TetraExpress™ encompasses the core recombinant gene expression system that
enables the production of recombinant proteins in Tetrahymena thermophila.
The system is designed to include:
Tetrahymena thermophila harbors an AT rich genome with alternative codon usage. While not necessary in all cases, recombinant genes are typically optimized for expression in Tetrahymena.
TetraGenetics utilizes its own codon optimization table that was developed based on the analysis of highly expressed Tetrahymena genes. Following optimization genes are generally synthesized by third party vendors.
TetraGenetics uses proprietary ribosomal DNA vectors (pTRAS series) for transformation and expression in Tetrahymena. pTRAS vectors are reassembled into independent palindromic chromosomes in transformed cells that are amplified up to 9,000 times leading to transgene copy numbers of up to 18,000 per cell.
A number of inducible promoters are utilized in TetraExpress™ including robust inducible metallothionein promoters (e.g MTT5) that are activated by divalent metal cations (e.g. Cd2+, Ca2+). Additionally, TetraGenetics has developed Starvation based promoters that are activated following a shift of the cells from nutrient-rich to nutrient-deficient medium.
Tetrahymena are grown in complex proteose-peptone based medium and will achieve a cell density of approximately 1-2e6 cells/ml in shake flasks with strain-dependent doubling times typically ranging from 2-3 hours. Cells are harvested by low speed centrifugation prior to processing.