Bioengineered and functionalized silk proteins accelerate wound healing in rat and human dermal fibroblasts.

Wound healing is an intrinsic process directed towards the restoration of damaged or lost tissue. The development of a dressing material having the ability to control the multiple aspects of the wound environment would be an ideal strategy to improve wound healing. Though natural silk proteins, fibroin, and sericin have demonstrated tissue regenerative properties, the efficacy of bioengineered silk proteins on wound healing is seldom assessed. Furthermore, silk proteins sans contaminants, having low molecular masses, and combining with other bioactive factors can hasten the wound healing process. Herein, recombinant silk proteins, fibroin and sericin, and their fusions with cecropin B were evaluated for their wound-healing effects using in vivo rat model. The recombinant silk proteins demonstrated accelerated wound closure in comparison to untreated wounds and treatment with Povidone. Among all groups, the treatment with recombinant sericin-cecropin B (RSC) showed significantly faster healing, greater than 90% wound closure by Day 12 followed by recombinant fibroin-cecropin B (RFC) (88.86%). Furthermore, histological analysis and estimation of hydroxyproline showed complete epithelialization, neovascularization, and collagenisation in groups treated with recombinant silk proteins. The wound healing activity was further verified by in vitro scratch assay using HADF cells, where the recombinant silk proteins induced cell proliferation and cell migration to the wound area. Additionally, wound healing-related gene expression showed recombinant silk proteins stimulated the upregulation of EGF and VEGF and regulated the expression of TGF-ß1 and TGF-ß3. Our results demonstrated the enhanced healing effects of the recombinant silk fusion proteins in facilitating complete tissue regeneration with scar-free healing. Therefore, the recombinant silks and their fusion proteins have great potential to be developed as smart bandages for wound healing.

Transgenic Silkworms Overexpressing Relish and Expressing Drosomycin Confer Enhanced Immunity to Multiple Pathogens.

The sericulture industry faces substantial economic losses due to severe pathogenic infections caused by fungi, viruses, and bacteria. The development of transgenic silkworms against specific pathogens has been shown to enhance disease resistance against a particular infection. A single gene or its products that can confer protection against multiple pathogens is required. In an attempt to develop silkworms with enhanced immunity against multiple pathogens, we generated transgenic silkworm lines with an overexpressed NF-kB transcription factor, Relish 1, under two different promoters. Separately, a potent anti-fungal gene, Drosomycin, was also expressed in transgenic silkworms. Both Relish 1 and Drosomycin transgenic silkworms had single copy genomic integration, and their mRNA expression levels were highly increased after infection with silkworm pathogens. The overexpression of the Relish 1 in transgenic silkworms resulted in the upregulation of several defense-related genes, Cecropin B, Attacin, and Lebocin, and showed enhanced resistance to Nosema bombycis (microsporidian fungus), Nucleopolyhedrovirus (BmNPV), and bacteria. The Drosomycin expressing transgenic silkworms showed elevated resistance to N. bombycis and bacteria. These findings demonstrate the role of Relish 1 in long-lasting protection against multiple pathogens in silkworms. Further, the successful introduction of a foreign gene, Drosomycin, also led to improved disease resistance in silkworms.

Recombinant expression of sericin-cecropin fusion protein and its functional activity.

OBJECTIVE: Silk sericin is a natural polymer with potential utility in biomedical and biotechnological applications. Recombinantly expressed sericin ensures a source of pure protein with no contamination and with multiple properties when expressed as a fusion protein. Hence, the present paper aims to recombinantly express a functional silk sericin fusion protein. RESULTS: In order to develop a more effective sericin protein, we have attempted to recombinantly express a part of sericin sequence, which represents a highly conserved and internally repetitive unit of the sericin1 protein, and its fusion with cecropin B, a potent antimicrobial peptide. Both difficult-to-express proteins were expressed in Escherichia coli and purified by nickel-charged affinity resin. Further, functional assay demonstrated that both proteins were individually active against Gram-positive and negative bacteria, with enhanced bactericidal activity observed in sericin-cecropin B fusion protein. CONCLUSIONS: To our knowledge, this is the first report not only on the recombinant expression of sericin as a fusion protein but also the bactericidal possibility of the 38-amino acid serine-rich motif of sericin protein. We also discuss the potential biomedical and biotechnological applications of this sericin hybrid protein.

Molecular characterization and functional analysis of the vitellogenin receptor from eri silkworm, Samia ricini.

The vitellogenin receptor (VgR) plays a critical role in egg development by mediating endocytosis of the major yolk protein precursor vitellogenin (Vg). Therefore, identifying the VgR of beneficial insects and its characterization could lead to the development of novel egg production strategies to enhance their commercial values. Here, we present the cloning, expression, and functional characterization of the VgR from an economically important eri silkworm, Samia ricini. The complete mRNA sequence was 6002 bp with an ORF of 5484 bp, encoding a protein of 1827 amino acids. Sequence analyses revealed that the SrVgR contained all of the conservative structural motifs characteristics of LDLR family members. The SrVgR was specifically expressed in the ovary, and the mRNA level increased steadily in pupal stages, reached its peak on day 9, and then declined to a bare minimum in adults. RNA interference (RNAi) clearly reduced the VgR transcript levels, disrupted the ovarian development resulting in malformed ovarioles and abnormal development of eggs. Taken together, these data provide conclusive evidence for the essential roles of VgR in insect reproduction.