David O’Connell

University College Dublin

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Simplifying G Protein-Coupled Receptor Isolation with a Calcium-Dependent Fragment Complementation Affinity System

Biochemistry (2018)

Niamh Ní Mhurchú Lioudmila Zoubak Gavin McGauran Sara Linse Alexei Yeliseev

The process of isolating recombinant G protein-coupled receptors from membrane preparations is challenging because the process requires solubilization in detergent micelles and multistep affinity chromatography protocols. Solubilization buffers contain high concentrations of salts, detergents, and glycerol that create stringent conditions necessary to stabilize the receptor but in which affinity chromatography resins perform poorly, and these resins also require the addition of eluting agents that complicate downstream assays. To simplify this process we have developed a high affinity fragment complementation molecular switch as a highly specific system for receptor capture in solubilization buffer with a calcium chelation-based elution step releasing functional protein in a simple buffer. Here we describe in detail the design, methodology, interpretation, and limitations of this novel affinity chromatography system in the isolation and purification of the cannabinoid G protein-coupled receptor CB2, in comparison with commercially available systems. This powerful tool may be applied to any recombinant membrane bound protein and can be further optimized to enhance the yield and purity of the most challenging protein targets for study.

Protein purification

10.1021/acs.biochem.8b00469

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Citations (9)

Probing pathogen proliferation

Nature Reviews Microbiology (2006)

David O’Connell

10.1038/nrmicro1567

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Citations (0)

Spoilt for choice

Nature Reviews Microbiology (2004)

David O’Connell

Functional Genomics

10.1038/nrmicro805

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Biofilms to die for...

Nature Reviews Microbiology (2007)

David O’Connell

10.1038/nrmicro1684

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Base-ics of trypanosome immune evasion

Nature Reviews Microbiology (2005)

David O’Connell

Evasion (ethics)

Base (topology)

10.1038/nrmicro1154

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The Elegant Universe

Ploughshares (2022)

David O’Connell

The Winter 2022-23 Issue. Ploughshares is an award-winning journal of new writing. Since 1971, Ploughshares has discovered and cultivated the freshest voices in contemporary American literature, and now provides readers with thoughtful and entertaining literature in a variety of formats. Find out why the New York Times named Ploughshares "the Triton among minnows."

10.1353/plo.2022.0153

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Identification of Pathogenic Pathways for Recurrence of Focal Segmental Glomerulosclerosis after Kidney Transplantation

Diagnostics (2024)

Sahra Pajenda Daniela Gerges Ludwig Wagner David O’Connell Monika Aiad

Primary focal segmental glomerulosclerosis (FSGS) is a disease of the podocytes and glomerulus, leading to nephrotic syndrome and progressive loss of renal function. One of the most serious aspects is its recurrence of disease in over 30% of patients following allogeneic kidney transplantation, leading to early graft loss. This research investigates the individual genetic predispositions and differences in the immune responses leading to recurrence of FSGS after transplantation. We performed exome sequencing on six patients with recurrent FSGS to identify variants in fifty-one genes and found significant variations in the alpha-2-macroglobulin (A2M). Immunoblotting was used to investigate effects of specific gene variants at the protein level. Further expression analysis identified A2M, exophilin 5 (EXPH5) and plectin (PLEC) as specific proteins linked to podocytes, endothelial cells, and the glomerulus. Subsequent protein array screening revealed the presence of non-HLA-specific antibodies, including TRIM21, after transplantation. Using Metascape for pathway and process enrichment analysis, we focused on the IL-17 signaling and chemotaxis pathways. ELISA measurements showed significantly elevated IL-17 levels in patients with recurrent FSGS (32.30 ± 9.12 pg/mL) compared to individuals with other glomerular diseases (23.16 ± 2.49 pg/mL;

Glomerulus

10.3390/diagnostics14151591

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Citations (1)

Pump action

Nature Reviews Microbiology (2005)

David O’Connell

10.1038/nrmicro1190

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Changes induced by local injection of human lymphoid cell lymphokine into dermal metastases of breast carcinoma: A light and electron microscopical study

The Journal of Pathology (1982)

F.J. Paradinas Barbara M. Southcorr David O’Connell Franciscus den Hollander A.H.W.M. Schuurs

Lymphokines (LCL-LK) prepared from the human lymphoid cell line RPMI 1788 were injected into dermal nodular metastases of three patients with advanced breast carcinoma anergic to recall antigen (tuberculin). Three different injection schedules were employed. Ten such nodules were examined by excision biopsy and their histological appearances were compared with biopsies of four further nodules not injected with the lymphokine. Intranodular injection of LCL-LK resulted in clinical regression of tumour and histological evidence of tumour-cell necrosis with pleomorphic leucocytic infiltration by polymorphs, macrophages and lymphocytes. Electron microscopy of two such nodules failed to show close cell contact between leucocytes and tumour cells prior to tumour-cell necrosis. This study extends evidence that injection of inflammatory lymphokines into accessible tumour can result in local tumour regression and it suggests that such tumour-cell destruction may be the result of a variety of factors operating during local inflammatory response.

Breast carcinoma

Infiltration (HVAC)

10.1002/path.1711380403

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Citations (8)

Triggering GAS invasive disease

Nature Reviews Microbiology (2006)

David O’Connell

10.1038/nrmicro1504

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Citations (3)