Secretion of functional human enzymes by Tetrahymena thermophila

Weide, Thomas; Herrmann, Lutz; Bockau, Ulrike; Niebur, Nadine; Aldag, Ingo; Laroy, Wouter; Contreras, Roland; Tiedtke, Arno; Hartmann, Marcus W W

doi:10.1186/1472-6750-6-19

Cited by 26 publications

(16 citation statements)

References 35 publications

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“…In previous experiments we could show that fusion proteins of the T. thermophila PLA 1 signal peptide with the human DNase I became secreted and correctly processed [14]. Interestingly, the results we show here clearly indicate that also human signal peptides are sufficient for proper secretion.…”

Section: Discussionsupporting

confidence: 68%

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

Aldag¹,

Bockau²,

Rossdorf³

et al. 2011

BMC Biotechnol

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BackgroundTetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila 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 T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.ResultsFunctional and full length human intestinal alkaline phosphatase was expressed by T. thermophila 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.ConclusionsWith 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 T. thermophila 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.

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Section: Discussionsupporting

confidence: 68%

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

Aldag¹,

Bockau²,

Rossdorf³

et al. 2011

BMC Biotechnol

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“…Increasing interest in the industrial use of Tetrahymena (Ethuin et al, 1995; Jayaram et al, 2010; Kiy and Tiedtke, 1992; Weide et al, 2007) spurred the examination of less expensive media for large scale culture, in particular the use of skim milk based media (Table I). In a bioreactor under conditions of high cell density fermentation and cell retention, skim milk based medium has supported the culture of Tetrahymena at cell densities of more than 2.2 × 10 7 cells/ml, equivalent to 48 g dry weight (Weide et al, 2006). A more dilute skim milk based medium (MYE medium, 1%(w/v) skim milk, 1%(w/v) yeast extract) has also been successfully used in batch fermenters to support growth up to ~3 × 10 6 cells/ml (De Coninck et al, 2004).…”

Section: Cell Culture Mediamentioning

confidence: 99%

“…growth without food vacuoles and its implications. Exp Cell Res, 102 (1), 127–137. h (personal communication, P. Doerder) i Weide, T., Herrmann, L., Bockau, U., Niebur, N., Aldag, I., Laroy, W., et al (2006). Secretion of functional human enzymes by tetrahymena thermophila.…”

Section: Figurementioning

confidence: 99%

Tetrahymena in the Laboratory: Strain Resources, Methods for Culture, Maintenance, and Storage

Cassidy-Hanley

2012

Methods in Cell Biology

101

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“…An active form of the human enzyme DNaseI has been expressed in T. thermophila, and disulfide bridges were also correctly formed with a consistent glycosylation pattern. 121 Another example is the production of correctly folded P. falciparum proteins for use as vaccines, including a synthetic vaccine antigen composed of the N-and C-terminal regions of MSP-1 which was successfully secreted into culture medium and shown to be immunogenic in an MF1 mouse model. 122 Previously, the stable expression of full-length P. falciparum circumsporozoite protein was reported, with the expressed antigen containing the natural secretory signal sequence and the C-terminal glycophosphatidylinositol (GPI) anchor, as shown by immunofluorescence imaging.…”

mentioning

confidence: 99%

Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules

Legastelois

Buffin

Peubez

et al. 2016

Human Vaccines & Immunotherapeutics

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The increasing demand for recombinant vaccine antigens or immunotherapeutic molecules calls into question the universality of current protein expression systems. Vaccine production can require relatively low amounts of expressed materials, but represents an extremely diverse category consisting of different target antigens with marked structural differences. In contrast, monoclonal antibodies, by definition share key molecular characteristics and require a production system capable of very large outputs, which drives the quest for highly efficient and cost-effective systems. In discussing expression systems, the primary assumption is that a universal production platform for vaccines and immunotherapeutics will unlikely exist. This review provides an overview of the evolution of traditional expression systems, including mammalian cells, yeast and E.coli, but also alternative systems such as other bacteria than E. coli, transgenic animals, insect cells, plants and microalgae, Tetrahymena thermophila, Leishmania tarentolae, filamentous fungi, cell free systems, and the incorporation of non-natural amino acids.

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Secretion of functional human enzymes by Tetrahymena thermophila

Cited by 26 publications

References 35 publications

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

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

Tetrahymena in the Laboratory: Strain Resources, Methods for Culture, Maintenance, and Storage

Non-conventional expression systems for the production of vaccine proteins and immunotherapeutic molecules

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