Cloning and Expression in Escherichia-Coli of Clostridium-Thermocellum Dna Encoding Subcellulosomal Proteins

A genomic library of Clostridium thermocellum DNA was constructed in a lambda gt11 phage vector, and DNA fragments encoding subunits of the subcellulosome fraction (which is composed of six main protein subunits, named J1 to J6) were isolated by immunological screening. Five DNA fragments from the recombinant lambda phages were subcloned into vectors pBR325 and pUC8 to create recombinant plasmids which were subsequently transformed into Escherichia coli. Two of the five DNA fragments, encoding J2 and J5 subunits, were found to be identical by Southern blot hybridization and restriction mapping. Each recombinant protein was prepared by ion-exchange column chromatography from heat-treated crude E. coli cell-free extracts. Recombinant proteins, J2, J3, J5, and J6, showed CM Case activity; J2 and J5 were also able to hydrolyse PNPC and xylan. Recombinant protein J4 did not show any enzymatic activities.