生物化学技术

In eukaryotic cells, the reversible attachment of small ubiquitin-like modifi er (SUMO) protein is a post-translational modifi cation that has been demonstrated to play an important role in various cellular processes. Moreover, it has been found that SUMO as an N-terminal fusion partner e ...

We have previously demonstrated that Gam1, an avian adenoviral protein inhibits sumoylation. By counteracting the SUMO pathway, Gam1 has a significant impact on virus-infected cells, but in isolation the inhibitory effects of the Gam1 protein can be exploited to intentionally manip ...

In this chapter we describe a novel, sensitive, homogenous high throughput reporter-based in vitro assay for SUMO protease activity developed by Progenra, Inc. A reporter construct was created by fusing His6-tagged small ubiquitin-like modifi er (SUMO) to the amino terminus of the report ...

In humans cells three SUMO paralogues (SUMO-1, SUMO-2 and SUMO-3) and six SUMO specific proteases (SENP1-SENP3 and SENP5-SENP7) are expressed. Together the SUMO proteases perform three distinct functions. They: (1) process the immature pro-SUMO proteins into the active forms, (2) remove S ...

To measure rates of sumoylation and isopeptidase cleavage in vitro, we developed an enzyme assay that is based on fl uorescence resonance energy transfer (FRET). FRET is a process by which the excited state energy of a fl uorescent donor molecule is transferred to an acceptor molecule. Effi cient en ...

SUMO proteases catalyze two reactions, deconjugation of SUMO from substrates and processing of precursor SUMO isoforms to prepare SUMO for conjugation. The SUMO protease family includes two members in yeast (Ulp1 and Ulp2) and as many as six members in human (SENP1-3, SENP5-7). SENP/Ulp prot ...

Fluorous-based carbohydrate microarrays provide an alternative to traditional covalent microarray �platforms for probing protein–carbohydrate-binding interactions. The most studied plant lectin, concanavalin A (ConA), is known to bind to terminally α-linked mannos ...

Neoglycoproteins, such as BSA-glycosides, contain carbohydrates covalently attached to a protein carrier via nonnaturally occurring linkages. These conjugates have been used for decades to study carbohydrate–protein interactions and are frequently used as immunogens to ...

Our experience in coating of solid surfaces with glycans, mainly for obtaining routine glycoarrays based on immunological plates, is summarized. Three polystyrene coating techniques are described: direct physical adsorption, covalent binding, and immobilization using the b ...

Monitoring multiple biological interactions in a multiplexed array format has numerous advantages. However, converting well-developed surface chemistry for spectroscopic measurements to array-based, high-throughput screening is not a trivial process and often proves ...

Glycoarrays have become a powerful platform to investigate the interactions of many biological events involving carbohydrates. The carbohydrates immobilization on the surface of the substrates is a key step of glycoarray fabrication. Plenty of strategies have been applied to the i ...

Surface-based tools, such as microarrays and optical biosensors, are being increasingly applied to the analysis of carbohydrate–protein interactions. A key to these developments is the presentation of the carbohydrate to the protein target. Dual polarisation interferometry ( ...

The interaction of polysaccharides with proteins modulates or triggers many biological effects. In particular, heparan sulphate proteoglycans (HSPGs) have multiple regulatory interactions with growth factors, enzymes, enzyme inhibitors, and some components of the extra ...

This chapter describes one of my laboratory’s working protocols for carbohydrate-based microarrays. Using a standard microarray spotter, we print carbohydrate antigens directly on the nitrocellulose-coated bioarray substrates. Because these substrates support nonco ...

Glycan-processing enzymes such as glycosyltransferases and glycosidases are responsible for the makeup of the glycome. The definition of their substrate specificities is, therefore, a central task in glycomics. In addition, these enzymes are themselves useful synthetic tools f ...

Glycan microarrays are surfaces that contain immobilized oligosaccharides or glycoconjugates and have proven useful in probing the interactions between glycan-binding proteins (GBPs) and individual glycans. Such glycan microarrays have been especially important in st ...

Glycan microarrays, carrying hundreds of different sugars on chip surfaces, have become a standard tool for the study of interactions of biomolecules with carbohydrates. The chip-based format offers important advantages, including the ability to screen in parallel several thous ...

Glycan microarray technology enables the screening of large numbers of glycan samples for glycan–protein interactions, based on the presentation of immobilized glycans in a discrete pattern on a solid support. Here we describe a glycan microarray approach employing glycans enzym ...

Glycosaminoglycans (GAGs) perform numerous vital functions within the body. As major components of the extracellular matrix, these polysaccharides participate in a diverse array of cell-signaling events. We have developed a simple microarray assay for the evaluation of protein ...

Widespread bacterial resistance to antibiotics is a significant public health concern. To remain a step ahead of evolving bacteria, new methods to study resistance to antibacterials and to uncover novel antibiotics that evade resistance are urgently needed. Herein, microarray-b ...