An RGD-Containing Peptide Derived from Wild Silkworm Silk Fibroin Promotes Cell Adhesion and Spreading

Abstract

Arginine-Glycine-Aspartate (RGD) tripeptide can promote cell adhesion when present in the amino acid of proteins such as fibronectin. In order to demonstrate the bioactivity of an RGD-containing silk protein, a gene encoding the RGD motif-containing peptide GSGAGGRGDGGYGSGSS (–RGD–) derived from nonmulberry silk was designed and cloned, then multimerised and inserted into a commercial pGEX expression vector for recombinant expression of (–RGD–)n peptides. Herein, we focus on two glutathione-S-transferase (GST)-tagged fusion proteins, GST–(–RGD–)4 and GST–(–RGD–)8, which were expressed in Escherichia coli BL21, purified by GST affinity chromatography, and analyzed with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (MS). Target peptides (–RGD–)4 and (–RGD–)8 (6.03 and 11.5 kDa) were cleaved from the GST-tag by thrombin digestion, as verified with MS and SDS-PAGE. Isoelectric point analysis confirmed that target peptides were expressed and released in accordance with the original design. Target peptides self-assembled into a mainly α-helical structure, as determined by circular dichroism spectroscopy. Furthermore, (–RGD–)4 and (–RGD–)8 modified mulberry silk fibroin films were more effective for rapid cell adhesion, spreading and proliferative activity of L929 cells than some chemically synthesized RGD peptides modified and mulberry silk lacking the RGD motif.

Antibiotic‐based silk fibroin films for burn wound healing

“…Cell viability and cell adhesion studies were performed as per the published protocol, 15 but with slight modifications. The L929 mouse fibroblasts cells were obtained from NCCS‐Pune.…”

Silk Fibroin: An Ancient Material for Repairing the Injured Nervous System

“…The ECM proteins contain one or several short peptide motifs involved in several cellular processes such as cell adhesion, migration, and proliferation. The natural SF from various species of insects such as Antheraea yamamai and Antheraea pernyi has various RGD sites with the ability to improve cell adhesion, while the RGD content in the B. mori (silkworm) is lower [ 163 ]. Consequently, the integration of more informational molecules within the silkworm SF sequence is a strategy for increasing the performance of the silk fibroin.…”

“…Consequently, the integration of more informational molecules within the silkworm SF sequence is a strategy for increasing the performance of the silk fibroin. Kang et al (2018) have produced recombinant SF films with RGD sequences integrated in the cloned gene of B. mori [ 163 ]. Furthermore, the peptide sequence IKVAV derived from laminin has shown a significant impact on the improvement of neurite growth and cell adhesion.…”

Bioinspired Materials for Wound Healing Application: The Potential of Silk Fibroin

“…Some wild silkworm species, which secrete a silk fibroin with higher cell affinity and adhesion due to the motif arginine-glycine-aspartate (RGD) in the protein chain, also result unsuitable for domestication, thus resulting in them being less indicated for biomaterial applications [ 29 ]. Silk represents a valuable biomaterial for various medical and pharmaceutical applications [ 30 ] and, beyond its well-known use as a suture material, the intense research and in vivo and in vitro studies on silk have revealed its huge potential for different clinical treatments [ 31 ], and for many biomedical applications due to its tunable properties and the possibility for it to be combined with other materials such as proteins, polymers and ceramics for improved properties and functions [ 30 , 31 ].…”