Biomedical subjects

Eugenie Hainsworth

Publications and source records attributed to Eugenie Hainsworth.

4 recordsLinked to original sources

PubMed2006

On-chip protein synthesis for making microarrays.

Protein microarrays are a miniaturized format for displaying in close spatial density hundreds or thousands of purified proteins that provide a powerful platform for the high-throughput assay of protein function. The traditional method of producing them requires the high-throughput production and printing of proteins, a laborious method that raises concerns about the stability of the proteins and the shelf life of the arrays. A novel method of producing protein microarrays, called nucleic acid programmable protein array (NAPPA), overcomes these limitations by synthesizing proteins in situ. NAPPA entails spotting plasmid DNA encoding the relevant proteins, which are then simultaneously transcribed and translated by a cell-free system. The expressed proteins are captured and oriented at the site of expression by a capture reagent that targets a fusion protein on either the N- or C-terminus of the protein. Using a mammalian extract, NAPPA expresses and captures 1000-fold more protein per feature than conventional protein-printing arrays. Moreover, this approach minimizes concerns about protein stability and integrity, because proteins are produced just in time for assaying. NAPPA has already proven to be a robust tool for protein functional assays.

Animals↗

PubMed2005

Emerging tools for real-time label-free detection of interactions on functional protein microarrays.

The availability of extensive genomic information and content has spawned an era of high-throughput screening that is generating large sets of functional genomic data. In particular, the need to understand the biochemical wiring within a cell has introduced novel approaches to map the intricate networks of biological interactions arising from the interactions of proteins. The current technologies for assaying protein interactions--yeast two-hybrid and immunoprecipitation with mass spectrometric detection--have met with considerable success. However, the parallel use of these approaches has identified only a small fraction of physiologically relevant interactions among proteins, neglecting all nonprotein interactions, such as with metabolites, lipids, DNA and small molecules. This highlights the need for further development of proteome scale technologies that enable the study of protein function. Here we discuss recent advances in high-throughput technologies for displaying proteins on functional protein microarrays and the real-time label-free detection of interactions using probes of the local index of refraction, carbon nanotubes and nanowires, or microelectromechanical systems cantilevers. The combination of these technologies will facilitate the large-scale study of protein interactions with proteins as well as with other biomolecules.

Animals↗

PubMed2004

Self-assembling protein microarrays.

Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.

Cell Cycle Proteins↗

PubMed2004

The Pseudomonas aeruginosa PA01 gene collection.

Pseudomonas aeruginosa, a common inhabitant of soil and water, is an opportunistic pathogen of growing clinical relevance. Its genome, one of the largest among bacteria [5570 open reading frames (ORFs)] approaches that of simple eukaryotes. We have constructed a comprehensive gene collection for this organism utilizing the annotated genome of P. aeruginosa PA01 and a highly automated and laboratory information management system (LIMS)-supported production line. All the individual ORFs have been successfully PCR-amplified and cloned into a recombination-based cloning system. We have isolated and archived four independent isolates of each individual ORF. Full sequence analysis of the first isolate for one-third of the ORFs in the collection has been completed. We used two sets of genes from this repository for high-throughput expression and purification of recombinant proteins in different systems. The purified proteins have been used to set up biochemical and immunological assays directed towards characterization of histidine kinases and identification of bacterial proteins involved in the immune response of cystic fibrosis patients. This gene repository provides a powerful tool for proteome- and genome-scale research of this organism, and the strategies adopted to generate this repository serve as a model for building clone sets for other bacteria.

Bacterial Proteins↗