A method for global protein expression and antibody screening on high- density filters of an arrayed cDNA library

Büssow, Konrad; Cahill, Dolores J.; Nietfeld, Wilfried; Bancroft, David; Scherzinger, Eberhard; Lehrach, Hans; Walter, Gerald

doi:10.1093/nar/26.21.5007

Cited by 275 publications

(210 citation statements)

References 0 publications

Supporting

Mentioning

206

Contrasting

Unclassified

Order By: Relevance

“…As the control clone, we used a cDNA expression clone from a human fetal brain library [23] expressing protein Drebrin F (Swiss-Prot accession: Q9UJU6) as an N-terminal fusion protein with an RGS-His 6 tag.…”

Section: Control Clonementioning

confidence: 99%

Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

Kersten

Possling

Blaesing

et al. 2004

Analytical Biochemistry

View full text Add to dashboard Cite

To gain insights into complex biological processes, such as transcription and replication, the analysis of protein-DNA interactions and the determination of their sequence requirements are of central importance. In this study, we probed protein microarray technology and ultraviolet crosslinking combined with mass spectrometry (MS) for their practicability to study protein-DNA interactions. We chose as a model system the well-characterized interaction of bacterial replication initiator DnaA with its cognate binding site, the DnaA box. Interactions of DnaA domain 4 with a high-aYnity DnaA box (R4) and with a low-aYnity DnaA box (R3) were compared. A mutant DnaA domain 4, A440V, was included in the study. DnaA domain 4, wt, spotted onto FAST slides, revealed a strong signal only with a Cy5-labeled, double-stranded, 21-mer oligonucleotide containing DnaA box R4. No signals were obtained when applying the mutant protein. Ultraviolet crosslinking combined with nanoLC/MALDI-TOF MS located the site of interaction to a peptide spanning amino acids 433-442 of Escherichia coli DnaA. This fragment contains six residues that were identiWed as being involved in DNA binding by recently published crystal structure and nuclear magnetic resonance (NMR) analysis. In the future, the technologies applied in this study will become important tools for studying protein-DNA interactions.  2004 Elsevier Inc. All rights reserved.

show abstract

Section: Control Clonementioning

confidence: 99%

Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

Kersten

Possling

Blaesing

et al. 2004

Analytical Biochemistry

View full text Add to dashboard Cite

show abstract

“…Our laboratory has developed protein array based technologies and methodologies since first developing the hEx1 protein expression library [18] with which we have performed studies on the binding of autoantibodies to arrayed proteins in Alopecia areata and Dilated Cardiomyopathy [19,20] , binding of antibodies to proteins identified from tumour neovasculature in humans [21],context independent motif identification in the human proteome [22] and of identification of novel proteinprotein interaction networks [23,24]. We have employed this library screening method as part of an ovarian cancer research consortium, Discovary, performing a pilot study on autoantibody identification screening the hEx1 protein library with ovarian cancer serum samples from a well characterised patient cohort with stage I ovarian cancer of mixed histology, stage III serous papillary adenocarcinoma, primary peritoneal carcinoma and normal/healthy individuals.…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Epitope presentation is an important determinant of the utility of antigens identified from protein arrays in the development of autoantibody diagnostic assays

Murphy

O’Connell

O’Kane

et al. 2012

Journal of Proteomics

View full text Add to dashboard Cite

* These authors contributed equally.Autoantibodies represent an attractive biomarker for diagnostic assays principally due to the stability of immunoglobulin in patient serum facilitating measurement with conventional assays. Immune responses to tumorigenesis may facilitate detection of ovarian cancer in the early stages of the disease with identification of a panel of tumour specific autoantibodies. Despite the reporting of many tumour associated autoantibodies using arrays of tumour antigens, this has not led to the advance in diagnostic capability as rapidly as was initially expected. Here we examine the potential diagnostic utility of candidate autoantibody biomarkers identified via screening of serum samples on a high content human protein array from a unique cohort of early stage and late stage ovarian cancer patients. We analyse the performance of autoantibodies to the tumour suppressor protein p53 and the novel autoantigens alpha adducin and endosulfine alpha identified in our array screen. Each antigen has different performance characteristics using conventional ELISA format and Western blot immunoassay. The high attrition rate of promising autoantigens identified by array screening can in part be explained by the presentation of the epitope of the antigen in the subsequent method of validation and this study provides directions on maximising the potential of candidate biomarkers. The most commonly used biomarker for ovarian cancer is Cancer Antigen 125 (CA125) or Mucin16 which is thought to provide a protective, lubricating barrier against particles and infectious agents at mucosal surfaces [9]. Elevated levels of this antigen are detected in over 90% of sera of disseminated ovarian cancer cases but only in 50% of patients in the early stages of the disease [10]. CA 125 screening is used with the addition of ultrasound screening. Although these combined approaches A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Materials and Methods Serum Sample DetailsSerum samples and clinical information was obtained with informed consent and study approval was obtained from St. James's Hospital and Adelaide and Meath incorporating the National Childerns Hospital research ethics committee.Pre-operative bloods were obtained from patients undergoing cytoreductive surgery for possible ovarian neoplasm. Blood was collected into a non-heparinised tube and allowed to clot, then centrifuged at 400 x g for 15 min. The serum supernatant was removed and dispensed into labelled cryovial tubes. Serum was stored at -80 o C until further use. hEx1 Serum Screening of Ovarian Cancer SamplesThe hEx1 array PVDF membranes (each hEx1 array is made up of 2 PVDF membranes, pt 8 and pt 9) were activated in ethanol for 1 min, rinsed in deionised water and washed in tris-buffered saline-T-T (500mM NaCl, 10mM Tris-HCl pH 7.5, 0.05% v/v Tween, 0.5% v/v Triton X ). Dessicated bacterial colonies were removed with tissue. The arrays washed in TBS-T-T for 10 min, TBS-T (500mM NaCl, 10mMTris-HCl pH 7.5, 0.05% v/v Tween) for 10 min and TB...

show abstract

“…Further, the positive effect of solubility tags was confirmed, with Thioredoxin (Trx) and maltose binding protein (MBP) leading the list (Dyson et al, 2004). By allowing denaturation, protein expression in E. coli is more efficient and, for example, expression of¨50,000 individual clones of human proteins in parallel was achieved based on clone filters (Bü ssow et al, 1998). This arrayed cDNA expression library has already been used as a resource of proteins for the in vitro generation of recombinant antibodies (Walter et al, 2001).…”

Section: Recombinant Protein Expression From Cdna Librariesmentioning

confidence: 99%

Perspectives for systematic in vitro antibody generation

Konthur

Hust

Dübel

2005

Gene

View full text Add to dashboard Cite

After the completion and refinement of the human genome, the characterization of individual gene products in respect of their functions, their modifications, their cellular localization and regulation in both space and time has generated an increased demand for antibodies for their analysis. Taking into account that the human genome contains¨25,000 genes, and that their products are found in different splice variants and produce proteins with post-translational modifications, it can be estimated that at least 100,000 different protein products have to be investigated to gain a complete picture of what's going on in the proteome of a cell.Antibodies are preferred tools helping with the characterization and detection of proteins as well as with elucidating their individual functions. The generation of antibodies to all available human protein products by immunization and/or the hybridoma technology is not only logistically and financially enduring, but may prove to be a difficult task, as quite a number of interesting targets may evade the immune response of experimental animals, for example, allosteric variants dependent on fragile interactions to cofactors, highly conserved antigens etc. For this reason, alternative methods for the generation of antibodies have to supplement these approaches. In vitro methods for antibody generation are seen to offer this capability. In addition, they may provide a cost effective and large scale production alternative for detection reagents for the research community in their own right. Among in vitro techniques, phage display has been evolved as the most efficient option for tackling this problem and approaches optimised for automation are emerging.Maximum benefit for proteomic research could be generated by judicious and preferably international coordination of the ongoing efforts to combine the strengths of the well established animal based approaches and the novel opportunities offered by in vitro methods. D

show abstract

A method for global protein expression and antibody screening on high- density filters of an arrayed cDNA library

Cited by 275 publications

References 0 publications

Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

Protein microarray technology and ultraviolet crosslinking combined with mass spectrometry for the analysis of protein–DNA interactions

Epitope presentation is an important determinant of the utility of antigens identified from protein arrays in the development of autoantibody diagnostic assays

Perspectives for systematic in vitro antibody generation

Contact Info

Product

Resources

About