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Biomedical subjects

Grace Y J Chen

Publications and source records attributed to Grace Y J Chen.

At least 19 recordsLinked to original sources
PubMed2005

Site-specific immobilization of proteins in a microarray using intein-mediated protein splicing.

One of the critical issues in the generation of a protein microarray lies in the choice of immobilization strategies, which ensure proteins are adhered to the glass surface while properly retaining their native biological activities. Herein, we report a bacterium-based, intein-mediated strategy to generate N-terminal cysteine-containing proteins which are then chemoselectively immobilized to a thioester-functionalized glass slide to generate the corresponding protein microarray. We also showed preliminary data of the strategy in a yeast host system.

Electrophoresis, Polyacrylamide Gel↗
PubMed2005

Nanodroplet profiling of enzymatic activities in a microarray.

We describe a generic method for the large-scale functional characterization of enzymes in a microarray. Poly-l-lysine and amine reactive slides were coated with fluorogenic substrates sensitive to proteases and phosphatases. Patterning enzymes on the slides by robotic printing produced spatially addressable, segregated droplets that were simultaneously exposed to the on-chip sensors. Multiple enzymes were profiled using this system that provided fluorescence readouts across temporal and stoichiometric dimensions concurrently on a single microarray substrate. This integrated microarray platform is applicable not only for the functional annotation of proteins, but also for the rapid agonist and antagonist discovery and in performing on-chip kinetics.

Microarray Analysis↗
PubMed2004

Improving the intein-mediated, site-specific protein biotinylation strategies both in vitro and in vivo.

One of the critical issues in the generation of a protein microarray lies in the choice of immobilization strategies, which ensure proteins are adhered to the glass surface while properly retaining their native biological activities. We previously developed intein-mediated strategies for protein biotinylation and site-specific protein microarray generation. Herein, we report new findings of these strategies, which improve the biotinylation efficiency of proteins by up to 10-folds.

Biotin↗
PubMed2004

Expanding the scope of site-specific protein biotinylation strategies using small molecules.

We present a new approach to site-specifically biotinylate protein in a cell-free protein synthesis system with puromycin-containing small molecules. With this new method, biotinylated proteins were generated from the DNA templates in a matter of hours, making it useful for protein microarray generation. We also validated that the method is compatible with other high-throughput cloning/proteomics methods.

Binding Sites↗
PubMed2004

Expanded utility of the native chemical ligation reaction.

The post-genomic era heralds a multitude of challenges for chemists and biologists alike, with the study of protein functions at the heart of much research. The elucidation of protein structure, localization, stability, post-translational modifications, and protein interactions will steadily unveil the role of each protein and its associated biological function in the cell. The push to develop new technologies has necessitated the integration of various disciplines in science. Consequently, the role of chemistry has never been so profound in the study of biological processes. By combining the strengths of recombinant DNA technology, protein splicing, organic chemistry, and the chemoselective chemistry of native chemical ligation, various strategies have been successfully developed and applied to chemoselectively label proteins, both in vitro and in live cells, with biotin, fluorescent, and other small molecule probes. The site-specific incorporation of molecular entities with unique chemical functionalities in proteins has many potential applications in chemical and biological studies of proteins. In this article, we highlight recent progress of these strategies in several areas related to proteomics and chemical biology, namely, in vitro and in vivo protein biotinylation, protein microarray technologies for large-scale protein analysis, and live-cell bioimaging.

Biochemical Phenomena↗
PubMed2004

Versatile protein biotinylation strategies for potential high-throughput proteomics.

We present intein-mediated approaches for efficient biotinylation of proteins site-specifically. The reactive C-terminal thioester generated from intein-assisted protein splicing (either in vitro or in live cells) served as an attractive and exclusive site for attaching cysteine-containing biotin. Using these novel biotinylation strategies, we were able to efficiently biotinylate many proteins from different biological sources in a potentially high-throughput, high-content fashion. Some of these proteins were subsequently immobilized, in a very simple manner, onto different avidin-functionalized solid surfaces for applications such as protein microarray and surface plasmon resonance (SPR) spectroscopy, highlighting the numerous advantages of using biotin over other tags (e.g., GST, His-tag, etc.) as the method of choice in protein purification/immobilization. In addition, our intein-mediated strategies provided critical advantages over other protein biotinylation strategies in a number of ways. For the first time, we also successfully demonstrated that intein-mediated protein biotinylation proceeded adequately inside both bacterial and mammalian living cells, as well as in a cell-free protein synthesis system. Taken together, our results indicate the versatility of these intein-mediated strategies for potential high-throughput proteomics applications. They may also serve as useful tools for various biochemical and biophysical studies of proteins both in vitro and in vivo.

Biotinylation↗
PubMed2004

Site-specific immobilization of biotinylated proteins for protein microarray analysis.

The postgenome era has led to a new frontier of proteomics that requires the development of protein microarray, which enables us to unravel the biological function of proteins in a massively parallel fashion. Several ways of immobilizing proteins onto surfaces have been reported, but many of these attachments are unspecific, resulting in the unfavorable orientation of the immobilized proteins. His6 tag has been used to site-specifically immobilize proteins onto nickel-coated slides, which presumably oriented proteins uniformly on the surface of the slide. However, the binding between Ni-NTA and His tag proteins is not strong, causing the immobilized proteins to dissociate from the slide even under simple wash conditions. The authors have developed a novel strategy of using an intein-mediated expression system to generate biotinylated proteins suitable for immobilization onto avidin-functionalized glass slides. Array-scan results not only show successful immobilization of proteins onto slides by antibody detection method but also full retention of biological activities of the immobilized proteins. The strong and specific interaction between biotin and avidin also permits the use of stringent incubation and washing conditions on the protein microchip, thus making it a highly robust method for array studies.

Avidin↗
PubMed2004

Site-specific peptide immobilization strategies for the rapid detection of kinase activity on microarrays.

The massive throughput offered by array-based technologies can only be realized with the development of equally powerful strategies that offer reproducible consistency. The competence of arrays and efficacy of screening come under scrutiny, with most existing immobilization schemes that do not site-specifically ligate peptides on the arrays. Thus, it is crucial in array-based experiments to orientate peptides in an ordered and uniform fashion. Two new approaches were developed for the directed immobilization of peptides on a microarray, by exploiting measures involving native chemical ligation reactions as well as biotin-streptavidin interactions. This makes it possible to stably immobilize peptides in a consistent manner and in a predetermined orientation on the microarray. The first scheme employs glass slides that are functionalized with avidin for attachment of terminally biotinylated peptides. The second uses slides containing thioester moieties to ligate N-terminal cysteine containing peptides. The authors successfully immobilized peptides on chip using these strategies, and, in extending their method to the study of kinase activity on microarrays, they also developed a novel detection scheme that abrogates the dependence on traditional radioactivity-based kinase screening assays. This method employs fluorescently labeled antiphosphoserine and antiphosphotyrosine antibodies in assessing and monitoring kinase activity on arrays. The above methodologies provide for a fast and sensitive approach with which to conveniently assess kinase activity using peptide microarrays.

Antibodies↗
PubMed2004

Recent advances in gel-based proteome profiling techniques.

This review focuses on recent developments in gel-based proteomics techniques. By combining traditional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional gel electrophoretic techniques with recent advances in protein labeling using different classes of molecules (i.e., fluorescent dyes, chemical probes, radioisotopes), new technologies have been developed that allow for high-throughput studies of proteins at the whole-proteome scale.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2003

Cell-permeable small molecule probes for site-specific labeling of proteins.

We have successfully synthesized a number of small molecule probes designed for site-specific labeling of N-terminal cysteine-containing proteins expressed in live cells. Their utility for site-specific, covalent modifications of proteins was successfully demonstrated with purified proteins in vitro, and with live bacterial cells in vivo.

Cell Membrane Permeability↗
PubMed2003

Combinatorial peptide microarrays for the rapid determination of kinase specificity.

We report a rapid method for profiling of kinases using a strategy that couples the merits of combinatorics (in rapid diversity generation) with the throughput attainable using microarrays (in parallel screening). Alanine-scanning, deletion and positional-scanning peptide libraries of a kinase substrate were synthesized and site-specifically arrayed onto glass slides. The phosphorylation pattern of target sequences detected using fluorescently-labeled antiphosphoamino acid antibodies revealed the substrate preference of the kinase through its activity profile.

Amino Acid Sequence↗
PubMed2003

Solid-phase synthesis of peptide vinyl sulfones as potential inhibitors and activity-based probes of cysteine proteases.

Peptide vinyl sulfones were prepared from 2-chlorotrityl resin-bound phenolic amino vinyl sulfones in high yield and purity. This method enables the convenient synthesis of peptide vinyl sulfones having different amino acids at the P(1) position. It also allows efficient synthesis of vinyl sulfone-containing, activity-based probes of cysteine proteases used in a proteomic experiment.

Amino Acids↗
PubMed2003

Proteome analysis of Saccharomyces cerevisiae under metal stress by two-dimensional differential gel electrophoresis.

The defense mechanism by which cells combat metal stress remains poorly understood. By utilizing a newly developed technique - the differential gel electrophoresis (DIGE) - we evaluated the biological alterations of metal stress on Saccharomyces cerevisiae at its translational level. By simultaneously comparing the differential expression profiles of thousands of proteins as results of 15 different metal treatments, we were able to closely examine the response of a large number of proteins within the yeast proteome towards individual metals, as well as the response of the same proteins towards different metals. This, to our knowledge, is the first case which demonstrates the potential of DIGE as a high-throughput tool for large-scale proteome analysis. From our studies, where yeast cells were exhaustively treated with exogenous metals, 20-30% of all proteins detected showed statistically significant changes. According to different effects (up-/downregulation) of protein expression levels observed, we were able to tentatively divide the 15 metals into three groups. By mass spectrometric analysis, more than 50 protein spots were positively identified, both quantitatively and qualitatively. One of the proteins was identified to be Cu/Zn superoxide dismutase (SOD1), and its expression levels as a result of 15 different metal treatments was further examined in greater details. Significant changes in SOD1 expression were observed throughout all 15 DIGE gels.

Electrophoresis, Gel, Two-Dimensional↗