权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

产蛋白菌及其分泌载体高效生产有用蛋白

基本信息

批准号：
61860009
负责人：
UDAKA Shigezo
金额：
$ 5.06万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

项目摘要

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.

短芽孢杆菌是一种潜在的蛋白质生产菌。由于主要的外蛋白与细胞表面蛋白相同，因此将控制外蛋白表达和分泌的DNA区域插入高拷贝数质粒以构建表达和分泌载体PNU 200。利用该载体在B中生产各种有用的蛋白质。简记1.人白细胞介素2（IL 2）的产生。将IL 2基因插入到B的信号序列下游。pNU 200中的brevis主要细胞表面基因。将该质粒导入B。brevis 47.细胞外产生1 - 2 mg/l的IL 2，但IL 2在2个更长的培养中被部分降解。然后，将相同的质粒转移至B。brevis HPD 31，其不具有可检测的外切蛋白酶。通过优化培养条件，现在产生约20-30 mg/l的IL 2，而没有显著的IL 2.2降解。细菌β-淀粉酶的产生。将诺粘芽孢杆菌的β-淀粉酶基因连接到pNU 200的信号序列下游。通过改造B。brevis 47中，在适当的培养条件下，该菌株产生大量的β-淀粉酶（约0.5g/l）。甚至通过删除编码该酶的大C-末端区域的DNA，揭示β-淀粉酶的必需区域，获得了具有酶活性的β-淀粉酶。