Synaptic Research, LLC

Other

University of California

Johns Hopkins University

Tufts College

Ipsen Pharma S.a.s.

George A Oyler

Yien Che Tsai

Julian N Rosenberg

Yung-Nien Chang

Paul S Fishman

Randall L Kincaid

Charles Bix Shoemaker

Michael J Betenbaugh

Marc D Donohue

Lynette B Corbeil

Rachel Schrier

Mark Hildebrand

Roshan Shrestha

Chuehling Kuo

Aubrey Davis

Yen Che Tsai

A system for the identification of proteases and protease inhibitors is provided. The system has at least two components. The first component is a reporter construct with at least one binding site, a transcriptional promoter, an inducible promoter region, and at least one reporter gene, all functionally connected for expression of the reporter gene(s) in functional coordination with a transcriptional activation agent. The second component is a transcriptional activation agent comprising a nucleic acid binding domain, at least one protease substrate domain, and at least one transcriptional activation domain for an inducible promoter. The system allows detection and evaluation of agents affecting protease activity directed to the protease substrate domain. The system also allows for the detection of the presence of proteases in environmental samples.

System for the assessment of protease activity

A cell based assay for detection for protease activity is disclosed. In the assay a cell is engineered to express a protease substrate with at least one label, preferably on its C-terminus. Cleavage of the substrate by the protease that recognizes it results in a C-terminal fragment and a N-terminal fragment, where the fragment having the label is subject to ubiquitin proteasome degradation. The assay measures the disappearance of the label due to degradation of the fragment to which it is attached. A cell free assay is also described for detection of protease activity. In the cell free assay, the protease substrate is expressed in a solution that includes the elements of the ubiquitin proteasome pathway for degradation of the fragment. The assay measures the disappearance of the label attached to the fragment that results from cleavage by the protease.

N-end rule protease activity indication methods and uses thereof

This invention provides diatom-based vaccines.

Diatom-based vaccines

The present invention provides methods for the isolation of oils from intact or lysed microorganisms in aqueous media with pressurized carbon dioxide as a solute. Such oils may be used for the production of biofuels. Also provided for are methods for harvesting and rupturing whole cell microorganisms in aqueous media with pressurized carbon dioxide as a solute.

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A novel protein delivery system to generate induced pluripotent stem (iPS) cells is described. The delivery system comprises a construct with a receptor binding domain that recognizes a receptor in a somatic cell, a translocation domain that allows the transfer of an inducer into the cytosolic space, and a cargo bearing domain to which the inducer is attached and facilitates transfer of the inducer into the cell.

Protein delivery system to generate induced pluripotent stem (iPS) cells or tissue-specific cells

The present invention relates to a designer or recombinant ubiquitin ligase molecule that includes a toxin binding domain that is specific for a toxin active fragment, wherein the toxin active fragment is an enzymatically active fragment of one or more toxins or toxin serotypes; and an E3-ligase domain that comprises an E3-ligase or polypeptide that facilitates E2-mediated ubiquitination of the toxin active fragment. In an embodiment, the composition further includes a delivery system that allow the designer ubiquitin ligase to enter the cell. The present invention further includes methods for treating an individual intoxicated with a toxin by administering the designer ubiquitin ligase of the present invention.

Methods of degrading or inhibiting botulinum neurotoxin using designer ubiquitin ligases

A system and method for identifying aneurotoxin inhibitor employing aneurotoxin substrate complex having a peptide substrate, preferably SNAP-25, a reporter domain on one side of said peptide substrate and an immobilization domain on the opposite side of said peptide substrate. Theneurotoxin inhibitor is identified by its ability to decrease the relative amount of cleaved complex, detected through measuring a decrease in complex bound to a solid support. The method of the present invention also utilizes novel cells that express aneurotoxin substrate complex. Also provided are novel stable cell lines that express thetoxin substrate complex and viral vectors capable of efficiently expressing an active light chain of the BoNT within mammalian cells.

Methods for identifying inhibitors of botulinum neurotoxins

The invention provides eukaryotic unicellular algae engineered to express a nucleosome alteration protein fused to a protein with affinity to the DNA binding site acting in coordination. An example is a LexA-p300 fusion protein, where the p300 is derived from. The LexA binding domain guides the p300 to the binding site and the p300 loosens the nucleosome structure by acetylating histones within proximity of the transgene, thus remodeling the local chromatin structure to allow for high-level expression.

Enhanced gene expression in algae

The present invention relates to methods, systems, and kits for intoxicating cells, neuronal and non-neuronal cells, with a toxin or fragment thereof. This is done by subjecting toxin substrate and a lipid or polymeric carrier (e.g., DNA uptake facilitating agent) to one or more cells for use in cell based assays. In an aspect, the methods of the present invention allow for high throughput assays and, as such, for the evaluation of drug candidates.

Methods for the delivery of toxins or enzymatically active portions thereof

The invention provides a system of enhancing the expression of transgenes in algae. Transgenes are engineered to have a binding site for certain proteins in proximity to their promoter, for example a LexA binding site. The algae is also engineered to express a nucleosome alteration protein fused to a protein with affinity to the DNA binding site acting in coordination. An example is a LexA-p300 fusion protein, where the p300 is derived from. The LexA binding domain guides the p300 to the binding site and the p300 loosens the nucleosome structure by acetylating histones within proximity of the transgene, thus remodeling the local chromatin structure to allow for high-level expression.

Protein delivery system to generate pluripotent stem (iPS) cells or tissue specific cells

Designer ubiquitin ligases having a non-cleavable SNAP25 domain and E3-ligase domain

A system and method for identifying a botulinum neurotoxin inhibitor employing a botulinum neurotoxin substrate complex having a peptide substrate, preferably SNAP-25, a reporter domain on one side of said peptide substrate and an immobilization domain on the opposite side of said peptide substrate. The botulinum neurotoxin inhibitor is identified by its ability to decrease the relative amount of cleaved complex, detected through measuring a decrease in complex bound to a solid support. The method of the present invention also utilizes novel cells that express a botulinum neurotoxin substrate complex. The methods of the present invention are adapted for cell based screening to monitor the catalytic activity of a BoNT in living cells and to identify molecules that inhibit the catalytic activity of a BoNT in living cells. Also provided are novel stable cell lines that express the botulinum toxin substrate complex and viral vectors capable of efficiently expressing an active light chain of the BoNT within mammalian cells.

A system and method for identifying aneurotoxin inhibitor employing aneurotoxin substrate complex having a peptide substrate, preferably SNAP-25, a reporter domain on one side of said peptide substrate and an immobilization domain on the opposite side of said peptide substrate. Theneurotoxin inhibitor is identified by its ability to decrease the relative amount of cleaved complex, detected through measuring a decrease in complex bound to a solid support. The method of the present invention also utilizes novel cells that express aneurotoxin substrate complex. The methods of the present invention are adapted for cell based screening to monitor the catalytic activity of a BoNT in living cells and to identify molecules that inhibit the catalytic activity of a BoNT in living cells. Also provided are novel stable cell lines that express thetoxin substrate complex and viral vectors capable of efficiently expressing an active light chain of the BoNT within mammalian cells.

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Baltimore, MD, United States of America

George A Oyler