Abstract
The domesticated silkworm, Bombyx mori, is an economically important insect that synthesizes large amounts of silk proteins in its silk gland to make cocoons. In recent years, germline transformation strategies advanced the bioengineering of the silk gland as an ideal bioreactor for mass production of recombinant proteins. However, the yield of exogenous proteins varied largely due to the random insertion and gene drift caused by canonical transposon-based transformation, calling for site-specific and stable expression systems. In the current study, we established a targeted in-fusion expression system by using the transcription activator-like effector nuclease (TALEN)-mediated targeted insertion to target genomic locus of sericin, one of the major silk proteins. We successfully generated chimeric Sericin1-EGFP (Ser-2A-EGFP) transformant, producing up to 3.1% (w/w) of EGFP protein in the cocoon shell. With this strategy, we further expressed the medically important human epidermal growth factor (hEGF) and the protein yield in both middle silk glands, and cocoon shells reached to more than 15-fold higher than the canonical piggyBac-based transgenesis. This natural Sericin1 expression system provides a new strategy for producing recombinant proteins by using the silkworm silk gland as the bioreactor.
Highlights
The lepidopteran model insect Bombyx mori is an important economic insect and possesses a highly specialized larval tissue, the silk gland, to synthesize and secret massive silk proteins in a few days of the late final larval instar
Fibroin protein is synthesized in the posterior silk gland (PSG), assembled in the lumen of the middle silk gland (MSG) with sericin, and secreted into the anterior silk gland (ASG) to spin and form cocoon shells
In vitro synthesized transcription activator-like effector nuclease (TALEN) mRNA and Homologous Recombination (HR) donors were coinjected into 640 silkworm preblastoderm eggs in each group to generate Ser–2A–enhanced green fluorescent protein (EGFP) transgenic line
Summary
The lepidopteran model insect Bombyx mori is an important economic insect and possesses a highly specialized larval tissue, the silk gland, to synthesize and secret massive silk proteins in a few days of the late final larval instar. Each silkworm eats 20 g of mulberry leaves and produces about 0.5 g of pure silk protein, holding the great promise to be a cost-effective system for mass production of recombinant proteins (Ma et al, 2014). This efficient protein production capacity has been described as an important model for tissue-specific gene regulation and exogenous protein synthesis (Tomita et al, 2003; Takasu et al, 2016). Fibroin proteins account for 70%–80% of the total silk proteins, being composed by the heavy chain protein (FibH),
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