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

Wu, Meiyu; Ruan, Jinghua; Ye, Xiaogang; Zhao, Shuo; Tang, Xiaoli; Wang, Xiaoxiao; Li, Huiping

doi:10.3390/ijms22052709

Cited by 8 publications

(8 citation statements)

References 30 publications

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“…2 ). We demonstrated that P25, a major component of the ancient cocoon silk structure, is substitutable, as also demonstrated by a recent report of knocking out the P25 coding gene in Bombyx mori 8 , 33 . P25 is a glycoprotein containing Asn-linked oligosaccharide chains that forms a compact structure because of intramolecular disulfide linkages but associates with the H–L complex through non-covalent interactions 34 .…”

Section: Discussionsupporting

confidence: 83%

Ectopic expression of sericin enables efficient production of ancient silk with structural changes in silkworm

Chen

Wang

et al. 2022

Nat Commun

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Bombyx mori silk is a super-long natural protein fiber with a unique structure and excellent performance. Innovative silk structures with high performance are in great demand, thus resulting in an industrial bottleneck. Herein, the outer layer sericin SER3 is ectopically expressed in the posterior silk gland (PSG) in silkworms via a piggyBac-mediated transgenic approach, then secreted into the inner fibroin layer, thus generating a fiber with sericin microsomes dispersed in fibroin fibrils. The water-soluble SER3 protein secreted by PSG causes P25’s detachment from the fibroin unit of the Fib-H/Fib-L/P25 polymer, and accumulation between the fibroin layer and the sericin layer. Consequently, the water solubility and stability of the fibroin-colloid in the silk glandular cavity, and the crystallinity increase, and the mechanical properties of cocoon fibers, moisture absorption and moisture liberation of the silk also improve. Meanwhile, the mutant overcomes the problems of low survival and abnormal silk gland development, thus enabling higher production efficiency of cocoon silk. In summary, we describe a silk gland transgenic target protein selection strategy to alter the silk fiber structure and to innovate its properties. This work provides an efficient and green method to produce silk fibers with new functions.

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Section: Discussionsupporting

confidence: 83%

Ectopic expression of sericin enables efficient production of ancient silk with structural changes in silkworm

Chen

Wang

et al. 2022

Nat Commun

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“…Proteins were extracted according to a previously described method with some modifications ( Wu et al, 2021 ). Briefly, silk gland and cocoon shell proteins were extracted with sodium dodecyl sulfate (SDS) buffer (1:40, wt/vol) containing 5% (vol/vol) β-mercaptoethanol, and the supernatants were collected by centrifuging after incubation at 37 °C for 8 h. The extracted proteins were quantified using a Modified BCA Protein Assay Kit (Sangon Biotech); then, western blotting analysis was performed.…”

Section: Methodsmentioning

confidence: 99%

“…Expression systems based on FibH, FibL, and ser1 promoters were often used because they could express relatively high levels of exogenous proteins ( Xu, 2014 ). Since 2003, when the FibL promoter was first used to specifically express recombinant proteins in silkworm silk glands via piggyBac transposon-mediated transgenes ( Tomita et al, 2003 ), the production of recombinant proteins using transgenic silkworm silk gland bioreactors has entered a period of rapid development ( Long et al, 2021 ; Wu et al, 2021 ; Chen et al, 2022 ). In addition, modification of piggyBac transposon-based transgene regulatory elements has been found to improve the expression levels of exogenous proteins ( Iizuka et al, 2008 ; Zhao et al, 2021 ), and a recent study showed that the use of native regulatory elements could increase the production of exogenous proteins up to 15-fold compared with an artificially truncated promoter ( Li et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

“…Although the piggyBac transposon-mediated transgenic silkworm silk gland has been gradually developed as a bioreactor in recent years ( Tomita et al, 2003 ; Long et al, 2021 ; Wu et al, 2021 ), piggyBac -mediated transgenes have some limitations such as uncertain insertion sites, low expression, and gene drift ( Fraser, 2012 ). However, the advent of targeted gene editing, which creates double-stranded breaks (DSBs) at genomic DNA, allows researchers to modify specific locations in the genome of the organism of interest; it can also enable more precise gene modification and more stable inheritance.…”

Section: Introductionmentioning

confidence: 99%

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TALEN-mediated homologous-recombination-based fibroin light chain in-fusion expression system in Bombyx mori

Yu,

Zheng,

et al. 2024

Front. Bioeng. Biotechnol.

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Silkworm was the first domesticated insect and has important economic value. It has also become an ideal model organism with applications in genetic and expression studies. In recent years, the use of transgenic strategies has made the silkworm silk gland an attractive bioreactor for the production of recombinant proteins, in particular, piggyBac-mediated transgenes. However, owing to differences in regulatory elements such as promoters, the expression levels of exogenous proteins have not reached expectations. Here, we used targeted gene editing to achieve site-specific integration of exogenous genes on genomic DNA and established the fibroin light chain (FibL) in-fusion expression system by TALEN-mediated homology-directed recombination. First, the histidine-rich cuticular protein (CP) was successfully site-directed inserted into the native FibL, and the FibL–CP fusion gene was correctly transcribed and expressed in the posterior silk gland under the control of the endogenous FibL promoter, with a protein expression level comparable with that of the native FibL protein. Moreover, we showed based on molecular docking that the fusion of FibL with cuticular protein may have a negative effect on disulfide bond formation between the C-terminal domain of fibroin heavy chain (FibH) and FibL–CP, resulting in abnormal spinning and cocoon in homozygotes, indicating a significant role of FibL in silk protein formation and secretion. Our results demonstrate the feasibility of using the FibL fusion system to express exogenous proteins in silkworm. We expect that this bioreactor system will be used to produce more proteins of interest, expanding the application value of the silk gland bioreactor.

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“…It has also been shown that production of human epidermal growth factor (hEGF) of about 8 kDa at 2.2 times higher amounts than normal silkworms was also possible by knocking out the P25 gene in silkworms. Modified silkworms were similar in appearance and produced fibers with same properties compared to cocoons obtained from unmodified silkworms (Wu et al, 2021). Increase in production of exogenous proteins by knocking out endogenous proteins such as P25 were suggested to be viable approaches for large scale production of proteins and proteins containing biomolecules.…”

Section: Production Of Growth Factors By Transgenic Silkwormsmentioning

confidence: 99%

Transgenic modifications of silkworms as a means to obtain therapeutic biomolecules and protein fibers with exceptional properties

Aramwit,

Jiang,

Muppuri

et al. 2023

Biotech & Bioengineering

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Transgenic modification of Bombyx mori silkworms is a benign approach for the production of silk fibers with extraordinary properties and also to generate therapeutic proteins and other biomolecules for various applications. Silk fibers with fluorescence lasting more than a year, natural protein fibers with strength and toughness exceeding that of spider silk, proteins and therapeutic biomolecules with exceptional properties have been developed using transgenic technology. The transgenic modifications have been done primarily by modifying the silk sericin and fibroin genes and also the silk producing glands. Although the genetic modifications were typically performed using the sericin 1 and other genes, newer techniques such as CRISPR/Cas9 have enabled successful modifications of both the fibroin H‐chain and L‐chain. Such modifications have led to the production of therapeutic proteins and other biomolecules in reasonable quantities at affordable costs for tissue engineering and other medical applications. Transgenically modified silkworms also have distinct and long‐lasting fluorescence useful for bioimaging applications. This review presents an overview of the transgenic techniques for modifications of B. mori silkworms and the properties obtained due to such modifications with particular focus on production of growth factors, fluorescent proteins, and high performance protein fibers.

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

Cited by 8 publications

References 30 publications

Ectopic expression of sericin enables efficient production of ancient silk with structural changes in silkworm

Ectopic expression of sericin enables efficient production of ancient silk with structural changes in silkworm

TALEN-mediated homologous-recombination-based fibroin light chain in-fusion expression system in Bombyx mori

Transgenic modifications of silkworms as a means to obtain therapeutic biomolecules and protein fibers with exceptional properties

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