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Genome editing of BmFib-H gene provides an empty Bombyx mori silk gland for a highly efficient bioreactor.

Ma S, Shi R, Wang X, Liu Y, Chang J, Gao J, Lu W, Zhang J, Zhao P, Xia Q - Sci Rep (2014)

Bottom Line: The impressive ability to secrete huge amount of pure silk protein, and to store proteins at an extremely high concentration (up to 25%) make the silk gland of Bombyx mori hold great promise to be a cost-effective platform for production of recombinant proteins.Using customized zinc finger nucleases (ZFN), we successfully performed genome editing of Bmfib-H gene, which encodes the largest and most abundant silk protein, in B. mori with efficiency higher than any previously reported.Furthermore, we demonstrated that the bioreactor is suitable for large scale production of protein-based materials.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China.

ABSTRACT
Evolution has produced some remarkable creatures, of which silk gland is a fascinating organ that exists in a variety of insects and almost half of the 34,000 spider species. The impressive ability to secrete huge amount of pure silk protein, and to store proteins at an extremely high concentration (up to 25%) make the silk gland of Bombyx mori hold great promise to be a cost-effective platform for production of recombinant proteins. However, the extremely low production yields of the numerous reported expression systems greatly hindered the exploration and application of silk gland bioreactors. Using customized zinc finger nucleases (ZFN), we successfully performed genome editing of Bmfib-H gene, which encodes the largest and most abundant silk protein, in B. mori with efficiency higher than any previously reported. The resulted Bmfib-H knocked-out B. mori showed a smaller and empty silk gland, abnormally developed posterior silk gland cells, an extremely thin cocoon that contain only sericin proteins, and a slightly heavier pupae. We also showed that removal of endogenous Bmfib-H protein could significantly increase the expression level of exogenous protein. Furthermore, we demonstrated that the bioreactor is suitable for large scale production of protein-based materials.

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Related in: MedlinePlus

Production of EGFP fusion proteins in Fib-H-I1.(a, b) Schematic representation of the transgenic vector. The detailed information of the vector construction was as described as Zhao et al.16 (c) The microinjection results and the fluorescent images of 3xp3-DsRed transgenic marker expression in the nerves system of embryo and compound eyes of adult. (d) Fluorescent images of dissected silk glands of N4-R3, N4/Fib-H-I1-R3 and Fib-H-I1-R3 during the wandering stage. (e) SDS-PAGE of cocoon proteins of N4 (1), N4/Fib-H-I1 (2), Fib-H-I1 (3), N4-R3 (4), N4/Fib-H-I1-R3 (5), Fib-H-I1-R3 (6), and (f) Western blotting analysis of cocoon proteins of N4-R3, N4/Fib-H-I1-R3 and Fib-H-I1-R3 with EGFP polyclonal antibody. Proteins were run at a 6% gel. The scale bars represent 1 cm.
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f2: Production of EGFP fusion proteins in Fib-H-I1.(a, b) Schematic representation of the transgenic vector. The detailed information of the vector construction was as described as Zhao et al.16 (c) The microinjection results and the fluorescent images of 3xp3-DsRed transgenic marker expression in the nerves system of embryo and compound eyes of adult. (d) Fluorescent images of dissected silk glands of N4-R3, N4/Fib-H-I1-R3 and Fib-H-I1-R3 during the wandering stage. (e) SDS-PAGE of cocoon proteins of N4 (1), N4/Fib-H-I1 (2), Fib-H-I1 (3), N4-R3 (4), N4/Fib-H-I1-R3 (5), Fib-H-I1-R3 (6), and (f) Western blotting analysis of cocoon proteins of N4-R3, N4/Fib-H-I1-R3 and Fib-H-I1-R3 with EGFP polyclonal antibody. Proteins were run at a 6% gel. The scale bars represent 1 cm.

Mentions: Compared with wild-type Dazao, the posterior silk glands (PSG) of Fib-H-I1 were smaller, while the anterior and middle silk glands (ASG and MSG) were normal in size (Fig. 1f and supplementary Fig. 4). We counted the number of cells in MSG and PSG in the 1st and 5th larvae stages, and no differences were observed. However, the PSG cells of Fib-H-I1 were smaller and abnormal in shape (Fig. 1f). This phenotype was in accordance with the fact that silk gland is an endoreplicating tissue and its growth solely depends on cell size increase. It is unclear how the cell size increase in PSG is regulated. The results here may provide some important information to reveal this mystery, because we shorted the PSG just by removing its product. More importantly, despite of the size of PSG and PSG cell, the lumen of Fib-H-I1 PSG was hollow inside, and MSG was almost empty with a very small amount of sericin proteins secreted by MSG cells (Fig. 1f and supplementary Fig. 4). To confirm this observation, the cocoons of Fib-H-I1 were firstly analyzed using scanning electron microscopy (SEM) and protein electrophoresis. Compared with wild-type Dazao, the cocoons of Fib-H-I1 were much lighter and thinner, while the pupae were little heavier (Fig. 1e, g), indicating a nutrient resorption from cocoon to pupae. SEM photography showed that the silk filaments within the cocoons were much thinner and exhibited a sericin-cocoon like phenotype (Fig. 3c). SDS-PAGE and western blotting analysis showed that the cocoons contained only sericins and some small fibroin proteins (supplementary Fig. 7). Although the mechanism of many aforementioned phenotypes is not clear, we were confidential to conclude that we had successfully generated a BmFib-H knock-out B. mori line, which might be an ideal solution for our original hypothesis as its PSGs became empty, while the ability of protein synthesis was not affected.


Genome editing of BmFib-H gene provides an empty Bombyx mori silk gland for a highly efficient bioreactor.

Ma S, Shi R, Wang X, Liu Y, Chang J, Gao J, Lu W, Zhang J, Zhao P, Xia Q - Sci Rep (2014)

Production of EGFP fusion proteins in Fib-H-I1.(a, b) Schematic representation of the transgenic vector. The detailed information of the vector construction was as described as Zhao et al.16 (c) The microinjection results and the fluorescent images of 3xp3-DsRed transgenic marker expression in the nerves system of embryo and compound eyes of adult. (d) Fluorescent images of dissected silk glands of N4-R3, N4/Fib-H-I1-R3 and Fib-H-I1-R3 during the wandering stage. (e) SDS-PAGE of cocoon proteins of N4 (1), N4/Fib-H-I1 (2), Fib-H-I1 (3), N4-R3 (4), N4/Fib-H-I1-R3 (5), Fib-H-I1-R3 (6), and (f) Western blotting analysis of cocoon proteins of N4-R3, N4/Fib-H-I1-R3 and Fib-H-I1-R3 with EGFP polyclonal antibody. Proteins were run at a 6% gel. The scale bars represent 1 cm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215353&req=5

f2: Production of EGFP fusion proteins in Fib-H-I1.(a, b) Schematic representation of the transgenic vector. The detailed information of the vector construction was as described as Zhao et al.16 (c) The microinjection results and the fluorescent images of 3xp3-DsRed transgenic marker expression in the nerves system of embryo and compound eyes of adult. (d) Fluorescent images of dissected silk glands of N4-R3, N4/Fib-H-I1-R3 and Fib-H-I1-R3 during the wandering stage. (e) SDS-PAGE of cocoon proteins of N4 (1), N4/Fib-H-I1 (2), Fib-H-I1 (3), N4-R3 (4), N4/Fib-H-I1-R3 (5), Fib-H-I1-R3 (6), and (f) Western blotting analysis of cocoon proteins of N4-R3, N4/Fib-H-I1-R3 and Fib-H-I1-R3 with EGFP polyclonal antibody. Proteins were run at a 6% gel. The scale bars represent 1 cm.
Mentions: Compared with wild-type Dazao, the posterior silk glands (PSG) of Fib-H-I1 were smaller, while the anterior and middle silk glands (ASG and MSG) were normal in size (Fig. 1f and supplementary Fig. 4). We counted the number of cells in MSG and PSG in the 1st and 5th larvae stages, and no differences were observed. However, the PSG cells of Fib-H-I1 were smaller and abnormal in shape (Fig. 1f). This phenotype was in accordance with the fact that silk gland is an endoreplicating tissue and its growth solely depends on cell size increase. It is unclear how the cell size increase in PSG is regulated. The results here may provide some important information to reveal this mystery, because we shorted the PSG just by removing its product. More importantly, despite of the size of PSG and PSG cell, the lumen of Fib-H-I1 PSG was hollow inside, and MSG was almost empty with a very small amount of sericin proteins secreted by MSG cells (Fig. 1f and supplementary Fig. 4). To confirm this observation, the cocoons of Fib-H-I1 were firstly analyzed using scanning electron microscopy (SEM) and protein electrophoresis. Compared with wild-type Dazao, the cocoons of Fib-H-I1 were much lighter and thinner, while the pupae were little heavier (Fig. 1e, g), indicating a nutrient resorption from cocoon to pupae. SEM photography showed that the silk filaments within the cocoons were much thinner and exhibited a sericin-cocoon like phenotype (Fig. 3c). SDS-PAGE and western blotting analysis showed that the cocoons contained only sericins and some small fibroin proteins (supplementary Fig. 7). Although the mechanism of many aforementioned phenotypes is not clear, we were confidential to conclude that we had successfully generated a BmFib-H knock-out B. mori line, which might be an ideal solution for our original hypothesis as its PSGs became empty, while the ability of protein synthesis was not affected.

Bottom Line: The impressive ability to secrete huge amount of pure silk protein, and to store proteins at an extremely high concentration (up to 25%) make the silk gland of Bombyx mori hold great promise to be a cost-effective platform for production of recombinant proteins.Using customized zinc finger nucleases (ZFN), we successfully performed genome editing of Bmfib-H gene, which encodes the largest and most abundant silk protein, in B. mori with efficiency higher than any previously reported.Furthermore, we demonstrated that the bioreactor is suitable for large scale production of protein-based materials.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing, China.

ABSTRACT
Evolution has produced some remarkable creatures, of which silk gland is a fascinating organ that exists in a variety of insects and almost half of the 34,000 spider species. The impressive ability to secrete huge amount of pure silk protein, and to store proteins at an extremely high concentration (up to 25%) make the silk gland of Bombyx mori hold great promise to be a cost-effective platform for production of recombinant proteins. However, the extremely low production yields of the numerous reported expression systems greatly hindered the exploration and application of silk gland bioreactors. Using customized zinc finger nucleases (ZFN), we successfully performed genome editing of Bmfib-H gene, which encodes the largest and most abundant silk protein, in B. mori with efficiency higher than any previously reported. The resulted Bmfib-H knocked-out B. mori showed a smaller and empty silk gland, abnormally developed posterior silk gland cells, an extremely thin cocoon that contain only sericin proteins, and a slightly heavier pupae. We also showed that removal of endogenous Bmfib-H protein could significantly increase the expression level of exogenous protein. Furthermore, we demonstrated that the bioreactor is suitable for large scale production of protein-based materials.

Show MeSH
Related in: MedlinePlus