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

Research Project

Project/Area Number	62480046
Research Category	Grant-in-Aid for General Scientific Research (B)
Allocation Type	Single-year Grants
Research Field	蚕糸学
Research Institution	University of Osaka Prefecture (1988) Tokyo University of Agriculture and Technology (1987)
Principal Investigator	HIMENO Michio University of Osaka Prefecture, College of Agriculture, 農学部, 教授 (10026411)
Co-Investigator(Kenkyū-buntansha)	WADANO Akira University of Osaka Prefecture, College of Agriculture, 農学部, 講師 (40081575) SHIGEMATU Masanori Tokyo University of Agriculture and Technology,Faculty of Technol, 工学部, 助手 (90015032)
Project Period (FY)	1987 – 1988
Project Status	Completed (Fiscal Year 1988)
Budget Amount *help	¥6,400,000 (Direct Cost: ¥6,400,000) Fiscal Year 1988: ¥1,500,000 (Direct Cost: ¥1,500,000) Fiscal Year 1987: ¥4,900,000 (Direct Cost: ¥4,900,000)
Keywords	Fibroin / Fibroin Gene / fibroin Crystalline Fraction / Protein of Silk / Protein Engineering / Fussed Protein / フィブロイン / フィブロイン遺伝子 / フィブロイン結晶領域 / ベッター作成 / 遺伝子改変
Research Abstract	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.