Nucleotide sequence of the celG gene of Clostridium thermocellum and characterization of its product, endoglucanase CelG

The nucleotide sequence of the celG gene of Clostridium thermocellum, encoding endoglucanase CelG, was determined. The open reading frame extended over 1,698 bp and encoded a 566-amino-acid polypeptide (molecular weight of 63,128) similar to the C. thermocellum endoglucanase CelB (51.5% identical residues). The N terminus displayed a typical signal peptide, followed by a catalytic domain. The C terminus, which was separated from the catalytic domain by a 25-amino-acid segment rich in Pro, Thr, and Ser, contained two conserved stretches of 22 amino acids closely similar to those previously described in other cellulases from the same organism. Expression of the gene in Escherichia coli was increased by fusing the fragment coding for the catalytic domain in frame with the start of the lacZ' gene present in the vector. A low- and a high-M(r) form of the protein were purified. The two forms displayed identical enzymatic properties. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that both forms consist of a major polypeptide of M(r) 50,000 and two minor polypeptides of M(r)s 49,000 and 48,000, resulting from heterogeneous proteolytic cleavage at the C terminus. An antiserum raised against the forms purified from E. coli reacted with an immunoreactive polypeptide of M(r) 66,000, which was associated with the extracellular cellulolytic complex of C. thermocellum known as the cellulosome.