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.
Highlights
Due to a breeding and domestication history of over 4000 years, the silk gland of silkworm Bombyx mori possesses the huge ability to synthesize a large amount of silk proteins in its silk gland and secrete them as silk thread to build a cocoon, making it an ideal bioreactor mode organ for the mass production of valuable recombinant proteins[16]
The silk thread is composed of two types of silk protein, the major fibroin proteins, which are synthesized in the posterior silk gland cells and account for 70%– 80% of the silk thread weight, and the hydrophilic glue sericin proteins, which are synthesized in the middle silk gland cells and account for 20%–30% of the silk thread weight[17]
Immunohistochemical analysis of the cross sections of the middle silk gland (MSG) and silk of transgenic silkworm showed that the synthesized Human acidic fibroblast growth factor (haFGF) recombinant proteins were secreted into the sericin layer of the MSG lumen, and spun into the sericin layer of silk which was consistent with a previous report[27] (Fig. 1C,D)
Summary
Due to a breeding and domestication history of over 4000 years, the silk gland of silkworm Bombyx mori possesses the huge ability to synthesize a large amount of silk proteins in its silk gland and secrete them as silk thread to build a cocoon, making it an ideal bioreactor mode organ for the mass production of valuable recombinant proteins[16]. The fibroin and sericin expression systems, were developed and over ten foreign proteins, including model proteins, human or animal-derived pharmaceutical proteins and silk-based proteins, have been successfully expressed in transgenic silkworm silk glands using these expression systems in the past decade[22,23,24,25,26,27,28] These results showed that the silk gland is considered to be a cost-effective, easy-scale-up and processed bioreactor system for pharmaceutical protein production and silk genetically engineered proteins with improved mechanical properties and new biofunctionalities. Our results show that the silk gland of silkworm combined with jumpstarter-mediated remobilization could be an efficient bioreactor strategy for the large-scale production of bioactive recombinant haFGF in cocoons
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