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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

The strategy of PIG transposase-mediated transposon remobilization to improve the expression of haFGF in transgenic silkworm.(A) The procedure of the PIG transposase mediated transposon remobilization strategy. (B) Large-scale analysis of haFGF expression patterns in individuals of the offspring after hybridization and segregation by ELISA. (C) SDS–PAGE analysis of the haFGF proteins in cocoons from typical strains after hybridization with the jumpstarter. The asterisk indicates haFGF proteins. (D) Western blot analysis of haFGF proteins in the cocoons of typical strains after hybridization with the jumpstarter. The numbers represent the increased folds of haFGF expression levels in the remobilized mutants compared to that of the A11 line. (E) Genetic analysis of the insertion loci of the mutant strains afte r hybridization with the jumpstarter.
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f2: The strategy of PIG transposase-mediated transposon remobilization to improve the expression of haFGF in transgenic silkworm.(A) The procedure of the PIG transposase mediated transposon remobilization strategy. (B) Large-scale analysis of haFGF expression patterns in individuals of the offspring after hybridization and segregation by ELISA. (C) SDS–PAGE analysis of the haFGF proteins in cocoons from typical strains after hybridization with the jumpstarter. The asterisk indicates haFGF proteins. (D) Western blot analysis of haFGF proteins in the cocoons of typical strains after hybridization with the jumpstarter. The numbers represent the increased folds of haFGF expression levels in the remobilized mutants compared to that of the A11 line. (E) Genetic analysis of the insertion loci of the mutant strains afte r hybridization with the jumpstarter.

Mentions: Previous studies have shown that transgene expression will suffer host chromosome “position effect” due to the random insertion of the transposon, and higher transgene expression might occur preferably at the safe harbor loci323334. In silkworm, the phenomenon of transgene expression variation caused by the chromosome “position effect” was also observed in many reports2426293031. For higher expression of the transgene, we designed a jumpstart strategy of a piggyBac transposase-induced transposon remobilization to select the potential “safe harbor” loci in the silkworm genome, which permitted a higher expression of the exogenous gene (Fig. 2A). The original haFGF line 11 (L11) with a single transgene copy was hybridized with our previously constructed jumpstarter strain, which stably and universally expressed the piggyBac transposase35; the F1 moths with both 3xp3EGFP and 3xp3DsRed markers were then backcrossed with the wild type for segregation of the transposase. An ELISA assay of 96 randomly selected F2 progenies with only the 3xp3EGFP marker showed that the patterns of haFGF expression varied dramatically among those individuals, among which some significantly increased or decreased expression compared to the original L11 line (Fig. 2B). SDS–PAGE and western blot assays of the cocoon proteins from typical mutants further confirmed that haFGF expression levels significantly increased 4.2- to 5.6-fold in strains such as H9, G1, B10 and H2, or severely decreased in lines such as A1, A5, A6 and L9, which gave no detection (Fig. 2C,D). Inverse PCR analysis determined whether the PIG jumpstarter induced the remobilization of piggyBac in the genome, which caused a variation of haFGF expression among F2 progenies. The results showed that the original L11 strain contained one transgene insertion which located at nscaf2766: 342874 locus on Chr.17; after hybridization, the transgene remobilized to nscaf2888: 4013240 locus on Chr.15 in the A1 line, nscaf1681: 5840922 locus on Chr.22 in the B10 line, nscaf1681: 5840922 locus on Chr.22 and nscaf2529: 169295 locus on Chr.5 in the H2 line, respectively (Fig. 2E, Supplementary Table S1 and Figure S1). The results suggested that the transposase triggered the remobilization of transgene to another genomic locus in transgenic silkworm, and induced the expression variation of haFGF in the different strains. Genomic sequence analysis further indicated that the transgene insertions where the haFGF expression was increased located in regions containing no or few endogenous genes nearby (50 Kb upstream or downstream), while those insertions where the haFGF expression level was severely decreased located in intergenic regions containing many endogenous genes which might influence the expression of haFGF (Supplementary Figure S1), suggesting that genomic region containing no or few endogenous genes nearby (50 Kb upstream or downstream) could be favorable for exogenous gene expression. In consequence, the B10 line with a single transgene insertion and high-level expression of haFGF, which accounted for 26% of the totally extracted silk proteins and 5.2% of the cocoon shell weight, was maintained for further studies.


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)

The strategy of PIG transposase-mediated transposon remobilization to improve the expression of haFGF in transgenic silkworm.(A) The procedure of the PIG transposase mediated transposon remobilization strategy. (B) Large-scale analysis of haFGF expression patterns in individuals of the offspring after hybridization and segregation by ELISA. (C) SDS–PAGE analysis of the haFGF proteins in cocoons from typical strains after hybridization with the jumpstarter. The asterisk indicates haFGF proteins. (D) Western blot analysis of haFGF proteins in the cocoons of typical strains after hybridization with the jumpstarter. The numbers represent the increased folds of haFGF expression levels in the remobilized mutants compared to that of the A11 line. (E) Genetic analysis of the insertion loci of the mutant strains afte r hybridization with the jumpstarter.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: The strategy of PIG transposase-mediated transposon remobilization to improve the expression of haFGF in transgenic silkworm.(A) The procedure of the PIG transposase mediated transposon remobilization strategy. (B) Large-scale analysis of haFGF expression patterns in individuals of the offspring after hybridization and segregation by ELISA. (C) SDS–PAGE analysis of the haFGF proteins in cocoons from typical strains after hybridization with the jumpstarter. The asterisk indicates haFGF proteins. (D) Western blot analysis of haFGF proteins in the cocoons of typical strains after hybridization with the jumpstarter. The numbers represent the increased folds of haFGF expression levels in the remobilized mutants compared to that of the A11 line. (E) Genetic analysis of the insertion loci of the mutant strains afte r hybridization with the jumpstarter.
Mentions: Previous studies have shown that transgene expression will suffer host chromosome “position effect” due to the random insertion of the transposon, and higher transgene expression might occur preferably at the safe harbor loci323334. In silkworm, the phenomenon of transgene expression variation caused by the chromosome “position effect” was also observed in many reports2426293031. For higher expression of the transgene, we designed a jumpstart strategy of a piggyBac transposase-induced transposon remobilization to select the potential “safe harbor” loci in the silkworm genome, which permitted a higher expression of the exogenous gene (Fig. 2A). The original haFGF line 11 (L11) with a single transgene copy was hybridized with our previously constructed jumpstarter strain, which stably and universally expressed the piggyBac transposase35; the F1 moths with both 3xp3EGFP and 3xp3DsRed markers were then backcrossed with the wild type for segregation of the transposase. An ELISA assay of 96 randomly selected F2 progenies with only the 3xp3EGFP marker showed that the patterns of haFGF expression varied dramatically among those individuals, among which some significantly increased or decreased expression compared to the original L11 line (Fig. 2B). SDS–PAGE and western blot assays of the cocoon proteins from typical mutants further confirmed that haFGF expression levels significantly increased 4.2- to 5.6-fold in strains such as H9, G1, B10 and H2, or severely decreased in lines such as A1, A5, A6 and L9, which gave no detection (Fig. 2C,D). Inverse PCR analysis determined whether the PIG jumpstarter induced the remobilization of piggyBac in the genome, which caused a variation of haFGF expression among F2 progenies. The results showed that the original L11 strain contained one transgene insertion which located at nscaf2766: 342874 locus on Chr.17; after hybridization, the transgene remobilized to nscaf2888: 4013240 locus on Chr.15 in the A1 line, nscaf1681: 5840922 locus on Chr.22 in the B10 line, nscaf1681: 5840922 locus on Chr.22 and nscaf2529: 169295 locus on Chr.5 in the H2 line, respectively (Fig. 2E, Supplementary Table S1 and Figure S1). The results suggested that the transposase triggered the remobilization of transgene to another genomic locus in transgenic silkworm, and induced the expression variation of haFGF in the different strains. Genomic sequence analysis further indicated that the transgene insertions where the haFGF expression was increased located in regions containing no or few endogenous genes nearby (50 Kb upstream or downstream), while those insertions where the haFGF expression level was severely decreased located in intergenic regions containing many endogenous genes which might influence the expression of haFGF (Supplementary Figure S1), suggesting that genomic region containing no or few endogenous genes nearby (50 Kb upstream or downstream) could be favorable for exogenous gene expression. In consequence, the B10 line with a single transgene insertion and high-level expression of haFGF, which accounted for 26% of the totally extracted silk proteins and 5.2% of the cocoon shell weight, was maintained for further studies.

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