Synthesis and secretion of human nerve growth factor by Saccharomyces cerevisiae

Kanaya, Eiko; Higashizaki, Takako; Ozawa, Fumiko; Hirai, Keiko; Nishizawa, Masafumi; Tokunaga, Masao; Tsukui, Hiroko; Hatanaka, Hiroshi; Hishinuma, Fumio

doi:10.1016/0378-1119(89)90404-6

Cited by 33 publications

(2 citation statements)

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“…It has been shown that higher copy number of genes causes higher level of transcription and therefore more efficient production of (heterologous) proteins [ 14 ]. However, using high copy plasmids such as 2μ-based vectors is not the preferred option, since these plasmids are not mitotically stable and thereby lead to heterogeneous gene expression in a population of cells [ 15 , 16 ]. Therefore, integrating genes in multiple copies into the genome to maintain stable production of the desired molecule for many generations is of high interest.…”

Section: Introductionmentioning

confidence: 99%

EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae

et al. 2016

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Saccharomyces cerevisiae is widely used in the biotechnology industry for production of ethanol, recombinant proteins, food ingredients and other chemicals. In order to generate highly producing and stable strains, genome integration of genes encoding metabolic pathway enzymes is the preferred option. However, integration of pathway genes in single or few copies, especially those encoding rate-controlling steps, is often not sufficient to sustain high metabolic fluxes. By exploiting the sequence diversity in the long terminal repeats (LTR) of Ty retrotransposons, we developed a new set of integrative vectors, EasyCloneMulti, that enables multiple and simultaneous integration of genes in S. cerevisiae. By creating vector backbones that combine consensus sequences that aim at targeting subsets of Ty sequences and a quickly degrading selective marker, integrations at multiple genomic loci and a range of expression levels were obtained, as assessed with the green fluorescent protein (GFP) reporter system. The EasyCloneMulti vector set was applied to balance the expression of the rate-controlling step in the β-alanine pathway for biosynthesis of 3-hydroxypropionic acid (3HP). The best 3HP producing clone, with 5.45 g.L-1 of 3HP, produced 11 times more 3HP than the lowest producing clone, which demonstrates the capability of EasyCloneMulti vectors to impact metabolic pathway enzyme activity.

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Section: Introductionmentioning

confidence: 99%

EasyCloneMulti: A Set of Vectors for Simultaneous and Multiple Genomic Integrations in Saccharomyces cerevisiae

et al. 2016

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“…Several reports show in spite of the production of recombinant NGF-β protein in various eukaryotic systems such as yeast, insect, mammalian and plant cells, its expression level is low and in some hosts such as yeast is partially or fully non-functional. [6,[17][18][19][20]. The advantage of plant as a cheap system with eukaryotic PTM [21], enhanced the research on determination of new molecular pathways for production of active recombinant pharmaceutical proteins .The plant system has already been used to produce NGF-β.…”

Section: Discussionmentioning

confidence: 99%

Processing of Human Nerve Growth Factor β Proprotein in the Plant Cell Apoplast

Zangi

Ofoghi

Ehsani

et al. 2021

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The Human HNGF-β gene encodes a pre-pro-NGF-β precursor protein. The signal peptide is cleaved off in the endoplasmic reticulum and the resulting pro-protein is processed in the trans-Golgi network by the Furin enzyme.The processing of the proproteins in the plant cells is not clearly defined. In the present study, various changes were applied to the HNGF-β gene sequence to increase its expression in the plant cells. The construct containing the synthesized Plant NGF-β gene was agro-infiltrated into the Nicotiana benthamiana leaves. Thereafter, the expression of various forms of the recombinant NGF-β and its processing was evaluated using dot blot, western blot, and RP-HPLC analyses. Finally, the biological activity of the mature NGF-β purified from the apoplast was assessed on the differentiation of PC12 cells and the expression of tetanus toxin receptors on their surfaces.Dot blot and western blot results showed that the total soluble proteins extracted from the plant leaves and the apoplast extract contained pro-NGF-β and mature NGF-β proteins, respectively. The RP-HPLC results reconfirmed the presence of the mature NGF-β in the apoplast extract. The amount of the mature NGF-β produced in the plant leaves was estimated to be about 39µg/g of the fresh leaves. A gradual increase in the length and number of the neurites of the differentiated PC12 cells was showed upon treatment with the mature NGF-β. Immunofluorescence experiments showed that the FITC-labeled tetanus toxin strongly bound to PC12 cells treated with mature NGF-β. This modification shows a very important advantage for plants to produce valuable biosimilar pharmaceutical proteins from precursor without applying biochemical or co-expression of modifying enzymes.

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