Potentially functional polypeptides for modification of biomaterials
Mingyang Wu, Yunfei Liu, Fangfang Tu, Jiannan Wang*
National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, No. 199 Ren-ai Road, Suzhou Industrial Park, Suzhou, Jiangsu Province, 215123, China
*Corresponding author: Jiannan Wang, wangjn@suda.edu.cn
Silk fibroin is a native protein with regular amino acid sequence. Such as, Bombyx mori silk fibroin is composed of 12 repetitive and 11 non-repetitive regions, with the non-repetitive domain consisting of a hydrophilic polypeptide chain. The RGD tripeptides distribute in Antheraea yamamai and Antheraea pernyi silk fibroins up to 14 and 12 repeats, respectively. In order to determine the biomedical function of these typical peptides or potentially use them to modify materials, we cloned and extended a gene motif (f(1)) encoding the non-repetitive domain of Bombyx mori silk fibroin and a gene motif (-rgd-) encoding RGD-contained domain of Antheraea yamamai or Antheraea pernyi silk fibroins.
These motif and their multimers were inserted into a glutathione S-transferase (GST)-tagged fusion-protein expression vector. Motif (f(1)) and multimers (f(4), f(8), (-rgd-)4, and (-rgd-)8) were expressed in Escherichia coli BL21 cells following isopropyl β-D-1-thiogalactopyranoside induction, purified by GST-affinity chromatography. All express products (GST-F(1), GST-F(4), GST-F(8), GST-(-RGD-)4, and GST-(-RGD-)8) were successfully expressed confirmed by SDS-PAGE, mass spectrometry (MS), amino acid composition and charge assay. The peptides F(1), F(4), F(8), (-RGD-)4, and (-RGD-)8 were all cleaved clearly from the GST-fusion tag following thrombin digestion. The molecular weight by MS, amino acid composition and isoelectric points of released peptides were all consistent with the predicted values.
The peptides F(1), F(4), and F(8) were used to modify dacron membrane. Results showed F(1), F(4), and F(8) significantly improved water contact angle of dacron, and increased rapid adhesion and proliferation ability of cells. Cell adhesion and cell proliferation on (-RGD-)4 and (-RGD-)8 modified Bombyx mori silk fibroin films were better than that on unmodified film.
The study indicated that the express products of F(1), F(4), and F(8) could improve the hydrophilicity of hydrophobe materials for better application in biomaterials. F(1), F(4), and F(8) exhibited a negative ζ-potential and the measured pI was about 3.3, 3.2 and 3.0, respectively, expecting to be used in vascular tissue engineering. The express products (-RGD-)4 and (-RGD-)8 could be used to modify cellular interface materials to improve cell adhesion and growth on materials surface.
Acknowledgements
This work was supported by National Natural Science Foundation of China (Nos. 51173125 and 51473108), Natural Science Foundation of Jiangsu Province of China (No. BK2012633), College Natural Science Research Project of Jiangsu Province of China (No. 12KJA43004).
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