Efficient production of useful proteins by proteinproducing bacterium and its secretion vector

Research Project

Project/Area Number	61860009
Research Category	Grant-in-Aid for Developmental Scientific Research
Allocation Type	Single-year Grants
Research Field	発酵・醸造
Research Institution	Nagoya University
Principal Investigator	UDAKA Shigezo Nagoya University, Professor, 農学部, 教授 (70023463)
Co-Investigator(Kenkyū-buntansha)	TSUBOI Akio Nagoya Univ., Research associate, 農学部, 助手 (20163868) YAMAGATA Hideo Nagoya Univ., Associate Professor, 農学部, 助教授 (20023468) TSUKAGOSHI Norihiro Nagoya Univ., Associate Professor, 農学部, 助教授 (50115599)
Project Period (FY)	1986 – 1987
Project Status	Completed (Fiscal Year 1987)
Budget Amount *help	¥7,900,000 (Direct Cost: ¥7,900,000) Fiscal Year 1987: ¥1,600,000 (Direct Cost: ¥1,600,000) Fiscal Year 1986: ¥6,300,000 (Direct Cost: ¥6,300,000)
Keywords	Secretion of Useful proteins / Bacillus brevis / Production of interleukin 2 / Production of beta-amylase / バチルスブレビスの分泌ベクター / 蛋白質生産菌 / 有用蛋白質の高効率生産 / 分泌生産ベクター / Bacillus brevis / β-アミラーゼ
Research Abstract	Bacillus brevis was found to be a potential protein-producing bacterium. Since major exoprotein is identical to the cell surface proteins, the DNA region which controls expression and secretion of the exoprotein was inserted to a high-copy numberes plasmid to construct an expression and secretion vector, PNU 200. This vector was utilized to produce various usefull proteins in B. brevis. 1. production of human interleukin 2 (IL2). The IL2 gene was inserted to the downstream of signal sequence of B. brevis major cell surface gene in pNU 200. This plasmid was introduced into B. brevis 47. One - 2 mg/l of IL2 was produced extracellularly, but IL2 was partially degraded with 2 longer cultivation. Then, the same plasmid was transferred to B. brevis HPD 31 which has no detectable exoprotease. By optimizing culture conditions, now about 20-30 mg/l of IL2 was produced without noticiable degradation of IL2. 2. Production of bacterial beta-amylase. Beta-amylase gene of Bacillus nolymyxa was ligated to the downstream of signal sequence of pNU 200. By transforming B. brevis 47 with this plasmid, the transformant produced a large amount of beta-amylase (about 0.5 g/l) under appropriate cultural conditions. Enzymatically active beta-amylase was obtained even by deleting DNA coding for a large C-terminal region of the enzyme, revealing the necessary region of beta-amylase.