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Principal Investigator: Dr. Theodore Clark
Contact Information: E-mail: tgc3@cornell.edu - Phone: 607-253-4042
Sponsor: Tetragenetics, Inc.
Grant Number: W911NF06-C-0095
Title: Recombinant and Engineered Protein Accumulation Systems (REPAS)
Annual Direct Cost: $28,559
Project Period: 07/21/06-01/22/07
DESCRIPTION (provided by applicant): To demonstrate the utility of T. thermophila as a low-cost platform for rapid production of recombinant proteins on a large-scale we propose the following three specific aims: 1) The coding sequence for the hemagglutinin protein from the current H5N1 strain of avian flu will be cloned into a high-copy number rDNA-based vector and introduced into laboratory strains of T. thermophila. Gene constructs will prepared in such a way that resulting protein product (that is, HA) is targeted either to cilia, the constitutive secretory pathway, or an early step in the regulated secretory pathway. 2) Resulting transformed cell lines will be screened for protein synthesis using antibodies specific for the H5 hemagglutinin, and relative expression levels will be assessed. 3) Rapid methods of downstream purification will be applied to determine the most favorable strategies for large-scale production. The essential questions that will be addressed in this proposal are first, and foremost, will these proteins be expressed in high yield, and second, will they be targeted to cellular compartments that permit streamlined, low cost purification following expression. The short time line for completion of the project requires that we be able to clone genes and derive stably transformed cell lines reproducibly and rapidly. Based on previous work, we believe these latter demands should be relatively easy to address.
We chose HA as the target for these studies based on a number of criteria. On the one hand, it represents a model for the types of difficult to express proteins that can potentially be made in the Tetrahymena system. At the same time, production of a low cost, rapidly deployable vaccine against flu would have enormous benefit to mankind along with high commercial value in the marketplace. Finally, we believe the production of stably transformed cell lines that overexpress HA can be readily accomplished within the 6-month time frame of the award.
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