Signal peptide optimization tool for the secretion of recombinant protein from Saccharomyces cerevisiae

Mori, Akira; Hara, Shintaro; Sugahara, Tomohiro; Kojima, Takaaki; Iwasaki, Yoshinobu; Kawarasaki, Yasuaki; Sahara, Takehiko; Ohgiya, Satoru; Nakano, Hideo

doi:10.1016/j.jbiosc.2015.03.003

Cited by 40 publications

(36 citation statements)

References 21 publications

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“…To obtain optimal blockade, we first tested the signal peptides. Signal peptides, consisted of several amino acids, play determinant roles in protein distribution (Mori et al, 2015;Owji et al, 2018;Almagro Armenteros et al, 2019). Among the signal peptides tested, it was noticed that human IgK VIII enhanced the extracellular transportation of α-PD-1 scFv (Figure 1).…”

Section: Discussionmentioning

confidence: 99%

Augmenting the Effectiveness of CAR-T Cells by Enhanced Self-Delivery of PD-1-Neutralizing scFv

Yü

Nan

et al. 2020

Front. Cell Dev. Biol.

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Chimeric antigen receptor T (CAR-T) cell therapy is not satisfying in solid tumors. PD-1-mediated suppression greatly hinders CART cells in the microenvironment. It has been shown that PD-1 blockade improves the effectiveness of CART cells. Herein, we designed CART cells than could secret α-PD-1 scFv by themselves. To obtain optimal secretions of scFv, we screened several signal peptides. And the segment from human increased the extracellular production of PD-1-neutralizing proteins. The secreted neutralizing scFv efficiently blocked PD-1 and enhanced T cell activation when PD-L1 was present. Further analysis showed that CART cells themselves could secret α-PD-1 scFv with bioactivity. In contrast to the prototype, the scFv-producing CART cells demonstrated decreased PD-1 but increases expansion and toxicity against solid tumor cells. In the subcutaneous and orthotopic xenograft models, the self-delivered α-PD-1 scFv increased CART cell functionalities and tumor-suppressions. Our work suggested that engineering T cells to co-express antigen-responsive receptors and checkpoint inhibitors is effective to optimize CART cell therapy for solid tumors.

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Section: Discussionmentioning

confidence: 99%

Augmenting the Effectiveness of CAR-T Cells by Enhanced Self-Delivery of PD-1-Neutralizing scFv

Yü

Nan

et al. 2020

Front. Cell Dev. Biol.

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“…Indeed, the Lewis lab has elegantly demonstrated that deleting highly expressed proteins in CHO cells increases the yield of heterologous secreted proteins [25,26]. Modification of the secretory pathway, such as the optimization of signal sequences for protein targeting [71] and reducing the effect of the ERAD system [10], provides varying degrees of success and is contingent on the complexity and optimization of the protein product [72,73]. Our data and analysis may augment efforts by accounting for biogenesis capacity.…”

Section: Discussionmentioning

confidence: 93%

Protein Biogenesis Demands of the Early Secretory Pathway in Komagataella Phaffii

Alva¹,

Riera²,

Chartron³

2020

Preprint

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Background: Eukaryotes use distinct networks of biogenesis factors to synthesize, fold, monitor, traﬃc, and secrete proteins. During heterologous expression, saturation of any of these networks may bottleneck titer and yield. To understand the ﬂux through various routes into the early secretory pathway, we quantiﬁed the global and membrane-associated translatomes of Komagataella phaﬃi. Results: By coupling Ribo-seq with long-read mRNA sequencing, we generated a new annotation of protein-encoding genes. By using Ribo-seq with subcellular fractionation, we quantiﬁed demands on co- and posttranslational translocation pathways. During exponential growth in rich media, protein components of the cell-wall represent the greatest number of nascent chains entering the ER. Transcripts encoding the transmembrane protein PMA1 sequester more ribosomes at the ER membrane than any others. Comparison to Saccharomyces cerevisiae reveals conservation in the resources allocated by gene ontology, but variation in the diversity of gene products entering the secretory pathway. Conclusion: A subset of host proteins, particularly cell-wall components, impose the greatest biosynthetic demands in the early secretory pathway. These proteins are potential targets in strain engineering aimed at alleviating bottlenecks during heterologous protein production.

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“…Although some signal peptides (e.g., the sequence derived from the yeast mating factor) result in an overall higher yield in yeast, no secretion was obtained using this sequence for other proteins (Obst et al 2017). Suitable SPs may be obtained by identifying the secreted proteins of a fungal host and selecting the SP of the most abundant ones , although in vivo screening is needed as theoretical values generated by SignalP (a prediction software for SPs) are not associated with high levels of secretion in yeast (Mori et al 2015).…”

Section: Optimization Of Protein Secretion: Still a Black Boxmentioning

confidence: 99%

Strategies and Challenges for the Development of Industrial Enzymes Using Fungal Cell Factories

Arnau

Yaver

Hjort

2020

Grand Challenges in Biology and Biotechnology

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Signal peptide optimization tool for the secretion of recombinant protein from Saccharomyces cerevisiae

Cited by 40 publications

References 21 publications

Augmenting the Effectiveness of CAR-T Cells by Enhanced Self-Delivery of PD-1-Neutralizing scFv

Augmenting the Effectiveness of CAR-T Cells by Enhanced Self-Delivery of PD-1-Neutralizing scFv

Protein Biogenesis Demands of the Early Secretory Pathway in Komagataella Phaffii

Strategies and Challenges for the Development of Industrial Enzymes Using Fungal Cell Factories

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