A Trial for a Creation of a New Protein Fiber by Modification of Fibroin Gene, Bombyx mori L.

点击翻译按钮获取中文摘要

The silk fibroin is excellent materials as a fiber of cloth and it is expected as good materials for medical instrument and salso for immobilization of enzyme. This project is a trial for a ceration of new protein fiber by modification of fibroin gene, Bombyx mori L..Parts of a typical repeating amino acid sequence (- Ser - Gly - Ala - Gly - Ala - Gly -) in silk fibroin molecule are called a fibroin crystalline fraction (Fcp). If a radical amino acid be added in Fcp sequence, the character of fibroin protein will drastically changed. then in this project we attempt addition of the code of lysine and/or methionine in DNA of the Fcp sequence and expression of fibroin gene in escherichia coli.First, a expression vector of Fcp DNA sequence was made. When a passenger DNA sequence of 3n + 1 base pair is introduced into poly linker site of - galactosidase ( -gal) gene on plasmid p41 (made in this project) carring tac promoter. the introduced dna sequence will be express as a fussed protein with -gal in E.coli. Next, the Fcp sequence in plasmid pBmF6 harboring the fibroin gene (kindlly provided by Prof. Y. Suzuki) was obtained by Pst I, Hae II and Bal 31 treatment of the plasmid, and also by ALu I partially treatment, then we got plasmid pFCP and pFKB. Third, four DNA sequences were synthesized by a DNA synthesizer. The fragment were linked to p41 and also mixture of the fragments and polymer of one were introduced the plasmid, then plasmid pFMS was obtained. The pFCP, pFKB pr pFMS DNA easily deleted in E. coli. When E. coli JM109 carring pFCP, pFKB or pFMS was cultured on a plate containing IPTG and X-gal, the colony become blue. The blue colonies also reacted anti-Fcp rat serum. The protein extract isolated the blue colonies were applied on page, and determined by western blotting method using anti-Fcp rat serum and anti-rat IgG-HRPO conjugated rabbit IgG. The blue colonies produced a Fcp like protein but the molecular weight were not determined.