TetraExpress Technology

TetraExpress™ encompasses the core recombinant gene expression system that
enables the production of recombinant proteins in Tetrahymena thermophila.
The system is designed to include:

  • Synthetic genes, codon-optimized for high-level expression in Tetrahymena
  • Expression cassettes that place genes under the control of robust and inducible promoters
  • Very high-copy Tetrahymena ribosomal DNA (rDNA) expression vectors
  • Generation, storage and maintenance of transgenic Tetrahymena cell lines

tetrahymena

Gene Optimization

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.

gene-optimization

Vectors and Promoters

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.

vectors-and-promoters

Culture Growth and Harvest

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.

culture-growth-and-harvest-combined