Abstract
Human platelet derived growth factor (PDGF) is a major therapeutic protein with great demand in the clinical setting; however, its rate of supply is far from meeting needs. Here, we provide an effective strategy to produce PDGF-BB in large quantities using a transgenic silkworm. The codon-optimized PDGF-B gene regulated by the highly efficient sericin-1 expression system was integrated into the genome of a silkworm. The high transcriptional expression of the PDGF-BB gene in the transgenic silkworm competitively inhibited the transcription expression of the endogenous sericin-1 gene which caused a significant 37.5% decline. The PDGF-BB synthesized in the middle silk gland (MSG) of transgenic silkworms could form a homodimer through intermolecular disulfide bonds, which is then secreted into sericin lumen and finally, distributed in the sericin layer of the cocoon. In this study, a protein quantity of approximately 0.33 mg/g was found in the cocoon. Following a purification process, approximately 150.7 μg of recombinant PDGF-BB with a purity of 82% was purified from 1 g of cocoons. Furthermore, the bioactivity assays showed that the purified recombinant PDGF-BB was able to promote the growth, proliferation and migration of NIH/3T3 cells significantly. These results suggest that the silk gland bioreactor can produce active recombinant PDGF-BB as an efficient mitogen and wound healing agent.
Highlights
Human platelet derived growth factor (PDGF) belongs to the glycoprotein dimer family, and has four isoforms: PDGF-A, PDGF-B, PDGF-C, and PDGF-D [1]
The molecular weight of PDGF-BB is 14 kDa, and it plays an important role in many processes, such as cell proliferation and wound healing, due to it is a strong activity as a mitogen for various cell types, especially vascular endothelial cells (VECs) and bone marrow mesenchymal stem cell (BMSCs) [1]
We established an efficient sericin-1 expression system to produce the recombinant foreign proteins in the middle silk gland of larval silkworm [17]. This expression system was applied to the recombinant expression of PDGF-BB by the transgenic silkworm
Summary
Human platelet derived growth factor (PDGF) belongs to the glycoprotein dimer family, and has four isoforms: PDGF-A, PDGF-B, PDGF-C, and PDGF-D [1]. The PDGFs bind to the PDGF receptor (PDGFR) through a homodimer or heterodimer, such as PDGF-AA, PDGF-BB, or PDGF-AB, for molecular signal transition [2]. Among these compounds, PDGF-BB, constructed of two PDGF-B chains is the most commonly found dimer in the human body. The molecular weight of PDGF-BB is 14 kDa, and it plays an important role in many processes, such as cell proliferation and wound healing, due to it is a strong activity as a mitogen for various cell types, especially vascular endothelial cells (VECs) and bone marrow mesenchymal stem cell (BMSCs) [1]. The clinical demand for PDGF-BB for a wide range of applications in biomedicine and bio-cosmetics has increased, drawing much attention to the recombinant expression of PDGF-BB. As a result, constructing an efficient strategy for the cost effective and mass production of recombinant PDGF-BB with native bioactivity is urgent and necessary
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