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Biosynthesis of a potentially functional polypeptide derived from silk fibroin

Issue title: Frontiers in Biomedical Engineering and Biotechnology – Proceedings of the 3rd International Conference on Biomedical Engineering and Biotechnology, 25–28 September 2014, Beijing, China

Article type: Research Article

Authors: Zhao, Huanrong | Yang, Yunxing | Yi, Honggen | Yang, Gaoqiang | Wang, Jiannan;

Affiliations: National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China

Note: [] Corresponding author: Jiannan Wang, College of Textile and Clothing Engineering, Soochow University, No. 199 Ren-ai Road, Suzhou Industrial Park, Suzhou, 215123, P.R. China. Tel.: +86 512-67061153; Fax: +86 512-67061126; E-mail: [email protected].

Keywords: Silk fibroin, RGD, recombinant expression, amino acid composition, isoelectric point

DOI: 10.3233/BME-141015

Journal: Bio-Medical Materials and Engineering, vol. 24, no. 6, pp. 2057-2064, 2014

Published: 2014
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Abstract

In order to understand the relationship between sequences and biological functions of RGD-containing wild silkworm silk fibroin, it is important to purify the basic RGD-containing motif in large quantities. In this study, a gene monomer encoding RGD-contained motif GSGAGGRGDGGYGSGSS (-RGD-) derived from Antheraea pernyi (the same in Antheraea yamamai) was designed and cloned. (-RGD-)n in various degrees of polymerizations was obtained by gene monomer doubling-extension and expression. Two glutathione-S-transferase (GST)-tagged fusion proteins GST-(-RGD-)12 and GST-(-RGD-)24 were successfully expressed in Escherichiacoli (E. coli) BL21. The fusion proteins were isolated and purified by GST affinity chromatography, and the polypeptides (-RGD-)12 and (-RGD-)24 were cleaved from GST fusion proteins by thrombin digestion. Two-dimensional electrophoresis and amino acid composition analysis were performed to confirm the identity of the engineered polypeptides. Results indicated that this technology reliably obtained expected polypeptides (-RGD-)n for future research on structure and functions.

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