title: Biosynthesis and characterization of a non-repetitive polypeptide derived from silk fibroin heavy chain.
abstract: Silk fibroin heavy chain is the major protein component of Bombyx mori silk fibroin and is composed of 12 repetitive and 11 non-repetitive regions, with the non-repetitive domain consisting of a hydrophilic polypeptide chain. In order to determine the biomedical function of the non-repetitive domain or potentially use it to modify hydrophobic biomaterials, high-purity isolation is necessary. Previously, we cloned and extended a gene motif (f(1)) encoding the non-repetitive domain. Here, this motif and its multimers are inserted into a glutathione S-transferase (GST)-tagged fusion-protein expression vector. Motif f(1) and multimers f(4) and f(8) were expressed in Escherichia coli BL21 cells following isopropyl β-D-1-thiogalactopyranoside induction, purified by GST-affinity chromatography, and single bands of purified fusion proteins GST-F(1), GST-F(4), and GST-F(8), were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Target polypeptides F(1), F(4), and F(8), were cleaved clearly from the GST-fusion tag following thrombin digestion. Mass spectrometry results indicate that the molecular weights associated with fusion proteins GST-F(1), GST-F(4), and GST-F(8) are 31.5, 43.8, and 59.0kDa, respectively, and with the cleaved polypeptides F(1), F(4), and F(8) are 4.8, 16.8, and 32.8kDa, respectively. The F(1), F(4), and F(8) polypeptide chains are negatively charged with isoelectric points (pI) of 3.3, 3.2, and 3.0, respectively. The molecular weight and pI values of the polypeptide chains are consistent with the predicted values and the amino acid compositions similar to predicted sequences. FTIR and CD results show the molecular conformation of F(1) was mainly random coil, and more stable α-helix structure formed in longer molecular chain.
authors.affiliation: 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.
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, No. 199 Ren-ai Road, Suzhou Industrial Park, Suzhou, Jiangsu Province 215123, China.
authors.forename: Mingyang
authors.initials: M
authors.lastname: Wu
authors.affiliation: 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.
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, No. 199 Ren-ai Road, Suzhou Industrial Park, Suzhou, Jiangsu Province 215123, China. Electronic address: wangjn@suda.edu.cn.
authors.forename: Jiannan
authors.initials: J
authors.lastname: Wang
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D012831:Bombyx; D005349:Fibroins / Q000096:biosynthesis*
chemical_list: D005349:Fibroins
keywords: Charge property; Gene expression; Mass spectrometry; Molecular conformation; Non-repetitive domain; Purification; Silk fibroin