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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 artificial silk protein in BmFib-H knock-out B. mori line.(a) Schematic representation of the structure of artificial silk protein and fluorescent imaging of different subdivision of transgenic silk gland. (b) Mechanical properties of cocoon sheet of Fib-H-I1 and Fib-H-I1-R3. For all histogram, the test number was 10. (c) SEM observation of WT, Fib-H-I1 and Fib-H-I1-R3 cocoons. (d) Diameters of silk fibers from cocoons of Fib-H-I1 and Fib-H-I1-R3. Diameters were measured from the SEM photograph, and 50 samples were used for each test. (e, f) Cocoon weight and cocoon shell rate of Fib-H-I1 and Fib-H-I1-R3. The scale bars represent 1 cm unless specifically labeled within this figure.
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f3: Production of artificial silk protein in BmFib-H knock-out B. mori line.(a) Schematic representation of the structure of artificial silk protein and fluorescent imaging of different subdivision of transgenic silk gland. (b) Mechanical properties of cocoon sheet of Fib-H-I1 and Fib-H-I1-R3. For all histogram, the test number was 10. (c) SEM observation of WT, Fib-H-I1 and Fib-H-I1-R3 cocoons. (d) Diameters of silk fibers from cocoons of Fib-H-I1 and Fib-H-I1-R3. Diameters were measured from the SEM photograph, and 50 samples were used for each test. (e, f) Cocoon weight and cocoon shell rate of Fib-H-I1 and Fib-H-I1-R3. The scale bars represent 1 cm unless specifically labeled within this figure.

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 artificial silk protein in BmFib-H knock-out B. mori line.(a) Schematic representation of the structure of artificial silk protein and fluorescent imaging of different subdivision of transgenic silk gland. (b) Mechanical properties of cocoon sheet of Fib-H-I1 and Fib-H-I1-R3. For all histogram, the test number was 10. (c) SEM observation of WT, Fib-H-I1 and Fib-H-I1-R3 cocoons. (d) Diameters of silk fibers from cocoons of Fib-H-I1 and Fib-H-I1-R3. Diameters were measured from the SEM photograph, and 50 samples were used for each test. (e, f) Cocoon weight and cocoon shell rate of Fib-H-I1 and Fib-H-I1-R3. The scale bars represent 1 cm unless specifically labeled within this figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Production of artificial silk protein in BmFib-H knock-out B. mori line.(a) Schematic representation of the structure of artificial silk protein and fluorescent imaging of different subdivision of transgenic silk gland. (b) Mechanical properties of cocoon sheet of Fib-H-I1 and Fib-H-I1-R3. For all histogram, the test number was 10. (c) SEM observation of WT, Fib-H-I1 and Fib-H-I1-R3 cocoons. (d) Diameters of silk fibers from cocoons of Fib-H-I1 and Fib-H-I1-R3. Diameters were measured from the SEM photograph, and 50 samples were used for each test. (e, f) Cocoon weight and cocoon shell rate of Fib-H-I1 and Fib-H-I1-R3. The scale bars represent 1 cm unless specifically labeled within this figure.
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