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

Zhao Kang,Guangzhou Song,Jingjing Xu,Jiannan Wang,Mengyao Ding,Yining Wang,Huanrong Zhao

doi:10.3390/polym10111193

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.

Highlights

IntroductionBombyx mori silk fibroin has been extensively used for biomaterials because it is readily obtainable on large quantities ad displays good biocompatibility and biodegradability, resulting in practicable cell adhesion and cell spreading, high proliferative and anti-thrombosis activities, and potent ability to induce tissue repair [1,2,3,4,5,6,7]
Silk fibroin is a natural protein synthesized and secreted by silkworms and spiders
A few studies have reported that regenerated RGD tripeptide-containing silk fibroin materials from wild silkworm species are potentially useful biomaterials, with satisfactory cytocompatibility and the ability to promote tissue remodeling [15,16,17]

Summary

Introduction

Bombyx mori silk fibroin has been extensively used for biomaterials because it is readily obtainable on large quantities ad displays good biocompatibility and biodegradability, resulting in practicable cell adhesion and cell spreading, high proliferative and anti-thrombosis activities, and potent ability to induce tissue repair [1,2,3,4,5,6,7] Wild silkworm species such as Antheraea yamamai, Antheraea pernyi, Antheraea mylitta, Antheraea assama, and Philosamia ricini secrete another type of silk fibroin with arginine-glycine-aspartate (RGD) motif repeats in the protein chain [8,9,10,11,12], which is absent in mulberry (B. mori) silk fibroin [13]. The behaviors of wild silkworm species renders them unsuitable for domestication, resulting low production and minimal scope for biomaterial applications

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