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

Weide, Thomas; Bockau, Ulrike; Rave, Angelika; Herrmann, Lutz; Hartmann, Marcus W W

doi:10.1186/1471-2180-7-12

Cited by 13 publications

(24 citation statements)

References 31 publications

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“…DQ418790), the promoter and the terminator. The final expression cassette for the HC of the anti-CD20 antibody carried a 1 kb fragment of the MTT1 promoter-active region, the BTU2 terminator sequence of the neo2 cassette and was cloned into a pAX_vector derivate, which is based on the pAX-plasmid constructed by Weide et al 8 . The final expression cassette for the LC of the anti-CD20 antibody carried a 1.2 kb fragment of the MTT5 promoter-active region, the BTU2 terminator sequence of the neo2 cassette and was cloned into a pKOIX-based integrative expression vector by Cre/Lox recombination.…”

Section: Methodsmentioning

confidence: 99%

“…The final expression cassette for the LC of the anti-CD20 antibody carried a 1.2 kb fragment of the MTT5 promoter-active region, the BTU2 terminator sequence of the neo2 cassette and was cloned into a pKOIX-based integrative expression vector by Cre/Lox recombination. In the utilized pKOIX-vector-variant, the neo selection cassette was replaced by a bsd selection cassette (pKOIX_B_LC) 8 . The employed plasmids were cloned by standard methods (details on intermediate plasmids and primers are available from the authors).…”

Section: Methodsmentioning

confidence: 99%

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Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity

Calow¹,

Behrens

Mader³

et al. 2016

mAbs

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Antibody glycosylation is a key parameter in the optimization of antibody therapeutics. Here, we describe the production of the anti-cancer monoclonal antibody rituximab in the unicellular ciliate, Tetrahymena thermophila. The resulting antibody demonstrated enhanced antibody-dependent cell-mediated cytotoxicity, which we attribute to unusual N-linked glycosylation. Detailed chromatographic and mass spectrometric analysis revealed afucosylated, oligomannose-type glycans, which, as a whole, displayed isomeric structures that deviate from the typical human counterparts, but whose branches were equivalent to fragments of metabolic intermediates observed in human glycoproteins. From the analysis of deposited crystal structures, we predict that the ciliate glycans adopt protein-carbohydrate interactions with the Fc domain that closely mimic those of native complex-type glycans. In addition, terminal glucose structures were identified that match biosynthetic precursors of human glycosylation. Our results suggest that ciliate-based expression systems offer a route to large-scale production of monoclonal antibodies exhibiting glycosylation that imparts enhanced cell killing activity.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity

Calow¹,

Behrens

Mader³

et al. 2016

mAbs

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“…The final expression plasmid p22X-MSP-1-BBM was generated by the transfer of the expression cassette into the integrative acceptor plasmid p22X by Cre/Lox recombination [18], [47].…”

Section: Methodsmentioning

confidence: 99%

“…The resulting vector is ∼7.5 kb in size and contains a loxP site for Cre-mediated integration of the expression cassette downstream of the neo2 selection cassette [18], [48]. Further details of the constructs are available from the authors.…”

Section: Methodsmentioning

confidence: 99%

A Novel Malaria Vaccine Candidate Antigen Expressed in Tetrahymena thermophila

Cowan

Bockau²,

Eleni-Muus³

et al. 2014

PLoS ONE

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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.

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“…This constitutes the first report of the recognition of targeting and GPI anchoring signals in a heterologous expression host. This successful initiative led to further development of Tetrahymena as an expression system [57], although limited progress has been reported since then. Publication of the Tetrahymena genome should renew such interest by identifying secretion and organellar signals for secretion and targeting, regulatory elements for constructing transfection vectors, among others, in spite of a potential limitation of this system concerning the expression of plastid-derived genes, since no compelling evidence for its presence was found in the genome [58].…”

Section: Eukaryotic Systemsmentioning

confidence: 99%

Production of recombinant proteins from protozoan parasites

Fernández-Robledo¹,

Vasta²

2010

Trends in Parasitology

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Although the past decade has witnessed the sequencing from an increasing number of parasites, modern high-throughput DNA sequencing technologies have the potential to generate complete genome sequences at even higher rates. Along with the discovery of genes that might constitute potential targets for chemotherapy or vaccination, the need for novel protein expression platforms has become a pressing matter. In addition to reviewing the advantages and limitations of the currently available and emerging expression systems, we discuss novel approaches that could overcome the current limitations, including the 'pseudoparasite' concept, an expression platform in which the choice of the surrogate organism is based on its phylogenetic affinity to the target parasite, while taking advantage of the whole engineered organism as a vaccination adjuvant. Proteins from protozoan parasites as targets for diagnosis and interventionThe availability of the genome sequences of several parasites of medical importance has led to exponential progress in our understanding of their biology, and enabled the identification of potential targets for intervention [1]. Further, the continued advances in genome sequencing technologies holds great promise for the accomplishment of similar goals for virtually any parasite of interest [2]. When compared to other pathogens of human and veterinary importance, such as viruses and bacteria, large gaps exist in our knowledge of the virulence and pathogenesis mechanisms of protozoan parasites, and methodologies for eradication or management of parasitic diseases through vaccination or treatment are still in the distant future. Characterization of genes of interest for potential intervention identified by mining these genomes has been hindered because of the lack of suitable protein expression systems. A clear example is Plasmodium falciparum, the etiological agent of malaria, for which the lack of an effective vaccine, and the rapid emergence of drug-resistant strains, have made most intervention attempts extremely challenging [3]. Therefore, the development of innovative and efficient systems for the expression of recombinant proteins from protozoan parasites has become an urgent public health matter.

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

Cited by 13 publications

References 31 publications

Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity

Antibody production using a ciliate generates unusual antibody glycoforms displaying enhanced cell-killing activity

A Novel Malaria Vaccine Candidate Antigen Expressed in Tetrahymena thermophila

Production of recombinant proteins from protozoan parasites

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