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Introduction: Conventional hot-alkaline cocoon degumming techniques greatly weaken the physicochemical and mechanical properties of silk fibroin fiber, thus affecting the quality of silk fabric. Moreover, it causes massive energy waste and serious environmental pollution., Objective: This study aims to establish a novel cocoon self-degumming method by genetic modification of silkworm varieties and silk fibers., Methods: The self-degummed cocoon material was generated by specifically overexpressing trypsinogen protein in the sericin layer of silk thread; the effect of cocoon self-degumming method was evaluated by the degumming rate of sericin protein, the cleanliness and equivalent diameter of silk fibroin fiber; the basic characteristics of silk fibroin fiber degummed by cocoon self-degumming method and conventional hot-alkaline degumming technique were determined by electron microscopy, Fourier infrared spectroscopy, X-ray diffraction and tensile tests; the composition and biological activity of degummed sericin protein was respectively analyzed by liquid chromatograph-mass spectrometry and cytological experiments., Results: The genetically engineered self-degumming cocoon containing trypsinogen protein was successfully created, and the content of trypsinogen protein in silk was 47.14 ± 0.90 mg/g. The sericin protein in the self-degumming cocoon was removed out in water or 1 mM Tris-HCl buffer (pH = 8.0). Compared to alkaline-degummed silk fibroin, self-degummed silk fibroin had better cleanliness, thicker equivalent diameter, more complete silk structure and better mechanical property. In addition, sericin protein degummed from self-degumming cocoons significantly promoted cell proliferation and caused no obvious cytotoxicity., Conclusion: Compared to conventional hot-alkaline degumming technique, the cocoon self-degumming method by genetically overexpressing trypsinogen protein in sericin layer of silk thread can self-degummed in a mild degumming condition, and gain silk fiber with better quality and more biologically active sericin protein products. This strategy can not only reduce the environmental impact, but also generate greater economic value, which will accelerate its application in the silk and pharmaceutical industries., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Production and hosting by Elsevier B.V.)

Human connective tissue growth factor (CTGF) is a secreted cysteine-rich peptide that stimulates cell proliferation, migration, and extracellular matrix production during tissue development, differentiation, angiogenesis, implantation, wound healing, and fibrosis processes, with broad application in the medical and cosmetic medical fields. However, the production of CTGF is currently limited by its low yield and purity in current bioreactors. In this study, two genetically modified silkworm strains were generated harboring artificially designed CTGF-8ht and pepCTGF-8ht genes, respectively, that contain an enhanced His-tag with eight histidine residues with or without a transdermal peptide (pep). Both recombinant CTGF-8ht and pepCTGF-8ht proteins were successfully expressed in the silkworm silk gland and cocoon, and could be easily extracted and purified from the cocoon by single-affinity immunoprecipitation column chromatography, achieving a purity of more than 95%. Moreover, compared with CTGF-8ht protein, pepCTGF-8ht protein exhibited better cell proliferation activity by activating the extracellular signal-regulated kinase (ERK) pathway and enhanced hyaluronic acid synthesis activity by upregulating hyaluronan synthase 3 expression; moreover, the addition of pep significantly improved the transmembrane ability of CTGF-8ht protein. These results should help to promote the application prospects of CTGF and further guide the design and development of protein drugs from silkworm and other bioreactor systems. KEY POINTS : A silkworm bioreactor was optimized to produce connective tissue growth factor (CTGF) The transgene contained an enhanced 8-His-tag and transmembrane peptide (pep) Recombinant CTGF was easily purified with maintained or higher biological activity.

The multigene expression system is highly attractive to co-express multiple genes or multi-subunit complex-based genes for their functional studies, and in gene therapy and visual tracking of expressed proteins. However, the current multiple gene co-expression strategies usually suffer from severe inefficiency and unbalanced expression of multiple genes. Here, we report on an improved 2A self-cleaving peptide (2A)-based multigene expression system (2A-MGES), by introducing an optimized Kozak region (Ck) and altering the gene arrangement, both of which contributed to the efficient expression of two fluorescent protein genes in silkworm. By co-expressing DsRed and EGFP genes in insect cells and silkworms, the potent Ck was first found to improve the translation efficiency of downstream genes, and the expression of the flanking genes of 2A were improved by altering the gene arrangement in 2A-MGES. Moreover, we showed that combining Ck and an optimized gene arrangement in 2A-MGES could synergistically improve the expression of genes in the cell. Further, these two flanking genes, regulated by modified 2A-MGES, were further co-expressed in the middle silk gland and secreted into the cocoon, and both achieved efficient expression in the transgenic silkworms and their cocoons. These results suggested that the modified Ck-2A-MGES will be a potent tool for multiple gene expression, for studies of their functions, and their applications in insect species.

Sericin, as the major component of Bombyx mori silk, is a useful biomaterial for tissue engineering due to its hydrophilicity, biocompatibility and biodegradability. Here, we report the fabrication of a human acidic fibroblast growth factor (FGF1)-functionalized sericin hydrogel using a transgenic silkworm spun silk with FGF1 incorporated in its sericin layer. Sericin, together with FGF1, were simultaneously extracted from the silk fiber and then exposed to cold-induced hydrogel formation without additional crosslinking. The fabricated FGF1 sericin hydrogels demonstrated injectability, useful mechanical properties and a porous microstructure, which contributed to cell adhesion and survival. In addition, FGF1 achieved long-term storage in the sericin hydrogels over a wide range of temperatures. Further, the sericin-FGF1 demonstrated sustained release to promote cell proliferation and wound healing. Furthermore, cellular inflammatory responses showed that the FGF1 sericin hydrogels exhibited biocompatibility and no immunogenicity. This study revealed the successful exploration of FGF1-functionalized sericin hydrogels as a new protein-based biomaterial to expand applications of FGF1 and sericin in tissue and medical engineering. Further, we demonstrated a strategy for the predesign of exogenous protein-functionalized sericin hydrogels through genetically modifying silk fibers as sources for their cost effective production at a large scale., Statement of Significance: Sericin from the Bombyx mori silk, is regarded as a desirable biomaterial for tissue engineering due to its hydrophilicity, biocompatibility and biodegradability. Genetically engineering the sericin with functional exogenous proteins would enhance its biofunctions and further expand its application in tissue engineering. In this study, we demonstrated a method to fabricate a human acidic fibroblast growth factor (FGF1)-functionalized sericin hydrogel using a transgenic silkworm spun silk with FGF1 incorporated in its sericin layer. The fabricated FGF1 sericin hydrogels demonstrated injectability, porous microstructure, biocompatibility and no immunogenicity which contributed to cell adhesion and survival. Remarkably, FGF1 could achieve a long-term stability in the sericin hydrogels over a wide range of temperatures and sustained release to promote cell proliferation and wound healing. This study revealed the successful exploration of FGF1-functionalized sericin hydrogels as a new protein-based biomaterial in tissue and medical engineering application, and provided a strategy for the predesign of exogenous protein-functionalized sericin hydrogels through genetically modifying silk fibers as sources for their cost effective production at a large scale., (Copyright © 2018. Published by Elsevier Ltd.)

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.

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.

Fundamental and applied studies of silkworms have entered the functional genomics era. Here, we report a multi-gene expression system (MGES) based on 2A self-cleaving peptide (2A), which regulates the simultaneous expression and cleavage of multiple gene targets in the silk gland of transgenic silkworms. First, a glycine-serine-glycine spacer (GSG) was found to significantly improve the cleavage efficiency of 2A. Then, the cleavage efficiency of six types of 2As with GSG was analyzed. The shortest porcine teschovirus-1 2A (P2A-GSG) exhibited the highest cleavage efficiency in all insect cell lines that we tested. Next, P2A-GSG successfully cleaved the artificial human serum albumin (66 kDa) linked with human acidic fibroblast growth factor (20.2 kDa) fusion genes and vitellogenin receptor fragment (196 kD) of silkworm linked with EGFP fusion genes, importantly, vitellogenin receptor protein was secreted to the outside of cells. Furthermore, P2A-GSG successfully mediated the simultaneous expression and cleavage of a DsRed and EGFP fusion gene in silk glands and caused secretion into the cocoon of transgenic silkworms using our sericin1 expression system. We predicted that the MGES would be an efficient tool for gene function research and innovative research on various functional silk materials in medicine, cosmetics, and other biomedical areas.

Deletion of transposable elements post-genomic integration holds great promise for stability of the transgene in the host genome and has an essential role for the practical application of transgenic animals. In this study, a modified piggyBac vector that mediated deletion of the transposon sequence post-integration for transgene stability in the economically important silkworm Bombyx mori was constructed. The piggyBac vector architecture contains inversed terminal repeat sequences L1, L2 and R1, which can form L1/R1 and L2/R1 types of transposition cassettes. hsp70-PIG as the piggyBac transposase expression cassette for initial transposition, further remobilization and transgene stabilization test was transiently expressed in a helper vector or integrated into the modified vector to produce a transgenic silkworm. Shortening L2 increased the transformation frequency of L1/R1 into the silkworm genome compared to L2/R1. After the integration of L1/R1 into the genome, the remobilization of L2/R1 impaired the transposon structure and the resulting transgene linked with an impaired transposon was stable in the genome even in the presence of exogenously introduced transposase, whereas those flanked by the intact transposon were highly mobile in the genome. Our results demonstrated the feasibility of post-integration deletion of transposable elements to guarantee true transgene stabilization in silkworm. We suggest that the modified vector will be a useful resource for studies of transgenic silkworms and other piggyBac-transformed organisms.

Natural silk fiber spun by the silkworm Bombyx mori is widely used not only for textile materials, but also for biofunctional materials. In the present study, we genetically engineered an advanced silk material, named hSFSV, using a transgenic silkworm, in which the recombinant human acidic fibroblast growth factor (hFGF1) protein was specifically synthesized in the middle silk gland and secreted into the sericin layer to surround the silk fiber using our previously optimized sericin1 expression system. The content of the recombinant hFGF1 in the hSFSV silk was estimated to be approximate 0.07% of the cocoon shell weight. The mechanical properties of hSFSV raw silk fiber were enhanced slightly compared to those of the wild-type raw silk fiber, probably due to the presence of the recombinant of hFGF1 in the sericin layer. Remarkably, the hSFSV raw silk significantly stimulated the cell growth and proliferation of NIH/3T3 mouse embryonic fibroblast cells, suggesting that the mitogenic activity of recombinant hFGF1 was well maintained and functioned in the sericin layer of hSFSV raw silk. These results show that the genetically engineered raw silk hSFSV could be used directly as a fine biomedical material for mass application. In addition, the strategy whereby functional recombinant proteins are expressed in the sericin layer of silk might be used to create more genetically engineered silks with various biofunctions and applications., (Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Infectious bursal disease virus (IBDV) is the causative agent of a highly contagious disease affecting young chickens and causes serious economic losses to the poultry industry worldwide. Development of subunit vaccine using its major caspid protein, VP2, is one of the promising strategies to protect against IBDV. This study aim to test the feasibility of using silkworm to produce recombinant VP2 protein (rVP2) derived from a very virulent strain of IBDV (vvIBDV). A total of 16 transgenic silkworm lines harboring a codon-optimized VP2 gene driven by the sericin1 promoter were generated and analyzed. The results showed that the rVP2 was synthesized in the middle silk gland of all lines and secreted into their cocoons. The content of rVP2 in the cocoon of each line was ranged from 0.07 to 16.10 % of the total soluble proteins. The rVP2 was purified from 30 g cocoon powders with a yield of 3.33 mg and a purity >90 %. Further analysis indicated that the rVP2 was able to tolerate high temperatures up to 80 °C, and exhibited specific immunogenic activity in mice. To our knowledge, this is the first report of overexpressing rVP2 in the middle silk gland of transgenic silkworm, which demonstrates the capability of silkworm as an efficient tool to produce recombinant immunogens for use in new vaccines against animal diseases.

30K proteins are a group of structurally related proteins that play important roles in the life cycle of the silkworm Bombyx mori and are largely synthesized and regulated in a time-dependent manner in the fat body. Little is known about the upstream regulatory elements associated with the genes encoding these proteins. In the present study, the promoter of Bmlp3, a fat body-specific gene encoding a 30K protein family member, was characterized by joining sequences containing the Bmlp3 promoter with various amounts of 5' upstream sequences to a luciferase reporter gene. The results indicated that the sequences from -150 to -250bp and -597 to -675bp upstream of the Bmlp3 transcription start site were necessary for high levels of luciferase activity. Further analysis showed that a 21-bp sequence located between -230 and -250 was specifically recognized by nuclear factors from silkworm fat bodies and BmE cells, and could enhance luciferase reporter-gene expression 2.8-fold in BmE cells. This study provides new insights into the Bmlp3 promoter and contributes to the further clarification of the function and developmental regulation of Bmlp3., (Copyright © 2014. Published by Elsevier B.V.)

In a previous study, we isolated 1,119 bp of upstream promoter sequence from Bmlp3, a gene encoding a member of the silkworm 30 K storage protein family, and demonstrated that it was sufficient to direct fat body-specific expression of a reporter gene in a transgenic silkworm, thus highlighting the potential use of this promoter for both functional genomics research and biotechnology applications. To test whether the Bmlp3 promoter can be used to produce recombinant proteins in the fat body of silkworm pupae, we generated a transgenic line of Bombyx mori which harbors a codon-optimized Aspergillus niger phytase gene (phyA) under the control of the Bmlp3 promoter. Here we show that the Bmlp3 promoter drives high levels of phyA expression in the fat body, and that the recombinant phyA protein is highly active (99.05 and 54.80 U/g in fat body extracts and fresh pupa, respectively). We also show that the recombinant phyA has two optimum pH ranges (1.5-2.0 and 5.5-6.0), and two optimum temperatures (55 and 37 °C). The activity of recombinant phyA was lost after high-temperature drying, but treating with boiling water was less harmful, its residual activity was approximately 84% of the level observed in untreated samples. These results offer an opportunity not only for better utilization of large amounts of silkworm pupae generated during silk production, but also provide a novel method for mass production of low-cost recombinant phytase using transgenic silkworms.

Transcription activator-like effector nuclease (TALEN) possesses the characteristics of ease design and precise DNA targeting. In the silkworm Bombyx mori, TALEN has been successfully used to knockout an endogenous Bombyx gene, and shown the huge potential in functional genes research and improvement of the economical characteristics of silkworm. Thus, there is an urgent need to develop an applicable system that permits the efficient construction of customized TALEN with high activity that could efficiently induce the hereditable mutagenesis in the silkworm. In this study, we constructed an efficient assembly and evaluation system of the customized TALEN especiallly for silkworm genome editing by combination of a modified Golden Gate ligation strategy, a luciferase (LUC) reporter system in insect cell culture for binding activity and a surveyor nuclease assay system in silkworm embryos for cleavage efficiency. We showed the reliability of this system by assembling a pair of TALENs targeting a silkworm genome locus and assaying their binding and cleavage activities. The assembly strategy was convenient and efficient which allows the rapid construction of customized TALEN in less than 1 week, and the evaluation system was reliable and necessary for screening of the customized TALEN pair with high binding and cleavage activities. The results showed this system is a reliable and efficient tool for the construction of customized TALEN with high activity for gene targeting of silkworm, and will contribute to the wide application of TALEN technology in the functional gene research of silkworm.

The middle silk gland (MSG) of silkworm is thought to be a potential host for mass-producing valuable recombinant proteins. Transgenic MSG expression systems based on the usage of promoter of sericin1 gene (sericin-1 expression system) have been established to produce various recombinant proteins in MSG. However, further modifying the activity of the sericin-1 expression system to yield higher amounts of recombinant proteins is still necessary. In this study, we provide an alternative modification strategy to construct an efficient sericin-1 expression system by using the hr3 enhancer (hr3 CQ) from a Chongqing strain of the Bombyx mori nuclear polyhedrosis virus (BmNPV) and the 3'UTRs of the fibroin heavy chain (Fib-HPA), the fibroin light chain (Fib-LPA), and Sericin1 (Ser1PA) genes. We first analyzed the effects of these DNA elements on expression of luciferase, and found that the combination of hr3 CQ and Ser1PA was most effective to increase the activity of luciferase. Then, hr3 CQ and Ser1PA were used to modify the sericin1 expression system. Transgenic silkworms bearing these modified sericin1 expression vectors were generated by a piggyBac transposon mediated genetic transformation method. Our results showed that mRNA level of DsRed reporter gene in transgenic silkworms containing hr3 CQ and Ser1PA significantly increased by 9 fold to approximately 83 % of that of endogenous sericin1. As the results of that, the production of recombinant RFP increased by 16 fold to 9.5 % (w/w) of cocoon shell weight. We conclude that this modified sericin-1 expression system is efficient and will contribute to the MSG as host to mass produce valuable recombinant proteins.