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Chi-huey Wong - One of the best experts on this subject based on the ideXlab platform.

  • chemistry and glycobiology
    Chemical Communications, 2011
    Co-Authors: Chi-huey Wong
    Abstract:

    Current interests in glycobiology have stimulated the development of new tools for use to tackle major problems in the field, including, for example, Glycoprotein Synthesis, glycan array development and post-translational glycosylation monitoring. Recent advances in the Synthesis of Glycoproteins involve Glycoprotein remodelling, native chemical ligation (NCL), expressed protein ligation (EPL), Staudinger ligation, sugar-assisted ligation and pathway engineering to effectively produce homogeneous Glycoproteins with well defined glycans for structural and functional studies. Moreover, the development of glycan Synthesis, such as one-pot, chemoenzymatic and solid-supported syntheses, has greatly simplified the process in creating various glycans for functional and array study. Glycan array requires little sample and is able to test and compare many carbohydrate–protein interactions simultaneously. Finally, the changes in post-translational glycosylation, which is an indicator of disease progression, can be monitored by bioorthogonal chemical reporters with the cell's metabolic machinery. The interdisciplinary cooperation in chemistry and biology has yielded new strategies and led to an explosion of research in this field.

  • chemoenzymatic approaches to Glycoprotein Synthesis
    ChemInform, 2007
    Co-Authors: Clay S. Bennett, Chi-huey Wong
    Abstract:

    The construction of homogeneous Glycoproteins presents a formidable challenge to the synthetic chemist. Over the past few years there has been an explosion in the number of methods developed to address this problem. These methods include the development of novel ligation technologies for the Synthesis of the protein backbone, as well chemical and enzymatic approaches for introducing complex glycans into the peptide backbone. This tutorial review discusses the application of these techniques to the Synthesis of peptides and proteins possessing well defined glycans.

  • sugar assisted ligation in Glycoprotein Synthesis
    Journal of the American Chemical Society, 2007
    Co-Authors: Yuying Yang, Simon Ficht, Ashraf Brik, Chi-huey Wong
    Abstract:

    Sugar-assisted ligation (SAL) presents an attractive strategy for the Synthesis of glycopeptides, including the Synthesis of cysteine-free beta-O-linked and N-linked glycopeptides. Here we extended the utility of SAL for the Synthesis of alpha-O-linked glycopeptides and Glycoproteins. In order to explore SAL in the context of Glycoprotein Synthesis, we developed a new chemical synthetic route for the alpha-O-linked Glycoprotein diptericin epsilon. In the first stage of our Synthesis, diptericin segment Cys(Acm)37-Gly(52) and segment Val(53)-Phe(82) were assembled by SAL through a Gly-Val ligation junction. Subsequently, after Acm deprotection, diptericin segment Cys(37)-Phe(82) was ligated to segment Asp(1)-Asn(36) by means of native chemical ligation (NCL) to give the full sequence of diptericin epsilon. In the final synthetic step, hydrogenolysis was applied to remove the thiol handle from the sugar moiety with the concomitant conversion of mutated Cys(37) into the native alanine residue. In addition, we extended the applicability of SAL to the Synthesis of glycopeptides containing cysteine residues by carrying out selective desulfurization of the sulfhydryl-modified sugar moiety in the presence of acetamidomethyl (Acm) protected cysteine residues. The results presented here demonstrated for the first time that SAL could be a general and useful tool in the chemical Synthesis of Glycoproteins.

  • Advances in Glycoprotein Synthesis
    Chemical Communications, 2005
    Co-Authors: Lei Liu, Clay S. Bennett, Chi-huey Wong
    Abstract:

    The development of chemical and enzymatic methods for the Synthesis of homogeneous Glycoproteins is a fascinating challenge at the interface between chemistry and biology. Discussed here are the currently available methods for preparation of homogeneous Glycoproteins. These methods include (1) glycopeptide ligation; (2) Glycoprotein remodeling; and (3) in vivo suppressor tRNA technology.

  • a new strategy for Glycoprotein Synthesis ligation of synthetic glycopeptides with truncated proteins expressed in e coli as tev protease cleavable fusion protein
    Bioorganic & Medicinal Chemistry, 2005
    Co-Authors: Thomas J Tolbert, Dirk Franke, Chi-huey Wong
    Abstract:

    We report here the use of TEV protease cleavable fusion proteins to produce glycosylated bioactive peptides and proteins. Bacterial expression was utilized to produce two fusion proteins, GPRT-C37-H6 and His-tagged interleukin-2 (amino acids 6-133), which when cleaved by the tobacco etch virus NIa protease (TEV protease) to generate HIV entry inhibitor peptide C37-H6 and a truncated version of the cytokine interleukin-2, both containing N-terminal cysteines. The N-terminal cysteine containing C37-H6 and truncated interleukin-2 were then joined to a synthetic glycopeptide thioester utilizing native chemical ligation under nondenaturing and denaturing conditions, respectively. The ligations of the glycopeptide to the C37-H6 peptide and the truncated interleukin-2 protein both proceeded in high yield, though the size, and physical properties of the two polypeptides differ greatly.

Michael C Jewett - One of the best experts on this subject based on the ideXlab platform.

  • improving cell free Glycoprotein Synthesis by characterizing and enriching native membrane vesicles
    Nature Communications, 2021
    Co-Authors: Jasmine M Hershewe, Katherine F Warfel, Matthew P Delisa, Shaelyn M Iyer, Justin A Peruzzi, Claretta J Sullivan, Eric W Roth, Neha P Kamat, Michael C Jewett
    Abstract:

    Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked Glycoprotein Synthesis. We anticipate that these methods will facilitate on-demand Glycoprotein production and enable new CFE systems with membrane-associated activities.

  • improving cell free Glycoprotein Synthesis by characterizing and enriching native membrane vesicles
    bioRxiv, 2020
    Co-Authors: Jasmine M Hershewe, Katherine F Warfel, Matthew P Delisa, Shaelyn M Iyer, Justin A Peruzzi, Claretta J Sullivan, Eric W Roth, Neha P Kamat, Michael C Jewett
    Abstract:

    Abstract Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for accelerating the design of cellular function, on-demand biomanufacturing, portable diagnostics, and educational kits. Many essential biological processes that could endow CFE systems with desired functions, such as protein glycosylation, rely on the activity of membrane-bound components. However, without the use of synthetic membrane mimics, activating membrane-dependent functionality in bacterial CFE systems remains largely unstudied. Here, we address this gap by characterizing native, cell-derived membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membranebound machinery. We first use nanocharacterization techniques to show that lipid vesicles in CFE extracts are tens to hundreds of nanometers across, and on the order of ~3×1012 particles/mL. We then determine how extract processing methods, such as post-lysis centrifugation, can be used to modulate concentrations of membrane vesicles in CFE systems. By tuning these methods, we show that increasing the number of vesicle particles to ~7×1012 particles/mL can be used to increase concentrations of heterologous membrane protein cargo expressed prior to lysis. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving N-linked and O-linked Glycoprotein Synthesis. We anticipate that our findings will facilitate in vitro gene expression systems that require membrane-dependent activities and open new opportunities in glycoengineering.

  • synthetic glycobiology parts systems and applications
    ACS Synthetic Biology, 2020
    Co-Authors: Weston Kightlinger, Katherine F Warfel, Matthew P Delisa, Michael C Jewett
    Abstract:

    Protein glycosylation, the attachment of sugars to amino acid side chains, can endow proteins with a wide variety of properties of great interest to the engineering biology community. However, natural glycosylation systems are limited in the diversity of Glycoproteins they can synthesize, the scale at which they can be harnessed for biotechnology, and the homogeneity of Glycoprotein structures they can produce. Here we provide an overview of the emerging field of synthetic glycobiology, the application of synthetic biology tools and design principles to better understand and engineer glycosylation. Specifically, we focus on how the biosynthetic and analytical tools of synthetic biology have been used to redesign glycosylation systems to obtain defined glycosylation structures on proteins for diverse applications in medicine, materials, and diagnostics. We review the key biological parts available to synthetic biologists interested in engineering Glycoproteins to solve compelling problems in glycoscience, describe recent efforts to construct synthetic Glycoprotein Synthesis systems, and outline exemplary applications as well as new opportunities in this emerging space.

  • single pot Glycoprotein bioSynthesis using a cell free transcription translation system enriched with glycosylation machinery
    Nature Communications, 2018
    Co-Authors: Thapakorn Jaroentomeechai, Michael C Jewett, Jessica C Stark, Aravind Natarajan, Cameron J Glasscock, Laura E Yates, Karen J Hsu, Milan Mrksich, Matthew P Delisa
    Abstract:

    The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous Glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free Glycoprotein Synthesis (CFGpS) technology that seamlessly integrates protein bioSynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of Glycoproteins.

Gerhard F Ecker - One of the best experts on this subject based on the ideXlab platform.

Andrew Scholey - One of the best experts on this subject based on the ideXlab platform.

  • two time windows of anisomycin induced amnesia for passive avoidance training in the day old chick
    Neurobiology of Learning and Memory, 1995
    Co-Authors: Fiona M Freeman, Steven P R Rose, Andrew Scholey
    Abstract:

    The antibiotic anisomycin (ANP), a protein Synthesis inhibitor, was used to investigate the time-related changes in protein Synthesis following passive avoidance training in the day-old chick. Retention of memory for this simple learning task is known to be prevented by protein Synthesis inhibitors within the first hour posttraining. Here we report a second, later time window during which inhibition of protein Synthesis results in amnesia following one-trial passive avoidance training. Birds were given bilateral intracranial injections of ANI (10 μl/hemisphere of a 30 mM solution) at various times relative to training and tested 24 h later. Injections given between 0.5 h prior to 1.5 h post-training or 4-5 h posttraining, but not at later or at intervening times, resulted in amnesia. These results are discussed in the context of earlier findings, using the inhibitor of Glycoprotein Synthesis 2-deoxygalactose, that memory formation shows two Glycoprotein-Synthesis-dependent periods of sensitivity (Scholey, Rose, Zamani, Beck, and Schachner, 1993). The time windows of susceptibility of ANI and 2-Dgal are consistent with a model in which there are two waves of neural activity following training; during the second, commencing 4 h after training, proteins are synthesized and then glycosylated as part of the establishment of an enduring memory trace.

  • a role for the neural cell adhesion molecule in a late consolidating phase of Glycoprotein Synthesis six hours following passive avoidance training of the young chick
    Neuroscience, 1993
    Co-Authors: Andrew Scholey, Steven P R Rose, Maryam Reza Zamani, Elisabeth Bock, Melitta Schachner
    Abstract:

    Abstract We have investigated the effect of intracranial injections of the amnestic anti-metabolite, 2-deoxygalactose, and antibodies to the neural cell adhesion molecule on retention of a one-trial passive avoidance task in chicks. Groups of chicks received bilateral intracranial injections of 10 μmol/hemisphere 2-deoxygalactose or 10 μ1/hemisphere anti-neural cell adhesion molecule and were tested 24 h following training. 2-Deoxygalactose injections were amnestic when administered at a previously established time (30 min pre-training). Here we show that the agent is also amnestic when injected within a second time window occurring specifically 6–8 h after training. Administration of 2-deoxygalactose between 2 and 6 h or after 8 h post-training was without effect on retention tested 24 h following training. Anti-neural cell adhesion molecule injections were amnestic only when performed at a time which coincided with the second phase of 2-deoxygalactose susceptibility. Further experiments demonstrated that the neural cell adhesion molecule is one of the molecules into which 2-deoxygalactose is incorporated. Additionally, we investigated the extent of diffusion of 2-deoxygalactose and anti-neural cell adhesion molecule following their injection, with respect to their residence in forebrain loci known to be involved in the memory for passive avoidance. We interpret these data as indicating that two waves of Glycoprotein Synthesis are necessary for the establishment of long-term memory for the experience of passive avoidance training. The evidence is discussed in the context of earlier results indicating that the two waves involve different Glycoprotein species and, possibly, different forebrain regions. We speculate that the late phase of Glycoprotein Synthesis coincides with, and is required for, modulation of cell-cell adhesion processes, reflecting the selection and stabilization of synapses which maintain an enduring representation of long-term memory.

Penpun Wetwitayaklung - One of the best experts on this subject based on the ideXlab platform.