42,321 research outputs found
Use of Vibrio spp. for expression of E. coli enterotoxin B subunit fusion proteins: purification and characterization of a chimera containing a C-terminal fragment of DNA polymerase from herpes virus type 1.
No full textThe non-toxic B subunit of E. coli heat-labile enterotoxin (EtxB) is a convenient carrier molecule for the attachment and delivery of heterologous peptides into eukaryotic cells. To evaluate the properties of such EtxB-based fusion proteins an efficient method for their production and purification is required. High level production and purification of native EtxB has been achieved using heterologous expression and secretion in a marine vibrio [Amin, T., and Hirst, T. R. (1994) Protein. Expr. Purif. 5, 198-204]. However, the use of this method to isolate EtxB-fusion proteins has been precluded because of their susceptibilty to degradation by extracellular proteases secreted by members of the Vibrionaceae. In this paper a method is described for production of EtxB-pol, comprising the enterotoxin B-subunit linked to a 27 residue C-terminal fragment of POL, the catalytic subunit of DNA polymerase of herpes simplex virus type 1 (HSV-1). Following assessment of the relative efficacy of different Vibrio strains as hosts for EtxB-pol expression, the chimera was produced at the highest level of 3.5 mg/l by cultures of Vibrio sp.60. Addition of 0.3 mM EDTA to the growth medium blocked proteolysis of the secreted EtxB-pol fusion protein, which was then purified to homogeneity using ammonium sulfate fractionation and hydrophobic interaction chromatography, with a yield of 57%. Purified EtxB-pol reacted with both anti-EtxB and anti-pol peptide antibodies, and was able to specifically bind UL42, a processivity factor which normally binds to the C-terminal region of HSV-1 POL. This modified method for expression and purification of EtxB-pol should be of general utility for the preparation of other EtxB-based fusion proteins
AI3SD Video: Machines Learning Chemistry
No full textReinvigorated by algorithmic developments, faster hardware and large data sets, machine learning is pervading many aspects of chemistry. We present two examples from our recent studies, one in the area of drug discovery, the other focused on protein spectroscopy. In the first, we consider pharmaceutical lead discovery as active search in a space of labelled graphs [1]. We extend a recent data-driven adaptive Markov chain approach, and evaluate it on a focused drug design problem, where we search for an antagonist of an 훂v integrin, the target protein that belongs to a group of Arg-Gly-Asp integrin receptors. In the second example, we present a novel machine learning protocol that uses a few key structural descriptors to predict amide I IR spectra of proteins and agrees well with experiment [2]. Its transferability enabled us to distinguish protein secondary structures, probe atomic structure variations with temperature, and monitor protein folding.[1] Oglic, D., Oatley, S.A., Macdonald, S.J.F., McInally, T., Garnett, R., Hirst, J.D. & Gärtner, T. Active search for computer-aided drug design. Mol. Inf., 2018, 37, 1700130. https://doi.org/10.1002/minf.201700130[2] Ye, S., Zhong, K., Zhang, J., Hu, W., Hirst, J.D., Zhang, G., Mukamel, S., Jiang, J. A transferable machine learning protocol for predicting protein amide-I infrared spectra. J. Am. Chem. Soc., 2020, 142, 19071. https://doi.org/10.1021/jacs.0c0653
Efficient extracellular production of hybrid escherichia-coli heat-labile enterotoxin-b subunits in a marine vibrio
No full textEscherichia coil heat-labile enterotoxin B subunit (EtxB) has been proposed as a potential protein carrier for the delivery of heterologous peptides to target cells, particularly for the oral delivery of epitopes to the mucosal immune system. In this study, two extensions to the C-terminus of EtxB were genetically engineered that correspond to a well-characterized neutralising epitope of glycoprotein D from herpes simplex virus (EtxB-gD) and to the C-terminal nine amino acids from the 38 kDa subunit of HSV-encoded ribonucleotide reductase (EtxB-R2). Here we describe the extracellular secretion of the two hybrid EtxBs from a marine Vibrio harbouring a broad-host range inducible expression vector containing the hybrid genes. Large amounts of intact fusion proteins (15-20 mg per liter of culture) were secreted into the medium upon induction. These hybrid proteins maintained the receptor-binding activity of the native toxin as well as being cross-reactive with anti-EtxB and anti-heterologous peptide monoclonal antibodies
EDTA protects E. coli heat-labile enterotoxin B subunit-based fusion proteins from proteolytic degradation during their production by Vibrio spp.
No full textE. coli heat-labile enterotoxin B subunit (EtxB) has been proposed as a potential protein carrier for the delivery of heterologous peptides into target cells. To thoroughly exploit the biotechnological potential of EtxB-based fusion proteins, a simple method has to be worked out for their expression and purification. Production of these chimeric toxins faces problems with regard to both their low yield and stability. In this study we describe a protocol for the optimal production and secretion of undegraded EtxB hybrids into the extracellular medium of cultures of non-toxinogenic Vibrio strains. The highest level of expression of two chimeric toxins, EtxB-R2 and EtxB-pol, was obtained by using Vibrio sp.60 cultures, reaching 52 mg/l and 8 mg/l, respectively. The presence of 0.3 mM EDTA in the culture medium totally preserved both chimeric toxins from proteolysis, also during a prolonged expression. This system may be useful for the preparation of other EtxB-based fusion proteins
1993-1994 T. R. Pearson
No full textT. R. Pearson, a.k.a. Rick Gavin, was born in Winston-Salem, North Carolina. He was a student at North Carolina State University, where he gained a B.A. and M.A. in English. He was the first recipient of the John and Renée Grisham Writer in Residence Fellowship. He is the acclaimed author of fourteen novels, including A Short History of a Small Place and Warwolf, and a dozen screenplays. Top of the Rock is his fifth nonfiction book. He lives in Virginia and Brooklyn, New York. (Photo credit: Marian Young)https://egrove.olemiss.edu/grisham_res/1026/thumbnail.jp
"Closing the R&D Gap, Evaluating the Sources of R&D Spending"
No full textBoth spending and tax policies have been implemented in the United States with the goal of stimulating private sector research and development (R&D). Karier questions whether current R&D policy, especially the research and experimentation tax credit, can contribute to closing the gap between nondefense expenditures on R&D in the United States and such expenditures in other countries, such as Japan and Germany. He also explores possible changes to our current R&D policy to make it more effective.
Letter from J. R. Eakin to Carl Hayden
No full textLetter from J. R. Eakin to Carl T. Hayden concerning access to Rowe Well and the canyon
Letter from J. R. Eakin to Stephen Mather
No full textLetter from J. R. Eakin to Stephen T. Mather about expenses and reconstruction of the Kaibab Trail
Letter from Carl Hayden to J. R. Eakin
No full textLetter from Carl T. Hayden to J. R. Eakin regarding changes to the Grand Canyon National Park boundaries and the purchase of lands from William Randolph Hearst
Letter from F. R. Goodman to Carl Hayden
No full textLetter from F. R. Goodman to Carl T. Hayden asking for clarification about the agreement to construct an approach road to the par
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