TY - JOUR
T1 - An RGD-containing peptide derived from wild silkworm silk fibroin promotes cell adhesion and spreading
AU - Kang, Zhao
AU - Wang, Yining
AU - Xu, Jingjing
AU - Song, Guangzhou
AU - Ding, Mengyao
AU - Zhao, Huanrong
AU - Wang, Jiannan
N1 - Funding Information:
Funding: This research was funded by National Natural Science Foundation of China [Nos. 51473108, 51873141], Natural Science Foundation of Jiangsu Province of China [No. BK20181192] and College Natural Science Research Project of Jiangsu Province of China [No. 18KJA540001].
Publisher Copyright:
© 2018 by the authors.
PY - 2018/10/26
Y1 - 2018/10/26
N2 - 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.
AB - 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.
KW - Biosynthesis
KW - Cell adhesion
KW - Cell proliferation
KW - RGD peptides
KW - Silk fibroin
KW - Wild silkworm
UR - http://www.scopus.com/inward/record.url?scp=85055715543&partnerID=8YFLogxK
U2 - 10.3390/polym10111193
DO - 10.3390/polym10111193
M3 - Article
AN - SCOPUS:85055715543
SN - 2073-4360
VL - 10
JO - Polymers
JF - Polymers
IS - 11
M1 - 1193
ER -