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Patrick S Daugherty

Publications and source records attributed to Patrick S Daugherty.

14 recordsLinked to original sources
PubMed2006

Intracellular protein interaction mapping with FRET hybrids.

A quantitative methodology was developed to identify protein interactions in a broad range of cell types by using FRET between fluorescent proteins. Genetic fusions of a target receptor to a FRET acceptor and a large library of candidate peptide ligands to a FRET donor enabled high-throughput optical screening for optimal interaction partners in the cytoplasm of Escherichia coli. Flow cytometric screening identified a panel of peptide ligands capable of recognizing the target receptors in the intracellular environment. For both SH3 and PDZ domain-type target receptors, physiologically meaningful consensus sequences were apparent among the isolated ligands. The relative dissociation constants of interacting partners could be measured directly by using a dilution series of cell lysates containing FRET hybrids, providing a previously undescribed high-throughput approach to rank the affinity of many interaction partners. FRET hybrid interaction screening provides a powerful tool to discover protein ligands in the cellular context with potential applications to a wide variety of eukaryotic cell types.

Cell Line↗
PubMed2006

Blue fluorescent proteins with enhanced brightness and photostability from a structurally targeted library.

The utility of blue fluorescent protein (BFP) has been limited by its low quantum yield and rapid photobleaching. A library targeting residues neighboring the chromophore yielded a variant with enhanced quantum yield (0.55 versus 0.34), reduced pH sensitivity and a 40-fold increase in photobleaching half-life. This BFP, named Azurite, is well expressed in bacterial and mammalian cells and extends the palette of fluorescent proteins that can be used for imaging.

Flow Cytometry↗
PubMed2006

Protease specificity determination by using cellular libraries of peptide substrates (CLiPS).

We report a general combinatorial approach to identify optimal substrates of a given protease by using quantitative kinetic screening of cellular libraries of peptide substrates (CLiPS). A whole-cell protease activity assay was developed by displaying fluorescent reporter substrates on the surface of Escherichia coli as N-terminal fusions. This approach enabled generation of substrate libraries of arbitrary amino acid composition and length that are self-renewing. Substrate hydrolysis by a target protease was measured quantitatively via changes in whole-cell fluorescence by using FACS. FACS enabled efficient screening to identify optimal substrates for a given protease and characterize their cleavage kinetics. The utility of CLiPS was demonstrated by determining the substrate specificity of two unrelated proteases, caspase-3 and enteropeptidase (or enterokinase). CLiPS unambiguously identified the caspase-3 consensus cleavage sequence DXVDG. Enteropeptidase was unexpectedly promiscuous, but exhibited a preference for substrates with the motif (D/E)RM, which were cleaved substantially faster than the canonical DDDDK recognition sequence, widely used for protein purification. CLiPS provides a straightforward and versatile approach to determine protease specificity and discover optimal substrates on the basis of cleavage kinetics.

Amino Acid Sequence↗
PubMed2006

Isolation of cell specific peptide ligands using fluorescent bacterial display libraries.

Methods for identifying and producing cell specific affinity reagents are critical in cell detection, separation, and therapeutic delivery applications, yet remain difficult and time consuming. To address these limitations, a rapid and quantitative screening approach was developed using intrinsically fluorescent bacterial display peptide libraries and fluorescence-activated cell sorting (FACS). High-throughput screening of fluorescent libraries yielded a panel of peptide ligands mediating specific recognition of human breast cancer tumor cells. Clonal populations of fluorescent, peptide-displaying bacteria enabled single-step, fluorescent labeling of the target cells for cytometry and microscopy analysis. Isolated peptides could be categorized into several distinct groups possessing strong consensus sequences with as many as six identities. Importantly, individual clones exhibited high specificity target cell binding, with more than 80-fold increased binding to tumor cells (ZR-75-1) relative to cell lines derived from healthy tissue (HMEC, MCF-10A). Fluorescent display libraries thus provide a powerful new methodology for parallel identification of cell specific affinity ligands.

Affinity Labels↗
PubMed2006

Microfluidic protein detection through genetically engineered bacterial cells.

Protein microarray technology, in which a large number of capture ligands are spatially arrayed at a high density, presents an attractive method for high-throughput proteomic analysis. Toward this end, we demonstrate the first cell-based protein detection in a microsystem, wherein Escherichia coli cells are genetically engineered to express the desired capture proteins on the membrane surface and are spatially arrayed as sensing elements in a microfluidic device. An E. coli clone expressing peptide ligands with high affinity and high specificity for target molecules was isolated a priori. Then these cells were electrokinetically immobilized on gold electrodes using dielectrophoresis, thus allowing each sensor element to be electrically addressable. Flow cytometry and subsequent fluorescence analysis verified the highly specific capture and detection of target molecules by the bacteria. Finally, through the coexpression of peptide-based capture ligands on the cell surface and fluorescent protein in the cytoplasm, we demonstrate an effective means of directly linking the fluorescence intensity to the density of capture ligands.

Escherichia coli↗
PubMed2006

Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands.

A bacterial display methodology was developed for N- and C-terminal display and demonstrated to enable rapid screening of very large peptide libraries with high precision and efficiency. To overcome limitations of insertional fusion display libraries, a new scaffold was developed through circular permutation of the Escherichia coli outer membrane protein OmpX that presents both N and C termini on the external cell surface. Circularly permuted OmpX (CPX) display was directly compared to insertional fusion display by screening comparable peptide libraries in each format using magnetic and fluorescence activated cell sorting. CPX display enabled in situ measurement of dissociation rate constants with improved accuracy and, consequently, improved affinity discrimination during screening and ranking of isolated clones. Using streptavidin as a model target, bacterial display yielded the well-characterized HP(Q)/(M) motif obtained previously using several alternative peptide display systems, as well as three additional motifs (L(I)/(V) CQNVCY, CGWMY(F)/(Y)xEC, ERCWYVMHWPCNA). Using CPX display, a very high affinity streptavidin-binding peptide was isolated having a dissociation rate constant k(off) = 0.002sec(-1) even after grafting to the C terminus of an unrelated protein. Comparison of individual clones obtained from insertional fusion and terminal fusion libraries suggests that the N-terminal display yields sequences with greater diversity, affinity, and modularity. CPX bacterial display thus provides a highly effective method for screening peptide libraries to rapidly generate ligands with high affinity and specificity.

Affinity Labels↗
PubMed2005

Enzymatic synthesis of layered titanium phosphates at low temperature and neutral pH by cell-surface display of silicatein-alpha.

We introduce a novel method of inorganic synthesis using the catalytic and structure-directing properties of the demosponge enzyme silicatein-alpha. Recombinant silicatein-alpha was displayed at the surface of Escherichia coli cells by fusion to outer membrane protein A and used to biocatalytically direct the formation of layered and amorphous titanium phosphates from a small water-soluble precursor at near-neutral pH at 16 degrees C. Synthesis of titanium phosphates, with potential applications in catalysis and separation technology, previously has required prolonged reactions with phosphoric acid at elevated temperatures. Additionally, we use library screening to isolate a 15-mer with affinity toward the silicatein active site (Kd ca. 50 nM) and introduce this new approach to demonstrate the success of our display strategy. Considering our previous findings with native silicatein filaments, we suggest that this scalable, efficient, cell-based system may have a broad utility for the synthesis of a range of structured metallophosphates and other inorganic materials.

Amino Acid Sequence↗
PubMed2005

Automated design of degenerate codon libraries.

Degenerate codon libraries are frequently used in protein engineering and evolution studies but are often limited to targeting a small number of positions to adequately limit the search space. To mitigate this, codon degeneracy can be limited using heuristics or previous knowledge of the targeted positions. To automate design of libraries given a set of amino acid sequences, an algorithm (LibDesign) was developed that generates a set of possible degenerate codon libraries, their resulting size, and their score relative to a user-defined scoring function. A gene library of a specified size can then be constructed that is representative of the given amino acid distribution or that includes specific sequences or combinations thereof. LibDesign provides a new tool for automated design of high-quality protein libraries that more effectively harness existing sequence-structure information derived from multiple sequence alignment or computational protein design data.

Algorithms↗
PubMed2005

Marker-specific sorting of rare cells using dielectrophoresis.

Current techniques in high-speed cell sorting are limited by the inherent coupling among three competing parameters of performance: throughput, purity, and rare cell recovery. Microfluidics provides an alternate strategy to decouple these parameters through the use of arrayed devices that operate in parallel. To efficiently isolate rare cells from complex mixtures, an electrokinetic sorting methodology was developed that exploits dielectrophoresis (DEP) in microfluidic channels. In this approach, the dielectrophoretic amplitude response of rare target cells is modulated by labeling cells with particles that differ in polarization response. Cell mixtures were interrogated in the DEP-activated cell sorter in a continuous-flow manner, wherein the electric fields were engineered to achieve efficient separation between the dielectrophoretically labeled and unlabeled cells. To demonstrate the efficiency of marker-specific cell separation, DEP-activated cell sorting (DACS) was applied for affinity-based enrichment of rare bacteria expressing a specific surface marker from an excess of nontarget bacteria that do not express this marker. Rare target cells were enriched by >200-fold in a single round of sorting at a single-channel throughput of 10,000 cells per second. DACS offers the potential for automated, surface marker-specific cell sorting in a disposable format that is capable of simultaneously achieving high throughput, purity, and rare cell recovery.

Affinity Labels↗
PubMed2005

Evolutionary optimization of fluorescent proteins for intracellular FRET.

Fluorescent proteins that exhibit Forster resonance energy transfer (FRET) have made a strong impact as they enable measurement of molecular-scale distances through changes in fluorescence. FRET-based approaches have enabled otherwise intractable measurements of molecular concentrations, binding interactions and catalytic activity, but are limited by the dynamic range and sensitivity of the donor-acceptor pair. To address this problem, we applied a quantitative evolutionary strategy using fluorescence-activated cell sorting to optimize a cyan-yellow fluorescent protein pair for FRET. The resulting pair, CyPet-YPet, exhibited a 20-fold ratiometric FRET signal change, as compared to threefold for the parental pair. The optimized FRET pair enabled high-throughput flow cytometric screening of cells undergoing caspase-3-dependent apoptosis. The CyPet-YPet energy transfer pair provides substantially improved sensitivity and dynamic range for a broad range of molecular imaging and screening applications.

Bacterial Proteins↗
PubMed2004

Rapid isolation of high-affinity protein binding peptides using bacterial display.

A robust bacterial display methodology was developed that allows the rapid isolation of peptides that bind to arbitrarily selected targets with high affinity. To demonstrate the utility of this approach, a large library (5 x 10(10) clones) was constructed composed of random 15-mer peptide insertions constrained within a flexible, surface exposed loop of the Escherichia coli outer membrane protein A (OmpA). The library was screened for binding to five unrelated proteins, including targets previously used in phage display selections: human serum albumin, anti-T7 epitope mAb, human C-reactive protein, HIV-1 GP120 and streptavidin. Two to four rounds of enrichment (2-4 days) were sufficient to enrich peptide ligands having high affinity for each of the target proteins. Strong amino acid consensus sequences were apparent for each of the targets tested, with up to seven consensus residues. Isolated peptide ligands remained functional when expressed as insertional fusions within a monomeric fluorescent protein. This bacterial display methodology provides an efficient process for identifying peptide affinity reagents and should be useful in a variety of molecular recognition applications.

Amino Acid Sequence↗
PubMedDate not supplied

Flow cytometric screening of cDNA expression libraries for fluorescent proteins.

Fluorescence-activated cell sorting (FACS) was applied for quantitative screening of cDNA expression libraries in bacteria for rare fluorescent protein encoding cDNAs. Rare fluorescent cells, observed at a frequency of 1 in 200,000 bacteria in a cDNA expression library constructed from Astrangia lajollaensis, were detected, enriched, and purified by sorting, yielding three distinct green fluorescent proteins. Two of the isolated fluorescent proteins were found to be 2.5-fold brighter in whole cell fluorescence than the widely used and already optimized EGFP variant and possessed a novel cysteine-containing chromophore. FACS can possess significant advantages in the screening of cDNA libraries in bacteria, since desired genes may occur at low frequencies and possess unexpected properties. This strategy provides a high-throughput, quantitative approach for isolating fluorescent proteins from a more diverse range of organisms and should be extendable to proteins that are not intrinsically fluorescent with the use of available fluorescent indicators.

Amino Acid Sequence↗