Development and Characterization of a Novel Fusion Protein of a Mutated Granulocyte Colony-Stimulating Factor and Human Serum Albumin in Pichia pastoris

Huang, Yian; Wen, Xiaofang; Yang, Zhiyu; Wu, Yiliang; Lu, Yongjie; Zhou, Linfu

doi:10.1371/journal.pone.0115840

Cited by 6 publications

(3 citation statements)

References 15 publications

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“…P. pastoris can effectively secrete large amounts of recombinant HSA and HSA fusion proteins 39 – 41 . We constructed a strain expressing two fusion proteins (pJC172): (i) HSA-hGH, consisting of an alpha-mating factor secretion signal, HSA, a tobacco etch virus (TEV) protease cleavage site, and hGH; and (ii) Golgi-TEV, consisting of a Golgi apparatus localization signal (the membrane-binding domain of alpha-1,2-mannosyltransferase) and TEV protease 26 , 42 .…”

Section: Resultsmentioning

confidence: 99%

Versatile and on-demand biologics co-production in yeast

et al. 2018

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Current limitations to on-demand drug manufacturing can be addressed by technologies that streamline manufacturing processes. Combining the production of two or more drugs into a single batch could not only be useful for research, clinical studies, and urgent therapies but also effective when combination therapies are needed or where resources are scarce. Here we propose strategies to concurrently produce multiple biologics from yeast in single batches by multiplexing strain development, cell culture, separation, and purification. We demonstrate proof-of-concept for three biologics co-production strategies: (i) inducible expression of multiple biologics and control over the ratio between biologic drugs produced together; (ii) consolidated bioprocessing; and (iii) co-expression and co-purification of a mixture of two monoclonal antibodies. We then use these basic strategies to produce drug mixtures as well as to separate drugs. These strategies offer a diverse array of options for on-demand, flexible, low-cost, and decentralized biomanufacturing applications without the need for specialized equipment.

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Section: Resultsmentioning

confidence: 99%

Versatile and on-demand biologics co-production in yeast

et al. 2018

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“…Studies have shown that single injections of PEG-rhG-CSF were at least as potent as multiple injections of rhG-CSF in treating neutropenia [42]. Fusion of rhG-CSF to an Fc receptor [43] or serum albumin [44,45] was effective in increasing half-life of rhG-CSF and thus mobilization efficiency. In all of these studies, plasma levels of rhG-CSF were considered to be a marker for effectiveness of the modification.…”

Section: Discussionmentioning

confidence: 99%

Albumin Modifies Responses to Hematopoietic Stem Cell Mobilizing Agents in Mice

Danner

Bönig

Wiercinska

2019

Cells

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Albumin, the most abundant plasma protein, not only controls osmotic blood pressure, but also serves as a carrier for various small molecules, including pharmaceuticals. Its impact on pharmacological properties of many drugs has been extensively studied over decades. Here, we focus on its interaction with the following mobilizing agents: Granulocyte-colony stimulating factor (G-CSF) and AMD3100, where such analyses are lacking. These compounds are widely used for hematopoietic stem cell mobilization of healthy donors or patients. Using albumin-deficient (Alb−/−) mice, we studied the contribution of albumin to mobilization outcomes. Mobilization with the bicyclam CXCR4 antagonist AMD3100 was attenuated in Alb−/− mice compared to wild-type littermates. By contrast, mobilization with recombinant human G-CSF (rhG-CSF), administered twice daily over a five-day course, was significantly increased in Alb−/− mice. In terms of a mechanism, we show that rhG-CSF bioavailability in the bone marrow is significantly improved in Alb−/− mice, compared to wild-type (WT) littermates, where rhG-CSF levels dramatically drop within a few hours of the injection. These observations likely explain the favorable mobilization outcomes with split-dose versus single-dose administration of rhG-CSF to healthy donors.

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“…However, accurate glycosylation cannot be ensured for recombinant proteins expressed in yeasts and this will require extensive structural studies for confirmation. The aberrant glycosylation pattern often causes antigenicity of recombinant proteins which may induce production of neutralizing antibodies in the subject and unpredictable pharmacokinetic manifestations . Highly mannosylated proteins are for example linked to mannose binding endocytic receptors in antigen‐presenting cells .…”

Section: Discussionmentioning

confidence: 99%

Secreted expression of an active human interferon‐beta (HuIFNβ) in Kluyveromyces lactis

Madhavan

Sukumaran

2016

Engineering in Life Sciences

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Interferon‐beta (IFNβ) is a cytokine involved in the antiviral, anti‐proliferative and immunomodulatory responses of cells, and a potent drug for multiple sclerosis. Human interferon beta (HuIFNβ) gene fused with the glucoamylase signal sequence in the N‐terminus and 6 His Tag in the C‐terminus was cloned into pKlac1 vector and introduced in Kluyveromyces lactis to allow secreted expression and one‐step purification of the protein. Recombinant yeast transformant with the highest level of HuIFNβ production was identified, and this secreted up to 1 mg/L of the cytokine after 72 h of incubation. Glycosylated and non‐glycosylated forms of the cytokine were elaborated by the yeast, the latter in higher quantities. His tag of the protein allowed easy one‐step purification by nickel‐nitriloacetic acid affinity chromatography, yielding close to 100% purity. SDS‐PAGE, western blot and MALDI‐TOF‐TOF confirmed the identity of the protein. The biological activity of the recombinant HuIFNβ was confirmed by its anti cell proliferative activity on HeLa cells. Expression of HuIFNβ in K. lactis is advantageous since it is a very safe organism to produce proteins for therapeutic applications, allows glycosylation and offers a cost effective method for large scale production as it can be grown in cheap carbon sources.

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Development and Characterization of a Novel Fusion Protein of a Mutated Granulocyte Colony-Stimulating Factor and Human Serum Albumin in Pichia pastoris

Cited by 6 publications

References 15 publications

Versatile and on-demand biologics co-production in yeast

Versatile and on-demand biologics co-production in yeast

Albumin Modifies Responses to Hematopoietic Stem Cell Mobilizing Agents in Mice

Secreted expression of an active human interferon‐beta (HuIFNβ) in Kluyveromyces lactis

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