title: Biosynthesis of a potentially functional polypeptide derived from silk fibroin.
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 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.
authors.affiliation: National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China.
authors.forename: Huanrong
authors.initials: H
authors.lastname: Zhao
authors.affiliation: National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China.
authors.forename: Yunxing
authors.initials: Y
authors.lastname: Yang
authors.affiliation: National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China.
authors.forename: Honggen
authors.initials: H
authors.lastname: Yi
authors.affiliation: National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China.
authors.forename: Gaoqiang
authors.initials: G
authors.lastname: Yang
authors.affiliation: National Engineering Laboratory for Modern Silk, College of Textile and Clothing Engineering, Soochow University, Suzhou 215123, China.
authors.forename: Jiannan
authors.initials: J
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005349:Fibroins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*
chemical_list: D009842:Oligopeptides; C482288:fibroin, silkworm; C047981:arginyl-glycyl-aspartic acid; D005349:Fibroins
keywords: RGD; Silk fibroin; amino acid composition; isoelectric point; recombinant expression