Recombinant Silk Proteins with Additional Polyalanine Have Excellent Mechanical Properties

Abstract

This paper explores the structures of exogenous protein molecules that can effectively improve the mechanical properties of silkworm silk. Several transgenic vectors fused with the silkworm fibroin light chain and type 3 repeats in different multiples of the ampullate dragline silk protein 1 (MaSp1) from black widow spider with different lengths of the polyalanine motifs were constructed for this study. Transgenic silkworms were successfully obtained by piggyBac-mediated microinjection. Molecular detection showed that foreign proteins were successfully secreted and contained within the cocoon shells. According to the prediction of PONDR® VSL2 and PONDR® VL-XT, the type 3 repeats and the polyalanine motif of the MaSp1 protein were amorphous. The results of FTIR analysis showed that the content of β-sheets in the silk of transgenic silkworms engineered with transgenic vectors with additional polyalanine was significantly higher than that of wild-type silkworm silk. Additionally, silk with a higher β-sheet content had better fracture strength and Young’s modulus. The mechanical properties of silk with longer chains of exogenous proteins were improved. In general, our results provide theoretical guidance and technical support for the large-scale production of excellent bionic silk.

Drosophila melanogaster resilin improves the mechanical properties of transgenic silk

“…Zhao et al expressed recombinant spider silk protein containing different numbers of polyalanine motifs in the silk gland of silkworm. The polyalanine structure improved the strength and modulus of the resulting silk fibers [23]. Tang et al fused spider pyriform silk and aggregate silk genes with the silk light chain gene.…”

“…These studies show that introducing foreign proteins into silk and optimizing the molecular structure by gene editing technology is an effective method to improve the mechanical properties of silk fibers. The fusion of a spider silk protein and silk fibroin enables the resulting protein to participate in the silk fibroin complex assembly and secondary structure formation, significantly affecting the silk's mechanical properties [23]. However, to our best knowledge, no studies that improve the mechanical properties of silk with proteins other than spider silk have been published to date.…”

Novel insights into construct toxicity, strain optimization, and primary sequence design for producing recombinant silk fibroin and elastin-like peptide in E. coli

“…Both monomer units were designed based on naturally occurring primary sequences found in the MaSp2 dragline spidroin of orb-weaving spiders ( Teulé et al, 2009 ; Guerette et al, 1996 ; Malay et al, 2017 ). Modulating the tandem alanine length and total construct length were chosen as focal points to study the effect of construct design on expression outcomes, as these are primary factors implicated in determining the material properties of recombinant silks ( Bowen et al, 2018 ; Zhao et al, 2021 ; Bratzel and Buehler, 2012 ; Heidebrecht et al, 2015 ). All constructs expressed have an identical starting sequence that is present on the pET-19b expression vector, which contains a 10x histidine tag for purification followed by an enterokinase cleavage sequence (cleavage of the 10x histidine tag using the enterokinase site was not employed in this work).…”

The Contribution of Silk Fibroin in Biomedical Engineering

“…Promotes cell proliferation and exhibits anti-inflammatory activity [97] Cecropin B and moricin Antimicrobial activity [98] Green fluorescent protein and cecropin Antibacterial activity and fluorescence [99] Enhanced green fluorescent protein, DsRed monomer fluorescent protein and monomeric Kusabira orange Exhibits fluorescence [100] Laminin and fibronectin peptide adhesive fragments Exhibits increased adhesive activity [101] Polyalanine motifs Displays improved mechanical properties [102] Collagen and fibronectin Improves cell adhesive properties [103]…”