Production of Antibody Derivatives in the Methylotrophic Yeast Pichia pastoris

Schoonooghe, Steve; Leoen, Jannick; Haustraete, Jurgen

doi:10.1007/978-1-61779-974-7_19

Cited by 6 publications

(5 citation statements)

References 9 publications

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“…The expression vector, which is a derivate of the pPICZα vector (Life Technologies) is supplemented with the AOX1 promoter fused to the α-mating factor pre-pro-signal sequence followed by the gene coding for the Nb construct. 57 Again, the Nb sequence included a C-terminal His 6 -tag. After selection of the appropriate expression clone, a 20-l production was performed in baffled shake flasks.…”

Section: Cloning Of Modified Nanobodies For Electroporationmentioning

confidence: 99%

Development and Validation of a Small Single-domain Antibody That Effectively Inhibits Matrix Metalloproteinase 8

et al. 2016

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A detrimental role for matrix metalloproteinase 8 (MMP8) has been identified in several pathological conditions, e.g., lethal hepatitis and the systemic inflammatory response syndrome. Since matrix MMP8-deficient mice are protected in the above-mentioned diseases, specific MMP8 inhibitors could be of clinical value. However, targeting a specific matrix metalloproteinase remains challenging due to the strong structural homology of matrix metalloproteinases, which form a family of 25 members in mammals. Single-domain antibodies, called nanobodies, offer a range of possibilities toward therapy since they are easy to generate, express, produce, and modify, e.g., by linkage to nanobodies directed against other target molecules. Hence, we generated small MMP8-binding nanobodies, and established a proof-of-principle for developing nanobodies that inhibit matrix metalloproteinase activity. Also, we demonstrated for the first time the possibility of expressing nanobodies systemically by in vivo electroporation of the muscle and its relevance as a potential therapy in inflammatory diseases.

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Section: Cloning Of Modified Nanobodies For Electroporationmentioning

confidence: 99%

Development and Validation of a Small Single-domain Antibody That Effectively Inhibits Matrix Metalloproteinase 8

et al. 2016

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“…ethylotrophic yeasts, such as Pichia pastoris, Hansenula polymorpha, and Candida boidinii, have been used as heterologous hosts for protein production (1)(2)(3)(4). Efficient production has been achieved with their strong and tightly regulated methanolinducible gene promoters in combination with high-cell-density cultures in methanol-containing medium.…”

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confidence: 99%

Molecular Characterization of Hap Complex Components Responsible for Methanol-Inducible Gene Expression in the Methylotrophic Yeast Candida boidinii

et al. 2015

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cWe identified genes encoding components of the Hap complex, CbHAP2, CbHAP3, and CbHAP5, as transcription factors regulating methanol-inducible gene expression in the methylotrophic yeast Candida boidinii. We found that the Cbhap2⌬, Cbhap3⌬, and Cbhap5⌬ gene-disrupted strains showed severe growth defects on methanol but not on glucose and nonfermentable carbon sources such as ethanol and glycerol. In these disruptants, the transcriptional activities of methanol-inducible promoters were significantly decreased compared to those of the wild-type strain, indicating that CbHap2p, CbHap3p, and CbHap5p play indispensable roles in methanol-inducible gene expression. Further molecular and biochemical analyses demonstrated that CbHap2p, CbHap3p, and CbHap5p localized to the nucleus and bound to the promoter regions of methanol-inducible genes regardless of the carbon source, and heterotrimer formation was suggested to be necessary for binding to DNA. Unexpectedly, distinct from Saccharomyces cerevisiae, the Hap complex functioned in methanol-specific induction rather than glucose derepression in C. boidinii. Our results shed light on a novel function of the Hap complex in methanol-inducible gene expression in methylotrophic yeasts.

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“…Sequences were each inserted separately into a vector pPICZalphaA (Invitrogen) using the XbaI/XhoI restriction sites, creating respectively construct A and construct B (see S1 Table for all constructs). A bicistronic vector was then created, as described by Schoonooghe and coworkers [ 22 ]. Briefly, the PmeI restriction site from construct B was removed by site-directed mutagenesis (Stratagene) using primers 1 and 2 (see S2 Table for all primers) to create construct C. Vectors were then digested, construct A with PciI and BamHI and construct C with BglII and PciI , and re-ligated together to form the bicistronic vector construct D.…”

Section: Methodsmentioning

confidence: 99%

Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies

Gagné,

Sarker,

Gingras

et al. 2023

PLoS ONE

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The importance and fast growth of therapeutic monoclonal antibodies, both innovator and biosimilar products, have triggered the need for the development of characterization methods at high resolution such as nuclear magnetic resonance (NMR) spectroscopy. However, the full power of NMR spectroscopy cannot be unleashed without labelling the mAb of interest with NMR-active isotopes. Here, we present strategies using either Komagataella phaffii (Pichia pastoris) or Escherichia coli that can be widely applied for the production of the antigen-binding fragment (Fab) of therapeutic antibodies of immunoglobulin G1 kappa isotype. The E. coli approach consists of expressing Fab fragments as a single polypeptide chain with a cleavable linker between the heavy and light chain in inclusion bodies, while K. phaffii secretes a properly folded fragment in the culture media. After optimization, the protocol yielded 10–45 mg of single chain adalimumab-Fab, trastuzumab-Fab, rituximab-Fab, and NISTmAb-Fab per liter of culture. Comparison of the 2D-1H-15N-HSQC spectra of each Fab fragment, without their polyhistidine tag and linker, with the corresponding Fab from the innovator product showed that all four fragments have folded into the correct conformation. Production of 2H-13C-15N-adalimumab-scFab and 2H-13C-15N-trastuzumab-scFab (>98% enrichment for all three isotopes) yielded NMR samples where all amide deuterons have completely exchanged back to proton during the refolding procedure.

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Production of Antibody Derivatives in the Methylotrophic Yeast Pichia pastoris

Cited by 6 publications

References 9 publications

Development and Validation of a Small Single-domain Antibody That Effectively Inhibits Matrix Metalloproteinase 8

Development and Validation of a Small Single-domain Antibody That Effectively Inhibits Matrix Metalloproteinase 8

Molecular Characterization of Hap Complex Components Responsible for Methanol-Inducible Gene Expression in the Methylotrophic Yeast Candida boidinii

Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies

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