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Ithaca, NY, United States

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Patent
Tetragenetics | Date: 2015-05-05

Methods are disclosed for the production of mammalian voltage-gated ion channels in ciliates. In other aspects, compositions comprising lipid bilayers containing mammalian voltage-gated ion channels are disclosed. In other aspects, compositions comprising purified and reconstituted mammalian voltage-gated ion channels are disclosed.


Tran E.J.,Vanderbilt University | Tran E.J.,Purdue University | Noble K.N.,Vanderbilt University | Noble K.N.,Meharry Medical College | And 7 more authors.
Genes and Development | Year: 2011

Nuclear export of messenger RNA (mRNA) occurs by translocation of mRNA/protein complexes (mRNPs) through nuclear pore complexes (NPCs). The DEAD-box protein Dbp5 mediates export by triggering removal of mRNP proteins in a spatially controlled manner. This requires Dbp5 interaction with Nup159 in NPC cytoplasmic filaments and activation of Dbp5's ATPase activity by Gle1 bound to inositol hexakisphosphate (IP 6). However, the precise sequence of events within this mechanism has not been fully defined. Here we analyze dbp5 mutants that alter ATP binding, ATP hydrolysis, or RNA binding. We found that ATP binding and hydrolysis are required for efficient Dbp5 association with NPCs. Interestingly, mutants defective for RNA binding are dominant-negative (DN) for mRNA export in yeast and human cells. We show that the DN phenotype stems from competition with wild-type Dbp5 for Gle1 at NPCs. The Dbp5-Gle1 interaction is limiting for export and, importantly, can be independent of Nup159. Fluorescence recovery after photobleaching experiments in yeast show a very dynamic association between Dbp5 and NPCs, averaging <1 sec, similar to reported NPC translocation rates for mRNPs. This work reveals critical steps in the Gle1-IP 6/Dbp5/Nup159 cycle, and suggests that the number of remodeling events mediated by a single Dbp5 is limited. © 2011 by Cold Spring Harbor Laboratory Press.


Choudhary V.,University of Fribourg | Wolyniak M.J.,Hampden-Sydney College | Scarcelli J.J.,Tetragenetics | Schneiter R.,University of Fribourg
Journal of Cell Science | Year: 2010

Cells of Saccharomyces cerevisiae lacking Apq12, a nuclear envelope (NE)-endoplasmic reticulum (ER) integral membrane protein, are defective in assembly of nuclear pore complexes (NPCs), possibly because of defects in regulating membrane fluidity. We identified BRR6, which encodes an essential integral membrane protein of the NE-ER, as a dosage suppressor of apq12Δ. Cells carrying the temperature-sensitive brr6-1 allele have been shown to have defects in nucleoporin localization, mRNA metabolism and nuclear transport. Electron microscopy revealed that brr6-1 cells have gross NE abnormalities and proliferation of the ER. brr6-1 cells were hypersensitive to compounds that affect membrane biophysical properties and to inhibitors of lipid biosynthetic pathways, and displayed strong genetic interactions with genes encoding non-essential lipid biosynthetic enzymes. Strikingly, brr6-1 cells accumulated, in or near the NE, elevated levels of the two classes of neutral lipids, steryl esters and triacylglycerols, and over-accumulated sterols when they were provided exogenously. Although neutral lipid synthesis is dispensable in wild-type cells, viability of brr6-1 cells was fully dependent on neutral lipid production. These data indicate that Brr6 has an essential function in regulating lipid homeostasis in the NE-ER, thereby impacting NPC formation and nucleocytoplasmic transport.


Parker S.A.,Cornell University | Diaz I.L.-C.,Cornell University | Anderson K.A.,Cornell University | Anderson K.A.,Tetragenetics | Batt C.A.,Cornell University
Protein Expression and Purification | Year: 2013

A single chain variable fragment (J591 ScFv) that recognizes the extracellular glyco-protein prostate specific membrane antigen (PSMA) was designed, constructed, and expressed in Pichia pastoris. Construction of the J591 ScFv was based on the reported complementarity-determining region (CDR) of the PSMA specific J591 monoclonal antibody (mAb). The nucleotide sequence encoding the J591-derived ScFv was codon-optimized for expression in P. pastoris and a 6× his-tag was added to facilitate affinity purification. A down-scale 2 L methanol-induced P. pastoris fermentation yielded 330 mg of total protein following a 96 h induction. Following Immobolized Metal Affinity Chromatography, functionality of the J591 ScFv was confirmed via Western blot, immunoblot, binding studies, and flow cytometry analysis. The J591 ScFv showed binding affinity and specificity to cell extracts containing PSMA and PSMA-expressing prostate cancer cells. Our results demonstrate that functional J591 ScFv can be produced in P. pastoris for use in diagnostic and targeted therapeutic applications. © 2013 Elsevier Ltd. All rights reserved.


Bisharyan Y.,Cornell University | Bisharyan Y.,Tetragenetics | Clark T.G.,Cornell University
Mitochondrion | Year: 2011

Here we demonstrate that ciliated protozoa can jettison mitochondria as intact organelles, releasing their contents to the extracellular space either in a soluble form, or in association with membrane vesicles at the cell periphery. The response is triggered by lateral clustering of GPI-anchored surface antigens, or by heat shock. In the first instance, extrusion is accompanied by elevated levels of intracellular calcium and is inhibited by Verapamil and BAPTA-AM arguing strongly for the involvement of calcium in triggering the response. Cells survive mitochondrial discharge raising the interesting possibility that extrusion is an early evolutionary adaptation to cell stress. © 2011 Elsevier B.V. and Mitochondria Research Society.


Madinger C.L.,New England Biolabs | Collins K.,University of California at Berkeley | Collins K.,Tetragenetics | Fields L.G.,New England Biolabs | And 2 more authors.
Eukaryotic Cell | Year: 2010

The growth, survival, and life cycle progression of the freshwater ciliated protozoan Tetrahymena thermophila are responsive to protein signals thought to be released by constitutive secretion. In addition to providing insights about ciliate communication, studies of constitutive secretion are of interest for evaluating the utility of T. thermophila as a platform for the expression of secreted protein therapeutics. For these reasons, we undertook an unbiased investigation of T. thermophila secreted proteins using wild-type and secretion mutant strains. Extensive tandem mass spectrometry analyses of secretome samples were performed. We identified a total of 207 secretome proteins, most of which were not detected in a set of abundant whole-cell protein identifications. Numerous proteases and other hydrolases were secreted from cells grown in rich medium but not cells transferred to a nutrient starvation condition. On the other hand, we detected the starvation-enhanced secretion of a small number of cytosolic proteins, suggestive of an exosome-like pathway in T. thermophila. Subsets of proteins from the T. thermophila regulated secretion pathway were detected with differential representation across strains and culture conditions. Finally, many secretome proteins had a predicted N-terminal signal sequence but no other annotated characteristic or functional classification. Our work provides the first comprehensive analysis of secreted proteins in T. thermophila and establishes the groundwork for future studies of constitutive protein secretion biology and biotechnology in ciliates. © 2010, American Society for Microbiology.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 1.58M | Year: 2015

DESCRIPTION provided by applicant Tetragenetics Inc an early stage biotechnology company in Cambridge MA has developed a powerful cross cutting technology for the overexpression of eukaryotic membrane and secreted proteins using Tetrahymena thermophila an important model organism with a unique features particularly well suited to the production of these important classes of proteins The companyandapos s proprietary SionXTM technology for example has enabled expression of properly folded and functional human ion channel at levels fold higher than has previously been possible with mammalian CHO or HEK cells the current gold standard for the production of these otherwise difficult to produce proteins These data have garnered considerable attention within the biotech and large pharma arenas and while the platform has numerous applications in basic research transfer of the technology to the broader academic community has challenges as well From a technical standpoint Tetrahymena is complex in terms of its genetics and not amenable to the kinds of andquot out of the boxandquot kit type solutions available for more conventional systems At the same time much of the technology for cell line development and expression optimization is proprietary and demands relatively high upfront licensing fees for commercial use that are beyond what academic researchers can afford To address this issue and to make the Tetrahymena platform accessible to the entire biomedical research community Tetragenetics Inc proposes to join forces with the NIH ORIP funded Tetrahymena Stock Center at Cornell University to provide overexpressing cell lines and or starting material for protein purification to not for profit academic and government research laboratories at reasonable costs The Stock Center has a well developed infrastructure for ordering storing and disseminating strains while Tetragenetics has the state of the art vectors cell lines and growth conditions that make this system so powerful The proposal describes how this public private partnership will work to bring the technology to the not for profit research community Additionally the proposal delineates plans to expand the capabilities of the current platform by developing new tools for heterologous gene expression including new vectors allowing andapos plug and playandapos options for construct design particularly with respect to epitope tag placement new promoters for driving inducible gene expression and new cell lines that will permit identification of positive transformants without direct drug selection These improvements will expand the flexibility of the platform for both academic and commercial use Finally Tetragenetics will explore use of the SionXTM platform for expression of historically challenging G protein coupled receptors in collaboration with Heptares Therapeutics a British company that has pioneered the development of stabilized GPCRs for structure based drug design The proposed studies are intended to remove a major stumbling block to the study of broad families of proteins that are critically important in normal functionin of the nervous respiratory endocrine and urinary and immune systems PUBLIC HEALTH RELEVANCE The proposed project seeks to put a new and powerful technology for the production of genetically engineered proteins into the hands of basic scientists in academic and government laboratories through the establishment of a public private partnership between Tetragenetics Inc an early stage biotechnology company in Cambridge MA and the NIH funded Tetrahymena Stock Center at Cornell University in Ithaca NY The underlying technology platform will permit in depth studies on proteins that were previously inaccessible with the long term goal of developing new and improved drugs for diseases ranging from neuropathic pain to cancer and autoimmunity


Patent
Tetragenetics | Date: 2014-03-04

This invention is directed to methods for recombinant polypeptide production and, in particular, methods and products for the production of recombinant polypeptides in ciliates.


This invention is directed to methods for the production of immunogenic granular particles. In certain embodiments, the invention is directed to methods and products for the production immunogenic granular particles produced in ciliates. In certain embodiments, the invention is directed to compositions comprising Grl/Ag fusion polypeptides.


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