COMPARISON OF 2 SCAFFOLDING POLYPEPTIDES FOR THE INTEGRATION OF DIFFERENT PROTEINS IN SYNTHETIC COMPLEXES DERIVED FROM THE CLOSTRIDIUM-THERMOCELLUM CELLULOSOME

The targeted integration of different polypeptides within artificial c omplexes derived from the Clostridium thermocellum cellulosome was inv estigated. Two scaffolding polypeptides, each containing two cohesin d omains, were compared. Cip6 consisted of a tandem duplication of a typ e I cohesin domain. Cip20 was constructed by fusing the same type I co hesin domain to the type II cohesin domain of SdbA. Cip6 was mixed wit h varying proportions of CelD, carrying a type I dockerin domain. and CelC-DSCelD, in which the dockerin domain of CelD was fused to the end oglucanase CelC. Likewise, Cip20 was mired with varying proportions of CelD and CelC-DSCipA-H, which carries the type II dockerin domain of CipA. Complex formation was analyzed by non-denaturing gel electrophor esis and densitometry: and the composition of the bands observed was c onfirmed by denaturing gel electrophoresis in a second dimension. With optimal proportions of the components, the ternary complex formed of Cip6, CelD, and CelC-DSCelD accounted for 36% of the proteins entering the gel. III contrast up to a 56% yield was obtained for the ternary complex formed of Cip20, CelD, and CelC-DSCipA-H. The results emphasiz e the advantage of using different types of cohesin-dockerin pairs in the design of cellulosome-derived complexes. (C) 1998 Elsevier Science Inc.