Cloning and expression of aClostridium thermocellum dna fragment that encodes a protein related to cellulosome component sL

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Abstract
Antibodies raised against the SL subunit of theClostridium thermocellum cellulosome were used to screen a library ofC. thermocellum chromosomal DNA fragments constructed in the vector λgt11. A DNA fragment that encoded a polypeptide that crossreacted with the anti-SL antibodies was isolated and its restriction map elucidated. No similarity with other previously cloned DNA fragments has been found. The anti-SL, crossreacting polypeptide was isolated from recombinantEscherichia coli and found to have a mol mass of 37,000 Da and to possess low levels of CMCase and Avicelase activity. Using CMC as the substrate, a temperature optimum of 55°C and a pH optimum of 6.6 were observed. These properties were compared to those ofC. thermocellum SL isolated by electroelution from an SDS gel, which was also found to possess low levels of CMCase and Avicelase activities. In addition, the SL proteins produced inC. thermocellum andE. coli were able to interact positively against Avicel with an endoglucanase (SS) purified from theC. thermocellum crude cellulase preparation, and with a recombinant protein that crossreacted with anti-SS antibodies.

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