微紫青霉CBHI酶的CBD蛋白编码区在大肠杆菌中的亚克隆及表达  

SUBCLONING AND EXPRESSION OF CODING REGION FOR CELLULASE BINDING DOMAIN OF CBH I FROM P.JANTHINELLUM IN E.COLI

作　　者：江天虹 王春卉[1] 高培基[1] 钟玲 邹玉霞[1] 

机构地区：[1]山东大学微生物技术国家重点实验室,济南250100

出　　处：《微生物学报》1998年第4期269-275,共7页Acta Microbiologica Sinica

基　　金：国家自然科学基金;山东省自然科学基金;国家教委博士点基金

摘　　要：对插入质粒pUC18－181上的微紫青霉（Penicilliumjanthinellum）CBHI酶的cDNA基因进行一系列DNA体外操作，包括进行序列定向缺失，最后将两末端修饰为平端后进行连接使质粒环化。用得到的产生序列定向缺失的重组质粒转化大肠杆菌JM109。利用CBD能吸附到结晶纤维素上的特性，从随机选取的24个缺失转化子中筛选到一株含CBD编码区的转化子JM109（pUC18C），所表达的CBD融合蛋白分子量为21kD．JM109（pUC18C）所产生的LacZ-CBD融合蛋白可通过对纤维素的吸附-解吸附过程一步纯化。其IPTG诱导的pNPC酶活力为零，表明该菌已不再具有CBHI酶活力。The in vitro DNA manipulations,included the nested deletions,of cbh1 from p.janthinellum inserted into pUC18-181 were carried out. The two ends of fragments were modified into blunt ends and the fragments were self-ligated. Then,the encircled plasmids were transformed to E.colt JM109.Utilizing the characterization of CBD binding to crystalline cellulose,one catalytic domain deletion transformant producing active LacZ-CBD fusion protein was isolated from 24 transformants randomly Picked from 400 transformants.The molecular weight of the LacZ-CBD fuSion protein is 21 kD. The plasmid was designated pUC 18C.The LacZ-CBD fusion protein produced by JM109(pUC18C)was able to be Purified by procedure of adsorption-desorption to cellulose.The pNPC activity of crude enzyme solution of JM109(pUC18C) induced by IPTG were zero,idenhfied that JM109(pUC18C)has no CBHI activity.

关 键 词：大肠杆菌 亚克隆 表达 CBHI酶 微紫青霉 

分 类 号：Q78[生物学—分子生物学]