| |
Tetrahymena thermophila, a sophisticated microbial eukaryote that combines ease of growth with biological properties not found in other systems, has emerged as a superior manufacturing platform for difficult to express eukaryotic membrane and secreted proteins. With a doubling time of 2-3 hrs, Tetrahymena reaches high biomass in a short time on a variety of inexpensive serum free media, and is fully scalable in bioreactors of 1,000 L or more. It is classified as a Biosafety Level 1 organism and maintains "Generally Regarded as Safe" (GRAS) status.
More importantly, our proprietary vectors and cell lines make possible stable transformation with heterologous genes at copy numbers approaching 18,000 per cell, resulting in high-level protein expression (TetraExpress™ Technology Platform). Unlike bacteria and yeast, Tetrahymena has no cell wall to impede downstream protein purification and is capable of mammalian-like post-translational modifications including simple core N-glycans that closely resemble those in humans. With hundreds of cilia that expand its surface, Tetrahymena devotes a large part of its metabolism to membrane protein production and is ideally suited to the expression of ion channels, GPCR’s, ABC-transporters, protein kinases, and surface antigens.
Furthermore, Tetrahymena is uniquely capable of both constitutive and regulated secretion. The organism stores ~ 20% of its total cell protein in several thousand dense core secretory granules that can be triggered to discharge at will. The material released from these granules takes the form of a proteinaceous gel (PRISM™) that can be readily harvested following granule discharge. Recombinant proteins can be targeted to this compartment and recovered in a few simple steps making PRISM™ a disruptive technology for production and purification of high value targets. Finally, PRISM™ has intrinsic immunostimulatory properties that make it a potent antigen-presenting vehicle for the delivery of low-cost, multi-subunit vaccines.
The first “animal-like” cell to be grown in pure culture, Tetrahymena has served as a key model for basic research in cellular biology and genetics since the 1950’s. Its development, physiology, biochemistry, and ultrastructure have been well studied, and its use as a model has yielded numerous insights into such fundamental processes as dynein-based motility; RNA self-splicing (Cech; Nobel Prize, 1989); the role of RNAi in chromatin remodeling (Allis, Gorovsky, 2002 Science “Breakthrough of the Year”) histone modifications and their role in chromatin dynamics (Allis; Gairdner International Award, 2007); and telomere structure and biosynthesis (Greider and Blackburn; Nobel Prize, 2009), to name a few. The large (~100Mb) T. thermophila macronuclear genome has been sequenced and annotated, greatly facilitating genetic manipulation in this organism and microarray expression analyses of the genome in different physiological and developmental stages are available.
While often likened to a metazoan in terms of its cellular complexity, T. thermophila is remarkably easy to grow. Vegetative cultures divide rapidly at 15ºC - 41ºC in a range of nutritional media including cGMP-compliant animal-free media; peptone-based media; non-fat milk; bacterized media; and nutritionally complete, totally defined synthetic media. Cells can be grown in microdrops, microtiter plates, shake flasks, or controlled growth fermentors to cell densities of 2 x 107 cell per ml (equivalent to a dry weight of ~50 g/L). Clones of interest can be easily frozen in liquid nitrogen for long-term storage.
TetraExpress™ is our toolkit of proprietary vectors and cell lines optimized for high-level expression of recombinant proteins in the Tetrahymena system. T. thermophila like other ciliates, has two functionally distinct nuclei - a polyploid macronucleus that is transcriptionally active, and a diploid micronucleus that is silent and functions only during sexual conjugation. Tetrahymena can be readily transformed with foreign DNA in either the macro- or micronucleus by homologous recombination. A variety of constitutive and inducible promoters can be used to drive heterologous gene expression. In addition to transformation at somatic gene loci, one can introduce foreign genes on novel ribosomal DNA-based vectors that become amplified as palindromic mini-chromosomes to very high copy number during conjugation. Following this process, transgenes are present at ~18,000 copies per cell resulting in robust expression at the protein level. Even difficult to express eukaryotic membrane proteins can be produced at >3-5% of total cell protein using this approach. Ribosomal DNA vectors will also accommodate multiple coding sequences and are ideal for the production of multi-subunit proteins, including fully assembled H+L chain monoclonal antibodies.
Engineering cis-telomerase RNAs that add telomeric repeats to themselves. (2010). Qiao F, Goodrich KJ, Cech TR. (2010). Proc Natl Acad Sci U S A. 1107(11):4914-8
Constitutive secretion in Tetrahymena thermophila. Madinger CL, Collins K, Fields LG, Taron CH, Benner JS. Eukaryot Cell. 2010 Mar 26.
Microarray analyses of gene expression during the Tetrahymena thermophila life cycle. (2009). Miao W, Xiong J, Bowen J, Wang W, Liu Y, Braguinets O, Grigull J, Pearlman RE, Orias E, Gorovsky MA. PLoS One. 4(2):e4429.
Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure. (2008). Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote. Eisen JA, Coyne RS, Wu M, Wu D, Thiagarajan M, Wortman JR, Badger JH, Ren Q, Amedeo P, Jones KM, Tallon LJ, Delcher AL, Salzberg SL, Silva JC, Haas BJ, Majoros WH, Farzad M, Carlton JM, Smith RK Jr, Garg J, Pearlman RE, Karrer KM, Sun L, Manning G, Elde NC, Turkewitz AP, Asai DJ, Wilkes DE, Wang Y, Cai H, Collins K, Stewart BA, Lee SR, Wilamowska K, Weinberg Z, Ruzzo WL, Wloga D, Gaertig J, Frankel J, Tsao CC, Gorovsky MA, Keeling PJ, Waller RF, Patron NJ, Cherry JM, Stover NA, Krieger CJ, del Toro C, Ryder HF, Williamson SC, Barbeau RA, Hamilton EP, Orias E. PLoS Biol. 2006 Sep;4(9):e286.
RNA-guided DNA deletion in Tetrahymena: an RNAi-based mechanism for programmed genome rearrangements. Yao MC, Chao JL. Annu Rev Genet. 2005;39:537-59
Functional genomics: the coming of age for Tetrahymena thermophila. Turkewitz AP, Orias E, Kapler G. Trends Genet. 2002 Jan;18 (1):35-40. Review.
A robust inducible-repressible promoter greatly facilitates gene knockouts, conditional expression, and overexpression of homologous and heterologous genes in Tetrahymena thermophila. (2002). Shang Y, Song X, Bowen J, Corstanje R, Gao Y, Gaertig J, Gorovsky MA. Proc Natl Acad Sci U S A. 99:3734-9.
|
|
|
