Limits...
Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons.

Wang F, Wang R, Wang Y, Zhao P, Xia Q - Sci Rep (2015)

Bottom Line: The high content of r-haFGF facilitated its purification and large-scald yields.Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing.These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons.

View Article: PubMed Central - PubMed

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

ABSTRACT
With an increasing clinical demand for functional therapeutic proteins every year, there is an increasing requirement for the massive production of bioactive recombinant human acidic fibroblast growth factor (r-haFGF). In this present study, we delicately explore a strategy for the mass production of r-haFGF protein with biological activity in the transgenic silkworm cocoons. The sequence-optimized haFGF was inserted into an enhanced sericin-1 expression system to generate the original transgenic silkworm strain, which was then further crossed with a PIG jumpstarter strain to achieve the remobilization of the expression cassette to a "safe harbor" locus in the genome for the efficient expression of r-haFGF. In consequence, the expression of r-haFGF protein in the mutant line achieved a 5.6-fold increase compared to the original strain. The high content of r-haFGF facilitated its purification and large-scald yields. Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing. These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons.

No MeSH data available.


Related in: MedlinePlus

(A) In vitro wound healing assay by treatment of the scratched NIH/3T3 with purified haFGF and a haFGF standard. Scale bar represents 200 μm. (B) The cell numbers in the scratched areas of different groups. Results are representative of three independent experiments. Asterisks indicate statistical significance based on Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4644950&req=5

f6: (A) In vitro wound healing assay by treatment of the scratched NIH/3T3 with purified haFGF and a haFGF standard. Scale bar represents 200 μm. (B) The cell numbers in the scratched areas of different groups. Results are representative of three independent experiments. Asterisks indicate statistical significance based on Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001).

Mentions: The bioactivity of the purified haFGF was investigated by cellular experiments. Firstly, the purified haFGF was used to cultivate NIH/3T3 cells and an equal hFGF1 standard was used as the positive control. Cell proliferation of NIH/3T3 could be significantly induced by purified haFGF with dosages of 100 ng/ml or 200 ng/ml (Fig. 4A). Thus the purified haFGF with dosage of 100 ng/ml was used to perform the further assay. The cell growth condition was checked at 24 h and 48 h time sites, respectively. The results showed that the NIH/3T3 cells without haFGF treatment grew poorly, and cellular apoptosis occurred. By contrast, the NIH/3T3 cells treated by purified haFGF or an equal hFGF1 standard grew well; they stretched on dishes and formed the typical morphology of cellular shape (Fig. 4B). NIH/3T3 cells treated with the purified haFGF and an equal hFGF1 standard also showed an increased absorbance at 450 nm at the 24 h, 48 h and 72 h time sites, respectively (Fig. 4C). The cell proliferation effects on NIH/3T3 cells induced by the purified haFGF were slightly lower at the 24 h and 48 h time sites, but higher at the 72 h time site than that of an equal hFGF1 standard, respectively. These results strongly suggested that the purified haFGF recombinant protein showed strong mitogenic activity to promote the cell proliferation of NIH/3T3 cells. Immunocytochemical analysis by EdU incorporation was used to monitor NIH/3T3 cell proliferation; the results showed that cells treated with purified haFGF and an equal hFGF1 standard exhibited strong RFP fluorescence signals comparing to a none-treated control (Fig. 5). A wound-healing assay was performed to investigate the effect of haFGF on NIH/3T3 cell migration. The NIH/3T3 cells treated by purified haFGF and a hFGF1 standard spread into the scratch regions (Fig. 6A), and their migrating cells were significantly higher than that of the control (Fig. 6B), suggesting the purified haFGF promoted chemotactic motility of NIH/3T3 cells and showed equivalent efficacy in wound healing. Furthermore, the economic characteristics of the transgenic silkworm were analyzed. The cocoon and pupa phenotypes of the transgenic silkworm were similar to that of the non-transgenic silkworm, and no obvious difference were found in the weights of the male and female cocoon and pupa between the transgenic silkworm and the wild-type silkworm (Supplementary Figure S3). These results suggest that overexpressing exogenous proteins in silk glands of transgenic silkworms did not influence the economic characteristics of the silkworm, and the silk gland of the transgenic silkworm could be a suitable bioreactor candidate for mass production of bioactive recombinant pharmaceutical proteins.


Large-scale production of bioactive recombinant human acidic fibroblast growth factor in transgenic silkworm cocoons.

Wang F, Wang R, Wang Y, Zhao P, Xia Q - Sci Rep (2015)

(A) In vitro wound healing assay by treatment of the scratched NIH/3T3 with purified haFGF and a haFGF standard. Scale bar represents 200 μm. (B) The cell numbers in the scratched areas of different groups. Results are representative of three independent experiments. Asterisks indicate statistical significance based on Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4644950&req=5

f6: (A) In vitro wound healing assay by treatment of the scratched NIH/3T3 with purified haFGF and a haFGF standard. Scale bar represents 200 μm. (B) The cell numbers in the scratched areas of different groups. Results are representative of three independent experiments. Asterisks indicate statistical significance based on Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001).
Mentions: The bioactivity of the purified haFGF was investigated by cellular experiments. Firstly, the purified haFGF was used to cultivate NIH/3T3 cells and an equal hFGF1 standard was used as the positive control. Cell proliferation of NIH/3T3 could be significantly induced by purified haFGF with dosages of 100 ng/ml or 200 ng/ml (Fig. 4A). Thus the purified haFGF with dosage of 100 ng/ml was used to perform the further assay. The cell growth condition was checked at 24 h and 48 h time sites, respectively. The results showed that the NIH/3T3 cells without haFGF treatment grew poorly, and cellular apoptosis occurred. By contrast, the NIH/3T3 cells treated by purified haFGF or an equal hFGF1 standard grew well; they stretched on dishes and formed the typical morphology of cellular shape (Fig. 4B). NIH/3T3 cells treated with the purified haFGF and an equal hFGF1 standard also showed an increased absorbance at 450 nm at the 24 h, 48 h and 72 h time sites, respectively (Fig. 4C). The cell proliferation effects on NIH/3T3 cells induced by the purified haFGF were slightly lower at the 24 h and 48 h time sites, but higher at the 72 h time site than that of an equal hFGF1 standard, respectively. These results strongly suggested that the purified haFGF recombinant protein showed strong mitogenic activity to promote the cell proliferation of NIH/3T3 cells. Immunocytochemical analysis by EdU incorporation was used to monitor NIH/3T3 cell proliferation; the results showed that cells treated with purified haFGF and an equal hFGF1 standard exhibited strong RFP fluorescence signals comparing to a none-treated control (Fig. 5). A wound-healing assay was performed to investigate the effect of haFGF on NIH/3T3 cell migration. The NIH/3T3 cells treated by purified haFGF and a hFGF1 standard spread into the scratch regions (Fig. 6A), and their migrating cells were significantly higher than that of the control (Fig. 6B), suggesting the purified haFGF promoted chemotactic motility of NIH/3T3 cells and showed equivalent efficacy in wound healing. Furthermore, the economic characteristics of the transgenic silkworm were analyzed. The cocoon and pupa phenotypes of the transgenic silkworm were similar to that of the non-transgenic silkworm, and no obvious difference were found in the weights of the male and female cocoon and pupa between the transgenic silkworm and the wild-type silkworm (Supplementary Figure S3). These results suggest that overexpressing exogenous proteins in silk glands of transgenic silkworms did not influence the economic characteristics of the silkworm, and the silk gland of the transgenic silkworm could be a suitable bioreactor candidate for mass production of bioactive recombinant pharmaceutical proteins.

Bottom Line: The high content of r-haFGF facilitated its purification and large-scald yields.Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing.These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons.

View Article: PubMed Central - PubMed

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

ABSTRACT
With an increasing clinical demand for functional therapeutic proteins every year, there is an increasing requirement for the massive production of bioactive recombinant human acidic fibroblast growth factor (r-haFGF). In this present study, we delicately explore a strategy for the mass production of r-haFGF protein with biological activity in the transgenic silkworm cocoons. The sequence-optimized haFGF was inserted into an enhanced sericin-1 expression system to generate the original transgenic silkworm strain, which was then further crossed with a PIG jumpstarter strain to achieve the remobilization of the expression cassette to a "safe harbor" locus in the genome for the efficient expression of r-haFGF. In consequence, the expression of r-haFGF protein in the mutant line achieved a 5.6-fold increase compared to the original strain. The high content of r-haFGF facilitated its purification and large-scald yields. Furthermore, the r-haFGF protein bioactively promoted the growth, proliferation and migration of NIH/3T3 cells, suggesting the r-haFGF protein possessed native mitogenic activity and the potential for wound healing. These results show that the silk gland of silkworm could be an efficient bioreactor strategy for recombinant production of bioactive haFGF in silkworm cocoons.

No MeSH data available.


Related in: MedlinePlus