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

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

A novel malaria vaccine candidate antigen expressed in Tetrahymena thermophila

Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens

Untersuchungen zur Stöchiometrie der Untereinheit c der ATP-Synthase aus Escherichia coli

Die mit der in dieser Arbeit entwickelten Rekonstitutionsmethode gemessenen Protonentranslokationsraten erreichen physiologisch relevante Werte und liegen ca. eine Größenordnung über Messungen, die mit vergleichbaren Methoden erzielt wurden. - Die Stöchiometrie der Untereinheit c ist in vitro vom Verhältnis der Untereinheiten a, b und c abhängig. Das bedeutet, dass bei einer Rekonstitution mit verringerter Menge an Untereinheit c Fo-Komplexe mit einheitlich weniger c-Untereinheiten entstehen. Das deutet darauf hin, dass der Fo-Komplex möglicherweise mit verschiedenen c-Stöchiometrien Protonen translozieren kann und dass die Stöchiometrie der Untereinheit c einen Einfluss auf die Protonentranslokationsrate des Fo-Komplexes haben könnte. - Die Stöchiometrie der Untereinheit c im FoF1-Komplex ist in vivo von der Expressionsrate ihres Gens weitgehend unabhängig. - Das unterschiedliche Verhalten der c-Stöchiometrie bei Variation der relativen c-Menge in vitro und in vivo legen einen Regulationsmechanismus in der Zelle nahe, der die Stöchiometrie der Untereinheit c je nach physiologischer Notwendigkeit einstellt und der unabhängig von der Expressionsrate der Untereinheit c arbeitet

Differential Modulation by Ca(2+) of Type III Secretion of Diffusely Adhering Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) strains are a common cause of persistent diarrhea among infants, primarily in developing countries. The pathogenicity of EPEC is associated with the expression and secretion of bacterial proteins encoded by the chromosomal locus of enterocyte effacement (LEE). The LEE-encoded type III-secreted proteins EspA, EspB, and EspD are part of a molecular syringe, which is used by EPEC to translocate effector proteins directly into the cytoplasm of host cells. The type III-secreted translocated intimin receptor (Tir) protein is thought to be delivered by an Esp-dependent mechanism into the host cell, and this is followed by insertion into the host plasma membrane, where the protein serves as the receptor for intimin, an afimbrial bacterial adhesin. Type III secretion is subject to environmental regulation, and secretion can be induced in vitro by growing bacteria in cell culture medium. In this study we found that Ca(2+) is involved in the regulation of type III secretion both in classical locally adherent EPEC and in atypical diffusely adherent EPEC. Interestingly, we observed contrasting secretion of Esp proteins and Tir in response to Ca(2+). While the secretion of Tir is clearly enhanced and the protein is integrated into HeLa membranes under calcium chelation conditions, Esp secretion is strongly reduced under these conditions. These data suggest that under Ca(2+)-depleted conditions Tir might be secreted into the medium and integrated into host membranes by an Esp-independent mechanism, without the need for a functional type III translocation machinery

Long term follow-up of a simplified and less burdened pancreatic duct ligation model of exocrine pancreatic insufficiency in Goettingen Minipigs

Background!#!Pancreatic duct ligation in a minipig model leads to exocrine pancreatic insufficiency (EPI). This allows the study of digestive processes and pancreatic enzyme replacement therapies. However, detailed descriptions of the surgical procedure, perioperative management, a determination of exocrine pancreatic insufficiency are scarce in the literature. Data of the long-term health status of minipigs upon EPI induction are still not available. Therefore, the present study describes in detail an experimental approach to the induction of exocrine pancreatic insufficiency via pancreatic duct ligation in minipigs and the long term follow up of the animal's health state.!##!Methods!#!14 Goettingen minipigs underwent pancreatic duct ligation via midline laparotomy for the induction of exocrine pancreatic insufficiency. Fecal fat content, fat absorption, chymotrypsin levels, body weight and blood vitamin and glucose levels were determined.!##!Results!#!Exocrine pancreatic insufficiency was successfully induced in 12 Goettingen minipigs. Two minipigs failed to develop exocrine insufficiency most likely due to undetected accessory pancreatic ducts. All animals tolerated the procedure very well and gained weight within 8 weeks after surgery without requiring pancreatic enzyme replacement therapy. The follow up for approx. 180 weeks showed a stable body weight and health state of the animals with normal blood glucose levels (Table 1). From approx. 130 weeks post pancreatic duct ligation, all animals were supplemented with pancreatic enzymes and vitamins resulting in blood concentrations almost within the reference range.!##!Conclusions!#!Pancreatic duct ligation in minipigs is an excellent method of inducing exocrine pancreatic insufficiency. It is important to identify and ligate accessory pancreatic ducts since persistence of accessory ducts will lead to maintenance of exocrine pancreatic function. The EPI model caused no persistent side effects in the animals and has the potential to be used in long-term EPI studies with up to 100 weeks post-OP without supplementation with enzymes and vitamins

Schematic diagram of the MSP-1-BBM construct which encodes the N-terminal MSP-1 Block 1 region, the K1 Block 2 synthetic sequence, the RO33 Block 2 sequence, the MAD20 Block 2 synthetic sequence [31] and a C-terminal fragment encoding residues 1571 to 1702 of MSP-1 precursor protein of <i>P. falciparum</i>.

<p>The Block 2 region is arranged similar to natural alleles. Synthetic Block 2 repeat sequences of the K1 and MAD20 serotypes are indicated by vertical and diagonal hatched markings, respectively.</p