ORCID Profile
0000-0002-5504-0929
Current Organisations
Karachi Institute of Economics and Technology
,
Limeburners Bay International AG
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Publisher: American Chemical Society (ACS)
Date: 11-01-2008
DOI: 10.1021/AC701688Q
Abstract: We demonstrate the use of surface-immobilized, oriented peptide aptamers for the detection of specific target proteins from complex biological solutions. These peptide aptamers are target-specific peptides expressed within a protein scaffold engineered from the human protease inhibitor stefin A. The scaffold provides stability to the inserted peptides and increases their binding affinity owing to the resulting three-dimensional constraints. A unique cysteine residue was introduced into the protein scaffold to allow orientation-specific surface immobilization of the peptide aptamer and to ensure exposure of the binding site to the target solution. Using dual-polarization interferometry, we demonstrate a strong relationship between binding affinity and aptamer orientation and determine the affinity constant KD for the interaction between an oriented peptide aptamer ST(cys+)_(pep9) and the target protein CDK2. Further, we demonstrate the high selectivity of the peptide aptamer STM_(pep9) by exposing surface-immobilized ST(cys+)_(pep9) to a complex biological solution containing small concentrations of the target protein CDK2.
Publisher: American Chemical Society (ACS)
Date: 03-01-2007
DOI: 10.1021/AC061863Z
Abstract: Protein microarray development is absolutely dependent upon the ability to construct interfaces capable of specific, stable, sensitive, and designable recognition of specific proteins. Peptide aptamers, being peptide recognition moieties presented and constrained by a robust scaffold protein, offer one possible solution. The relative uniformity of a scaffold protein across potentially many thousands of arrayed peptide aptamers is predicted to simplify the production of microarrays. This paper describes the generation and assaying characteristics of a scaffold protein adlayer. Orientational control of the scaffold protein STM, a triply mutated form of the stable intracellular protein inhibitor stefin A is achieved with a surface cysteine residue, which leads to the presentation of the scaffold recognition surface to solution. Operational stability of the system is excellent, with only a minor decrease in detection sensitivity over time (less than 1% h-1). We use this system to establish a surface plasmon resonance assay offering a limit of detection of 1 nM (150 ng mL-1) and determine the affinity constant of interaction of STM for a cognate antibody to be KD = 1.47 +/- 0.23 nM. Thus, we have established a solid foundation for the future creation of highly multiplexed peptide aptamer microarrays that will be compatible with a broad range of label-free detection technologies.
Publisher: Informa Healthcare
Date: 26-09-2007
DOI: 10.1517/17460441.2.10.1389
Abstract: Chemical genomics is a new and rapidly developing field. It refers to the use of cell-permeable small molecules, which are highly specific for their protein targets, in order to dissect biological pathways and to discover new drug leads. Small-molecule screening is usually limited to high-throughput approaches that use defined cell lines however, whole organism screening is gaining increasing attention. This review addresses the latter concept and highlights the advances in whole organism-based screening, with an emphasis on the use of the zebrafish (Danio rerio).
Publisher: Springer Science and Business Media LLC
Date: 31-07-2003
DOI: 10.1038/NBT852
Publisher: Elsevier BV
Date: 10-2008
DOI: 10.1016/J.BIOS.2008.03.036
Abstract: We report the investigation of label-free protein detection directly from lysed cells using microcantilever sensors. The integration of an internally referenced microcantilever sensor combined with peptide aptamer technology enables scalable and label-free detection of proteins from a complex biological environment (e.g. cell lysate). The internally referenced microcantilever sensor was found to be effective in minimizing both the effects of thermal drift and non-specific binding interactions with the backside of the cantilever, thereby allowing protein detection in a complex biological background. Highly specific peptide aptamers are used to modify the cantilever surface to specifically detect less than 80 nM CDK2 protein from yeast cell lysate. This binding of CDK2 on the microcantilever generates a tensile surface stress of average magnitude equal to 70+/-22 mN/m. Similar experiments conducted with quartz crystal microbalance (QCM) technology are consistent with the response observed using microcantilever sensors.
Publisher: Elsevier BV
Date: 09-2008
Publisher: American Chemical Society (ACS)
Date: 31-01-2014
DOI: 10.1021/LA403983B
Publisher: IEEE
Date: 12-2015
Publisher: Springer Science and Business Media LLC
Date: 2008
DOI: 10.1186/JBIOL62
Publisher: Elsevier BV
Date: 03-2011
Abstract: Protein microarrays represent an emerging technology that promises to facilitate high-throughput proteomics. The major goal of this technology is to employ peptides, full-length proteins, antibodies, and small molecules to simultaneously screen thousands of targets for potential protein-protein interactions or modifications of the proteome. This article describes the performance of a set of peptide aptamers specific for the human papillomavirus (HPV) type 16 oncoproteins E6 and E7 in a microarray format. E6 and E7 peptide aptamer microarrays were probed with fluorescence-labeled lysates generated from HPV-infected cervical keratinocytes expressing both E6 and E7 oncoproteins. Peptide aptamer microarrays are shown to detect low levels of E6 and E7 proteins. Peptide aptamers specific for cellular proteins included on these microarrays suggested that expression of CDK2, CDK4, and BCL-6 may be affected by HPV infection and genome integration. We conclude that peptide aptamer microarrays represent a promising tool for proteomics and may be of value in biological and clinical investigations of cervical carcinogenesis.
Publisher: Elsevier BV
Date: 10-2005
DOI: 10.1016/J.JMB.2005.08.001
Abstract: Peptide aptamers are peptides constrained and presented by a scaffold protein that are used to study protein function in cells. They are able to disrupt protein-protein interactions and to constitute recognition modules that allow the creation of a molecular toolkit for the intracellular analysis of protein function. The success of peptide aptamer technology is critically dependent on the performance of the scaffold. Here, we describe a rational approach to the design of a new peptide aptamer scaffold. We outline the qualities that an ideal scaffold would need to possess to be broadly useful for in vitro and in vivo studies and apply these criteria to the design of a new scaffold, called STM. Starting from the small, stable intracellular protease inhibitor stefin A, we have engineered a biologically neutral scaffold that retains the stable conformation of the parent protein. We show that STM is able to present peptides that bind to targets of interest, both in the context of known interactors and in library screens. Molecular tools based on our scaffold are likely to be used in a wide range of studies of biological pathways, and in the validation of drug targets.
Location: United Kingdom of Great Britain and Northern Ireland
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for Sophie Laurenson.