubiquitin-specific protease, UCH-L3, which cleaves only C-terminal extensions from ubiquitin. In this work, multigram yields were obtained of four ubiquitin fusions derived from cell paste generated in single 10-L fermentations. All were expressed intracellularly and remained soluble at extremely high levels of expression. Bacterial freeze--thaw lysates contained over 95% pure ubiquitin fusion protein. All four fusions were efficiently cleaved to ubiquitin and the peptide products. In one case, the final yield of peptide was 1.08 g from 3 L of low cell density bacterial culture. The combination of exceptional overexpression of the ubiquitin--peptide fusion proteins and a robust and specific protease are unique advantages contributing to a cost-effective, scaleable, and generic bioprocess for peptide production.

Escherichia coli↗
PubMedDate not supplied

Generation of an antibody with enhanced affinity and specificity for its antigen by protein engineering.

A detailed description of the interactions between an antibody and its epitope is necessary to allow an understanding of the way in which antibodies bind to antigenic surfaces presented by foreign molecules. Ideally this should be done by analysis of crystal structures of antibody-antigen complexes, but so far only two of these are available. An alternative strategy combines molecular modelling with site-directed mutagenesis (SDM) and using this we have generated a preliminary model of the complex between Gloop2, an antibody raised against a peptide containing the 'loop' determinant of hen egg-white lysozyme (HEL) which also binds the native protein, and its epitope on the protein surface. The main predictions from our model were; (1) that the surface of interaction between the antibody and the antigen is large (20 A X 15 A) and involves all the complementarity-determining regions (CDRs), (2) that electrostatic interactions were important in the formation of the complex, and (3) that conformational changes in either the loop or in the CDRs may occur during the formation of the complex. Here we report SDM studies which test some of these predictions; removal of two charged residues at the periphery of the combining site increases the affinity of the antibody for its antigen over 8-fold and decreases its ability to cross-react with closely-related antigens. This result is at variance with our original prediction but can be accommodated within our newly refined model; the role of electrostatics in antigen-antibody interactions is now questionable.

Animals↗
PubMedDate not supplied

The cloning and expression of an anti-peptide antibody: a system for rapid analysis of the binding properties of engineered antibodies.

The cloning of the light and heavy chain genes of an antilysozyme monoclonal antibody is described and the application of site-directed mutagenesis for reconstruction of complete cDNAs from incomplete cDNA clones is discussed. The behaviour of the RNA transcripts produced from these cDNAs after their subcloning into an appropriate Salmonella typhimurium, SP6, vector and subsequent injection into Xenopus oocytes has been analysed. The antibody secreted by oocytes after RNA injection has been quantitated and its antigen-binding properties compared with wild-type hybridoma antibody. The results show that this route for expression of antibodies, though limited in its capacity to produce more than a few micrograms of protein, is potentially a means for rapid evaluation of the antigen-binding properties of engineered antibodies.

Animals↗
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Sustaining care delivery--team nursing with intensive care assistants.

The Cardiothoracic Centre Liverpool faced an acute nursing recruitment problem, which is accentuated in the specialist area of intensive care. To ensure that activity through the 20-bedded unit remains broadly in line with contracted levels, a number of initiatives were implemented to help maximise the financial and non-financial resources available.

Critical Care↗
PubMedDate not supplied

Administrative issues related to addiction in the workplace.

Drug abuse is an important problem in the workplace. Seventy percent of current illegal drug users are employed, and approximately 7% of Americans employed in full-time work report heavy drinking. Drug-using employees are twice as likely to request time off, and 3.6 times more likely to be involved in a workplace accident. Individuals who use alcohol or other drugs in the workplace annually cost American business 81 billion dollars in lost productivity; 86% of these costs are attributed to drinking. Although alcohol is the primary source of problems for the workplace, other drugs such as amphetamines, cocaine, marijuana, and tobacco are also troublesome. Employee assistance programs and drug treatment hold great promise for coping with these problematic substances.

Employee Discipline↗
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Why are we waiting?

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Consumer Behavior↗
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DisplayWrite 2.

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Office Automation↗
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The transition from hospital to long-term institutional care.

The focus of this article is user and carer involvement in the transition of older people from acute hospital to continuing institutional long-term care. The policy context and research of relevant studies are reviewed. Findings from an action research project currently in progress further elucidate the concerns of older users and carers.

Aged↗