Biologically active human and mouse nerve growth factors secreted by the yeast Saccharomyces cerevisiae

Nishizawa, Masafumi; Ozawa, Fumiko; Higashizaki, Takako; Hirai, Keiko; Hishinuma, Fumio

doi:10.1007/bf00182801

Cited by 12 publications

(12 citation statements)

References 32 publications

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“…As a partial resolution, bacterial host engineering in the form of co-overexpression of Dsb disulfide-bonding machinery in bacteria could raise the level of soluble BDNF production to 35% (15). Similarly, despite the eukaryotic protein-folding and -processing machinery of yeast, NGF production in yeast yielded a low-fidelity product (17). Here we report that yeast also produces BDNF primarily in an inactive and misfolded form.…”

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confidence: 87%

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Directed Evolution of Brain-Derived Neurotrophic Factor for Improved Folding and Expression in Saccharomyces cerevisiae

Burns

Malott

Metcalf

et al. 2014

Appl Environ Microbiol

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cBrain-derived neurotrophic factor (BDNF) plays an important role in nervous system function and has therapeutic potential. Microbial production of BDNF has resulted in a low-fidelity protein product, often in the form of large, insoluble aggregates incapable of binding to cognate TrkB or p75 receptors. In this study, employing Saccharomyces cerevisiae display and secretion systems, it was found that BDNF was poorly expressed and partially inactive on the yeast surface and that BDNF was secreted at low levels in the form of disulfide-bonded aggregates. Thus, for the purpose of increasing the compatibility of yeast as an expression host for BDNF, directed-evolution approaches were employed to improve BDNF folding and expression levels. Yeast surface display was combined with two rounds of directed evolution employing random mutagenesis and shuffling to identify BDNF mutants that had 5-fold improvements in expression, 4-fold increases in specific TrkB binding activity, and restored p75 binding activity, both as displayed proteins and as secreted proteins. Secreted BDNF mutants were found largely in the form of soluble homodimers that could stimulate TrkB phosphorylation in transfected PC12 cells. Site-directed mutagenesis studies indicated that a particularly important mutational class involved the introduction of cysteines proximal to the native cysteines that participate in the BDNF cysteine knot architecture. Taken together, these findings show that yeast is now a viable alternative for both the production and the engineering of BDNF.

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confidence: 87%

“…Platforms for neurotrophin production include immortalized mammalian cell lines (13), bacteria (15), insect cell lines (16), and Saccharomyces cerevisiae (17). In particular, microbial hosts such as bacteria and yeast have the advantages of facile genetic modification, robust scaling, and comparatively low cost.…”

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confidence: 99%

Directed Evolution of Brain-Derived Neurotrophic Factor for Improved Folding and Expression in Saccharomyces cerevisiae

Burns

Malott

Metcalf

et al. 2014

Appl Environ Microbiol

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“…Nerve growth factor (NGF), the founder member of a protein family termed neurotrophins, is known to promote the survival and differentiation of primary sensory neurons, sympathetic neurons and cholinergic neurons of the basal forebrain [1–4]. Several attempts have been undertaken to produce a large quantity of recombinant hNGF from Escherichia coli [5], yeast [6], insect cells [7]and mammalian cell cultures [8, 9]. Preclinical studies using recombinant hNGF‐β to treat neuronal dysfunction of the central and peripheral nervous systems [10, 11]and clinical trials including the treatment of HIV‐related peripheral neuropathy carried out by Genentech Incorporated are potential indications for the need of a large quantity of biologically active hNGF‐β.…”

Section: Introductionmentioning

confidence: 99%

Human nerve growth factor beta (hNGF‐β): mammary gland specific expression and production in transgenic rabbits

Coulibaly¹,

Besenfelder²,

Fleischmann³

et al. 1999

FEBS Letters

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Transgenic rabbits carrying gene constructs encoding human nerve growth factor beta (hNGF-L L) cDNA were generated. Expression of hNGF-L L mRNA was restricted to the mammary gland of lactating rabbits. Western Blot analysis revealed a polypeptide of 13.2 kDa in the milk of transgenic animals. hNGF-L L was purified from the milk by a two-step chromatographic procedure. Electrospray mass spectroscopy analysis of purified hNGF-L L depicted a molecular weight of 13 261 Da per subunit. The biological activity of the hNGF-L L was tested using PC12W2 cells and cultures of dorsal root ganglion neurons from chicken embryos. Crude defatted milk from transgenic animals and purified hNGF-L L demonstrated full biological activity when compared to commercial recombinant hNGF-L L.z 1999 Federation of European Biochemical Societies.

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“…To address these concerns, hNGF has been produced in E. coli 3233, yeast34, insect cells35363738 and mammalian cells394041. Yet in these cell systems the yield of the hNGF protein is low, and some of them, such as the E. coli and the yeast systems might be unable to provide correct post-translational modifications for hNGF.…”

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confidence: 99%

Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors

Zeng

Zhu

et al. 2017

Sci Rep

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The salivary glands of animals have great potential to act as powerful bioreactors to produce human therapeutic proteins. Human nerve growth factor (hNGF) is an important pharmaceutical protein that is clinically effective in the treatment of many human neuronal and non-neuronal diseases. In this study, we generated 18 transgenic (TG) founder mice each carrying a salivary gland specific promoter-driven hNGF transgene. A TG mouse line secreting high levels of hNGF protein in its saliva (1.36 μg/mL) was selected. hNGF protein was successfully purified from the saliva of these TG mice and its identity was verified. The purified hNGF was highly functional as it displayed the ability to induce neuronal differentiation of PC12 cells. Furthermore, it strongly promoted proliferation of TF1 cells, above the levels observed with mouse NGF. Additionally, saliva collected from TG mice and containing unpurified hNGF was able to significantly enhance the growth of TF1 cells. This study not only provides a new and efficient approach for the synthesis of therapeutic hNGF but also supports the concept that salivary gland from TG animals is an efficient system for production of valuable foreign proteins.

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Biologically active human and mouse nerve growth factors secreted by the yeast Saccharomyces cerevisiae

Cited by 12 publications

References 32 publications

Directed Evolution of Brain-Derived Neurotrophic Factor for Improved Folding and Expression in Saccharomyces cerevisiae

Directed Evolution of Brain-Derived Neurotrophic Factor for Improved Folding and Expression in Saccharomyces cerevisiae

Human nerve growth factor beta (hNGF‐β): mammary gland specific expression and production in transgenic rabbits

Production of functional human nerve growth factor from the saliva of transgenic mice by using salivary glands as bioreactors

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