Identification of the endoglucanase encoded by the celB gene of Clostridium thermocellum

Abstract

The endoglucanase encoded by the celB gene of Clostridium thermocellum was purified from an E. coli strain carrying and expressing the C. thermocellum gene cloned in the plasmid pBR322. The preparation showed two active bands, with Mr 55,000 and 53,000, presumably derived from the primary translation product by proteolysis. Specific antiserum raised against these bands was used to identify the corresponding antigen in the culture supernatant of C. thermocellum: in a double immunodiffusion test (Ouchterlony), a precipitin line was observed which fused completely with that formed by an E. coli extract containing endoglucanase B expressed from the cloned gene. Proteins from C. thermocellum supernatant were further analyzed by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose sheet. After incubating the nitrocellulose blot with antiserum and subsequently with 125I-labeled protein A, a band with Mr 66,000, corresponding to the celB gene product expressed by C. thermocellum, was detected by autoradiography.