Production of an antibody Fab fragment using 2A peptide in insect cells

Mizote, Yu; Masumi-Koizumi, Kyoko; Katsuda, Tomohisa; Yamaji, Hideki

doi:10.1016/j.jbiosc.2020.03.009

Cited by 8 publications

(9 citation statements)

References 29 publications

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“…Therefore, the expression of the GOI is lost over time after cell division. In recent years, several studies have shown that suspension-adapted High Five and Sf9 cells are ideal hosts for the production of reporter proteins [15][16][17], antibodies [18][19][20] and surface proteins [21] in this BV-free environment. Still, the assessment of the insect cell/TGE system to produce more complex products such as VLPs remains to be investigated.…”

Section: Introductionmentioning

confidence: 99%

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production

et al. 2020

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High Five cells are an excellent host for the production of virus-like particles (VLPs) with the baculovirus expression vector system (BEVS). However, the concurrent production of high titers of baculovirus hinder the purification of these nanoparticles due to similarities in their physicochemical properties. In this study, first a transient gene expression (TGE) method based on the transfection reagent polyethylenimine (PEI) is optimized for the production of HIV-1 VLPs at shake flask level. Furthermore, VLP production by TGE in High Five cells is successfully demonstrated at bioreactor scale, resulting in a higher maximum viable cell concentration (5.1 × 106 cell/mL), the same transfection efficiency and a 1.8-fold increase in Gag-eGFP VLP production compared to shake flasks. Metabolism analysis of High Five cells indicates a reduction in the consumption of the main metabolites with respect to non-transfected cell cultures, and an increase in the uptake rate of several amino acids when asparagine is depleted. Quality assessment by nanoparticle tracking analysis and flow virometry of the VLPs produced shows an average size of 100–200 nm, in agreement with immature HIV-1 viruses reported in the literature. Overall, this work demonstrates that the High Five/TGE system is a suitable approach for the production of VLP-based vaccine candidates and other recombinant proteins.

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Section: Introductionmentioning

confidence: 99%

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production

et al. 2020

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“…There are different possibilities to achieve this coordinated expression, e.g. the introduction of a 2A peptide sequence [ 37 , 38 ]. Due to the polycistronic nature, small size and high “cleavage” efficiency, 2A peptides have received increased interest.…”

Section: Resultsmentioning

confidence: 99%

Expression of antibody fragments in Saccharomyces cerevisiae strains evolved for enhanced protein secretion

Wang

Chen

et al. 2021

Microb Cell Fact

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Monoclonal antibodies, antibody fragments and fusion proteins derived thereof have revolutionized the practice of medicine. Major challenges faced by the biopharmaceutical industry are however high production costs, long processing times and low productivities associated with their production in mammalian cell lines. The yeast Saccharomyces cerevisiae, a well-characterized eukaryotic cell factory possessing the capacity of posttranslational modifications, has been industrially exploited as a secretion host for production of a range of products, including pharmaceuticals. However, due to the incompatible surface glycosylation, few antibody molecules have been functionally expressed in S. cerevisiae. Here, three non-glycosylated antibody fragments from human and the Camelidae family were chosen for expression in a S. cerevisiae strain (HA) previously evolved for high α-amylase secretion. These included the Fab fragment Ranibizumab (Ran), the scFv peptide Pexelizumab (Pex), and a nanobody consisting of a single V-type domain (Nan). Both secretion and biological activities of the antibody fragments were confirmed. In addition, the secretion level of each protein was compared in the wild type (LA) and two evolved strains (HA and MA) with different secretory capacities. We found that the secretion of Ran and Nan was positively correlated with the strains’ secretory capacity, while Pex was most efficiently secreted in the parental strain. To investigate the mechanisms for different secretion abilities in these selected yeast strains for the different antibody fragments, RNA-seq analysis was performed. The results showed that several bioprocesses were significantly enriched for differentially expressed genes when comparing the enriched terms between HA.Nan vs. LA.Nan and HA.Pex vs. LA.Pex, including amino acid metabolism, protein synthesis, cell cycle and others, which indicates that there are unique physiological needs for each antibody fragment secretion.

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“…In their work, T2A preceded by a GSG linker (GT2A) exhibited the highest cleavage efficiency and mAb expression level. Beside antibody production in mammal cells [ 78 , 79 , 96 ], Mizote and colleagues [ 97 ] engineered a more complex expression cassette for the HC and LC genes (HC-GSG-furin signal-GSG-2A-LC) that exhibited high productivity of Fab fragments in Lepidoptera . Hence, this work demonstrated that bicistronic constructs using, in the appropriate order, GSG linkers, a furin cleavage site, and a 2 A peptide allow for the effective production of recombinant antibody molecules also in insect cells.…”

Section: Applicationsmentioning

confidence: 99%

Synthetic polycistronic sequences in eukaryotes

Wang

Marchisio

2021

Synthetic and Systems Biotechnology

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The need for co-ordinate, high-level, and stable expression of multiple genes is essential for the engineering of biosynthetic circuits and metabolic pathways. This work outlines the functionality and design of IRES- and 2 A-peptide-based constructs by comparing different strategies for co-expression in polycistronic vectors. In particular, 2 A sequences are small peptides, mostly derived from viral polyproteins, that mediate a ribosome-skipping event such that several, different, separate proteins can be generated from a single open reading frame. When applied to metabolic engineering and synthetic gene circuits, 2 A peptides permit to achieve co-regulated and reliable expression of various genes in eukaryotic cells.

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Production of an antibody Fab fragment using 2A peptide in insect cells

Cited by 8 publications

References 29 publications

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production

PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production

Expression of antibody fragments in Saccharomyces cerevisiae strains evolved for enhanced protein secretion

Synthetic polycistronic sequences in eukaryotes

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