[Subcloning and expression of coding region for cellulase binding domain of CBH I from P. janthinellum in E. coli].
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. coli 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, identified the JM109(pUC18C) has no CBHI activity.
Wang
T
T
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100.
Wang
C
C
Gao
P
P
Zhong
L
L
Zou
Y
Y
D000327:Adsorption; D002480:Cellulase / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D010407:Penicillium / Q000201:enzymology*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D011993:Recombinant Fusion Proteins; D002482:Cellulose; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase