Biosynthesis of a potentially functional polypeptide derived from silk fibroin.

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 Escherichia coli (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.