The bifunctional dihydrofolate reductase thymidylate synthase of Tetrahymena thermophila provides a tool for molecular and biotechnology applications

BACKGROUND: Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are crucial enzymes in DNA synthesis. In alveolata both enzymes are expressed as one bifunctional enzyme. RESULTS: Loss of this essential enzyme activities after successful allelic assortment of knock out alleles yields an auxotrophic marker in ciliates. Here the cloning, characterisation and functional analysis of Tetrahymena thermophila's DHFR-TS is presented. A first aspect of the presented work relates to destruction of DHFR-TS enzyme function in an alveolate thereby causing an auxotrophy for thymidine. A second aspect is to knock in an expression cassette encoding for a foreign gene with subsequent expression of the target protein. CONCLUSION: This system avoids the use of antibiotics or other drugs and therefore is of high interest for biotechnological applications

Biochemical and molecular characterisation of Tetrahymena thermophila extracellular cysteine proteases

BACKGROUND: Over the last decades molecular biologic techniques have been developed to alter the genome and proteome of Tetrahymena thermophila thereby providing the basis for recombinant protein expression including functional human enzymes. The biotechnological potential of Tetrahymena has been proved in numerous publications, demonstrating fast growth, high biomass, fermentation in ordinary bacterial/yeast equipment, up-scalability, existence of cheap and chemical defined media. For these reasons Tetrahymena offers promising opportunities for the development of a high expression system. Yet optimised high yield strains with protease deficiency such as commonly used in yeast and bacterial systems are not available. RESULTS: This work presents the molecular identification of predominant proteases secreted into the medium by Tetrahymena thermophila. A one-step purification of the proteolytic enzymes is described. CONCLUSION: The information provided will allow silencing of protease activity by either knock out methods or by Tetrahymena specific antisense-ribosome-techniques. This will facilitate the next step in the advancement of this exciting organism for recombinant protein production

A recombinase system facilitates cloning of expression cassettes in the ciliate Tetrahymena thermophila

BACKGROUND: Tetrahymena thermophila is one of the best characterized unicellular eukaryotes and its genome is sequenced in its entirety. However, the AT-richness of the genome and an unusual codon usage cause problems in cloning and expression of the ciliate DNA. To overcome these technical hiatuses we developed a Cre-dependent recombinase system. RESULTS: We created novel donor and acceptor vectors that facilitate the transfer of expression cassettes from the donor into novel acceptor plasmid. Expression vectors were used that encode the 19 kDa C-terminus of the MSP1 protein of Plasmodium falciparum and a blasticidin S (bsdR) resistance gene, respectively. The functional expression of these genes was demonstrated by western blot analysis with MSP1 specific antibodies and by a blasticidin growing assay. CONCLUSION: The Cre dependent recombinase system in combination with the modular structure of the donor vectors ease cloning and expression of foreign genes in the ciliate system, providing a powerful tool for protistology research in future

Expression, secretion and surface display of a human alkaline phosphatase by the ciliate Tetrahymena thermophila

<p>Abstract</p> <p>Background</p> <p><it>Tetrahymena thermophila </it>possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites <it>Ichthyophthirius multifiliis </it>and <it>Plasmodium falciparum </it>and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that <it>T. thermophila </it>is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using <it>T. thermophila </it>and thereby presents a powerful tool for the optimization of the ciliate-based expression system.</p> <p>Results</p> <p>Functional and full length human intestinal alkaline phosphatase was expressed by <it>T. thermophila </it>using a codon-adapted gene containing the native signal-peptide and GPI (Glycosylphosphatidylinositol) anchor attachment signal. HiAP activity in the cell extract of transformants suggested that the hiAP gene was successfully expressed. Furthermore, it was demonstrated that the enzyme was modified with N-glycosylation and localized on the surface membrane by the C-terminal GPI anchor. A C-terminally truncated version of hiAP lacking the GPI anchor signal peptide was secreted into the medium as an active enzyme. In a first approach to establish a high level expression system up to 14,000 U/liter were produced in a time frame of two days, which exceeds the production rate of other published expression systems for this enzyme.</p> <p>Conclusions</p> <p>With the expression of hiAP, not only a protein of commercial interest could be produced, but also a reporter enzyme that offers the possibility to analyze <it>T. thermophila </it>genes that play a role in the regulation of protein secretion. Additionally, the fact that ciliates do not secrete an endogenous alkaline phosphatase provides the possibility to use the truncated hiAP as a reporter enzyme, allowing the quantification of measures that will be necessary for further optimization of the host strains and the fermentation processes.</p

Secretion of functional human enzymes by Tetrahymena thermophila

BACKGROUND: The non-pathogenic ciliate Tetrahymena thermophila is one of the best-characterized unicellular eucaryotes used in various research fields. Previous work has shown that this unicellular organism provides many biological features to become a high-quality expression system, like multiplying to high cell densities with short generation times in bioreactors. In addition, the expression of surface antigens from the malaria parasite Plasmodium falciparum and the ciliate Ichthyophthirius multifiliis suggests that T. thermophila might play an important role in vaccine development. However, the expression of functional mammalian or human enzymes remains so far to be seen. RESULTS: We have been able to express a human enzyme in T. thermophila using expression modules that encode a fusion protein consisting of the endogenous phospholipase A(1 )precursor and mature human DNaseI. The recombinant human enzyme is active, indicating that also disulfide bridges are correctly formed. Furthermore, a detailed N-glycan structure of the recombinant enzyme is presented, illustrating a very consistent glycosylation pattern. CONCLUSION: The ciliate expression system has the potential to become an excellent expression system. However, additional optimisation steps including host strain improvement as wells as measures to increase the yield of expression are necessary to be able to provide an alternative to the common E. coli and yeast-based systems as well as to transformed mammalian cell lines

Astrocytes grown in Alvetex® 3 dimensional scaffolds retain a non-reactive phenotype

yesProtocols which permit the extraction of primary astrocytes from either embryonic or postnatal mice are well established however astrocytes in culture are different to those in the mature CNS. Three dimensional (3D) cultures, using a variety of scaffolds may enable better phenotypic properties to be developed in culture. We present data from embryonic (E15) and postnatal (P4) murine primary cortical astrocytes grown on coated coverslips or a 3D polystyrene scaffold, Alvetex. Growth of both embryonic and postnatal primary astrocytes in the 3D scaffold changed astrocyte morphology to a mature, protoplasmic phenotype. Embryonic-derived astrocytes in 3D expressed markers of mature astrocytes, namely the glutamate transporter GLT-1 with low levels of the chondroitin sulphate proteoglycans, NG2 and SMC3. Embroynic astrocytes derived in 3D show lower levels of markers of reactive astrocytes, namely GFAP and mRNA levels of LCN2, PTX3, Serpina3n and Cx43. Postnatal-derived astrocytes show few protein changes between 2D and 3D conditions. Our data shows that Alvetex is a suitable scaffold for growth of astrocytes, and with appropriate choice of cells allows the maintenance of astrocytes with the properties of mature cells and a non-reactive phenotype.BBSR

The bifunctional dihydrofolate reductase thymidylate synthase of provides a tool for molecular and biotechnology applications-4

<p><b>Copyright information:</b></p><p>Taken from "The bifunctional dihydrofolate reductase thymidylate synthase of provides a tool for molecular and biotechnology applications"</p><p>BMC Biotechnology 2006;6():21-21.</p><p>Published online 20 Mar 2006</p><p>PMCID:PMC1435751.</p><p>Copyright © 2006 Herrmann et al; licensee BioMed Central Ltd.</p>ut the presence of thymidine (CDM-T), whereas wildtype cells do. Addition of thymidine to the medium (CDM+T) recovers growth. Growth kinetics of DHFR-TS knock out cells compared to wildtype cells in media with or without thymidine show, that the knock out strain (pKOI) is growing as fast as wildtype cells on thymidine supplemented media (CDM+T). Knock out cells die without thymidine present (CDM-T). The curves are calculated by mean values of at least three independent experiments

The bifunctional dihydrofolate reductase thymidylate synthase of provides a tool for molecular and biotechnology applications-2

<p><b>Copyright information:</b></p><p>Taken from "The bifunctional dihydrofolate reductase thymidylate synthase of provides a tool for molecular and biotechnology applications"</p><p>BMC Biotechnology 2006;6():21-21.</p><p>Published online 20 Mar 2006</p><p>PMCID:PMC1435751.</p><p>Copyright © 2006 Herrmann et al; licensee BioMed Central Ltd.</p>g regions of the DHFR-TS gene and parts of its coding sequence (CDS), disrupted by a functional neomycin cassette conferring resistance to paromomycin. For more details refer to material and methods section