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

Abstract

Antibodies raised against the SL subunit of the Clostridium thermocellum cellulosome were used to screen a library of C. thermocellum chromosomal DNA fragments constructed in the vector lambda gt11. 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 recombinant Escherichia 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 degrees C and a pH optimum of 6.6 were observed. These properties were compared to those of C. 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 in C. thermocellum and E. coli were able to interact positively against Avicel with an endoglucanase (Ss) purified from the C. thermocellum crude cellulase preparation, and with a recombinant protein that crossreacted with anti-Ss antibodies.

Cellulase, Clostridia, and Ethanol

“…We and others devoted considerable efforts to the isolation and purification of a number of proteins of the C. thermocellum system as well as the cloning, expression, and sequencing of their relevant genes in Escherichia coli and other hosts (80,96,148,163,276,277). Of particular interest was our gene sequencing of S L (96,277) and the gene sequencing of S S (341).…”

“…Of particular interest was our gene sequencing of S L (96,277) and the gene sequencing of S S (341).…”

“…In addition to its function of binding other members of the cellulase complex to itself, it also binds to cellulose (33). Our first cloning and sequencing of cipA involved a truncated 5Ј region (277). Later, the remainder of the gene was cloned by chromosome walking and the entire sequence was determined (96).…”

Induction of the celC operon of Clostridium thermocellum by laminaribiose

“…In addition, many free glycosyl hydrolases are produced. The core of the cellulosome is CipA, a 250-kDa noncatalytic, scaffold protein (2)(3)(4)(5). CipA contains nine cohesin domains.…”

Bacterial genomes: what they teach us about cellulose degradation

“…Using these shotgun cloning techniques, cellulases were cloned from Cellulomonas fimi [24,25], Clostridium cellulolyticum [26] and F. succinogenes [27,28], as well as many of the individual components of the C. thermocellum cellulosome [29][30][31][32][33]. A similar approach was utilized to clone cellulases from samples of metagenomic DNA prepared from an environmental sample of microbes.…”