New tools for high‐throughput expression of fungal secretory proteins in <i>Saccharomyces cerevisiae</i> and <i>Pichia pastoris</i>

González, Mario; Brito, Nélida; Hernández-Bolaños, Eduardo; González, Celedonio

doi:10.1111/1751-7915.13322

Cited by 7 publications

(6 citation statements)

References 38 publications

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“…With the increasing demand for recombinant proteins, and continued discovery of novel medically useful proteins, the improvement of production technology has become a focus of research exploration. Various excellent expression systems based on different hosts have gradually become the primary tools for protein production, including plant [ 1 ], Escherichia coli ( E. coli ) [ 2 ], insect [ 3 ], yeast [ 4 ], and mammals [ 5 ].…”

Section: Introductionmentioning

confidence: 99%

P25 Gene Knockout Contributes to Human Epidermal Growth Factor Production in Transgenic Silkworms

Ruan

et al. 2021

IJMS

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Transgenic silkworm expression systems have been applied for producing various recombinant proteins. Knocking out or downregulating an endogenous silk protein is considered a viable strategy for improving the ability of transgenic expression systems to produce exogenous proteins. Here, we report the expression of human epidermal growth factor (hEGF) in a P25 gene knockout silkworm. The hEGF gene regulated by the P25 gene promoter was integrated into a silkworm’s genome. Five transgenic positive silkworm lineages were generated with different insertion sites on silkworm chromosomes and the ability to synthesize and secrete proteins into cocoons. Then, a cross-strategy was used to produce transgenic silkworms with a P25 gene knockout background. The results of the protein analysis showed that the loss of an endogenous P25 protein can increase the hEGF production to about 2.2-fold more than normal silkworms. Compared to those of transgenic silkworms with wild type (non-knockout) background, the morphology and secondary structure of cocoon silks were barely changed in transgenic silkworms with a P25 gene knockout background, indicating their similar physical properties of cocoon silks. In conclusion, P25 gene knockout silkworms may become an efficient bioreactor for the production of exogenous proteins and a promising tool for producing various protein-containing silk biomaterials.

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Section: Introductionmentioning

confidence: 99%

P25 Gene Knockout Contributes to Human Epidermal Growth Factor Production in Transgenic Silkworms

Ruan

et al. 2021

IJMS

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“…However, because of inefficient secretion, many recombinant proteins, particularly therapeutic proteins, have poor yields by P. pastoris [ 32 , 33 , 34 ]. Improving secretion levels through medium optimization or engineering strains may be a reasonable and promising strategy to increase the production of heterologous proteins by P. pastoris [ 35 , 36 , 37 ]. Our previous study has reported that NAC can promote HSA-pFSHβ secretion by increasing intracellular GSH pools [ 20 ].…”

Section: Discussionmentioning

confidence: 99%

Effect of N-acetyl-l-cysteine on Cell Phenotype and Autophagy in Pichia pastoris Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein

Geng

Yang

et al. 2023

Molecules

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Pichia pastoris is widely used for the production of recombinant proteins, but the low secretion efficiency hinders its wide application in biopharmaceuticals. Our previous study had shown that N-acetyl-l-cysteine (NAC) promotes human serum albumin and porcine follicle-stimulating hormone fusion protein (HSA-pFSHβ) secretion by increasing intracellular GSH levels, but the downstream impact mechanism is not clear. In this study, we investigated the roles of autophagy as well as cell phenotype in NAC promoting HSA-pFSHβ secretion. Our results showed that NAC slowed down the cell growth rate, and its effects were unaffected by Congo Red and Calcofluor White. Moreover, NAC affected cell wall composition by increasing chitin content and decreasing β-1,3-glucan content. In addition, the expressions of vesicular pathway and autophagy-related genes were significantly decreased after NAC treatment. Further studies revealed that autophagy, especially the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy and pexophagy, was significantly increased with time, and NAC has a promoting effect on autophagy, especially at 48 h and 72 h of NAC treatment. However, the disruption of mitophagy receptor Atg32, but not pexophagy receptor Atg30, inhibited HSA-pFSHβ production, and neither of them inhibited the NAC-promoted effect of HSA-pFSHβ. In conclusion, vesicular transport, autophagy and cell wall are all involved in the NAC-promoted HSA-pFSHβ secretion and that disruption of the autophagy receptor alone does not inhibit the effect of NAC.

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“…A novel system was reported for fast and easy expression of recombinant proteins in S. cerevisiae and P. pastoris. In S. cerevisiae, the gene needs only the transformation of yeast cells with an unpurified PCR product carrying the gene to be expressed, and in P. pastoris, it needs only the isolation of the plasmid generated in S. cerevisiae and its transformation into this second yeast, thus making this system suitable for HTP studies (González et al, 2018). A new, stable, autonomously replicating P. pastoris plasmid vector containing the full-length chromosome 2 centromeric DNA sequence was constructed that exhibits high stability for plasmid retention, facilitating genetic manipulation.…”

Section: Yeastmentioning

confidence: 99%

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

Tripathi

Shrivastava

2019

Front. Bioeng. Biotechnol.

369

243

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Infectious diseases, along with cancers, are among the main causes of death among humans worldwide. The production of therapeutic proteins for treating diseases at large scale for millions of individuals is one of the essential needs of mankind. Recent progress in the area of recombinant DNA technologies has paved the way to producing recombinant proteins that can be used as therapeutics, vaccines, and diagnostic reagents. Recombinant proteins for these applications are mainly produced using prokaryotic and eukaryotic expression host systems such as mammalian cells, bacteria, yeast, insect cells, and transgenic plants at laboratory scale as well as in large-scale settings. The development of efficient bioprocessing strategies is crucial for industrial production of recombinant proteins of therapeutic and prophylactic importance. Recently, advances have been made in the various areas of bioprocessing and are being utilized to develop effective processes for producing recombinant proteins. These include the use of high-throughput devices for effective bioprocess optimization and of disposable systems, continuous upstream processing, continuous chromatography, integrated continuous bioprocessing, Quality by Design, and process analytical technologies to achieve quality product with higher yield. This review summarizes recent developments in the bioprocessing of recombinant proteins, including in various expression systems, bioprocess development, and the upstream and downstream processing of recombinant proteins.

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New tools for high‐throughput expression of fungal secretory proteins in Saccharomyces cerevisiae and Pichia pastoris

Cited by 7 publications

References 38 publications

P25 Gene Knockout Contributes to Human Epidermal Growth Factor Production in Transgenic Silkworms

P25 Gene Knockout Contributes to Human Epidermal Growth Factor Production in Transgenic Silkworms

Effect of N-acetyl-l-cysteine on Cell Phenotype and Autophagy in Pichia pastoris Expressing Human Serum Albumin and Porcine Follicle-Stimulating Hormone Fusion Protein

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

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