Transgenic Silkworm-Based Silk Gland Bioreactor for Large Scale Production of Bioactive Human Platelet-Derived Growth Factor (PDGF-BB) in Silk Cocoons

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.

A silkworm based silk gland bioreactor for high-efficiency production of recombinant human lactoferrin with antibacterial and anti-inflammatory activities

“…Interestingly, we found that the expression level of rhLF achieved in this study was higher than that of growth factors [31, 33] produced in the silk glands of transgenic silkworms using a similar expression system. We speculated that the silk gland cells of silkworm likely preferred synthesis of proteins with higher molecular weights and higher abundance, such as human serum albumin [41] and antibodies [32, 39] that exhibit a pattern of high expression levels; these high abundant proteins with various biological activities are crucial for the organism.…”

“…With the development of genetic manipulation tools, in particular the successful establishment of the piggyBac transposon-mediated genetic transformation technology in silkworms [25], it is possible to genetically engineer the silkworm silk gland as an ideal bioreactor for the recombinant expression of more valuable foreign proteins along with synthesis of their silk proteins [26]. Therefore, two major expression systems associated with the usage of promoters from the genes for fibroins and sericins have been constructed [27–29], and more than ten recombinant proteins with various bio-functions and application potential have been successfully expressed in the silk glands of transgenic silkworms and cocoons, including human collagen, human serum albumin, antibodies, and human growth factors [30–33]. These efforts have greatly booted the process of massive production of recombinant pharmaceutical proteins by the silk gland of silkworm to meet the daily increased clinical demands.…”

Development of a Mucus Gland Bioreactor in Loach Paramisgurnus dabryanus

“…The development of highly effective bioreactors has always been a hot research topic due to an increasing market demand for protein/peptide drugs [ 40 ]. As of May 2017, 74 antibody-based molecules have been approved by a regulatory authority in a major market [ 41 ].…”

Transgenic and Diet-Enhanced Silk Production for Reinforced Biomaterials: A Metamaterial Perspective

“…An experimental animal model has also been widely used for recombinant silk protein production (154). Another advantage is that transgenic silkworms can provide high-level expression and efficient secretion of several recombinant proteins (96)(97)(98)(99)(100). Silkworm transgenesis can be an economical means of producing recombinant human type III procollagen, human serum albumin, human acid fibroblast growth factor, and antibodies (153,158,159).…”

“…Given this wealth of information, we focus primarily on silkworm-produced silk with properties reinforced using silkworm transgenesis and functional additive diets. Transgenic silkworms have been exploited to produce recombinant proteins of interest in a scalable manner (96)(97)(98)(99)(100).…”