摘要
链霉菌C3662的发酵液上清经 80 %硫酸铵沉淀 ,DEAE Sepharose和CM Sepharose层析分离后纯化出一种纤溶酶。SDS PAGE显示为单一的条带 ,分子量约为 30kD。以 pIJ699为载体 ,S .lividansTK2 4为宿主菌 ,鸟枪法克隆纤溶酶基因 ,从 30 0 0个转化子中挑选到 1个具活性转化子 ,经亚克隆 ,序列测定得到一个 90 3bp的完整ORF ,其GC %为 68 33% ,密码子第三位GC %为 95 6% ,符合链霉菌基因的典型特征。
A novel fibrinolytic protease from \%Streptomyces\% sp.C3662 was purified by (NH 4) 2SO 4 fractionation,DEAE\|Sepharose and CM\|Sepharose chromatography.The molecular weight of the protease was indicated to be 30 kD by SDS\|PAGE.Using a \%E.coli/Streptomyces\% shuttle plasmid pIJ699 as the vector,shot\|gun cloning was performed to clone the gene of the protease.One clone with fibrinolytic activity harboring a plasmid that contains a DNA fragment of 6kb was obtained from 3000 clones.Sequence analysis reveals that the open reading frame of the gene of the protease is 903bp in size,encoding a putative protein of 300 amino acids with 30kD.The overall GC% and the third codon GC% of the ORF were 68 33% and 956%,respectively.Comparison of homologue showed that the purative protein is highly homologous with other proteases of Streptomyces\sp.
出处
《微生物学报》
CAS
CSCD
北大核心
2001年第2期186-190,共5页
Acta Microbiologica Sinica
关键词
纤溶酶
纯化
鸟枪克隆
链霉菌
基因
Streptomyces sp., Fibrinolytic protease, Purification, Gene cloning
