Cloning and expression of the @entity1 XXXX in @entity526 . Characterization of the recombinant protein and a deletion mutant lacking the major interdomain linker.

@entity1 @entity71199 @entity12116 @entity1160 @entity492 @entity7391 @entity526
The @entity71199 catalyzes the first three steps in de novo @entity7391 biosynthesis in @entity1 cells. @entity1160 -dependent @entity12116 synthetase (CPSase), @entity492 transcarbamylase, and dihydroorotase activities are carried by a 243-kDa polypeptide chain that is organized into discrete functional domains connected by interdomain linkers. One of the connecting chain segments, the DA linker bridging the dihydroorotase and @entity492 transcarbamylase domains, is unusually long (109 residues) and conserved in length in all eukaryotic species. A plasmid (pCK-CAD10) that encodes the entire 243-kDa polypeptide was constructed and expressed in @entity526 . The recombinant protein was purified to homogeneity by ion exchange and gel filtration chromatography. The purified protein had kinetic parameters that were close to those obtained for native CAD. Moreover, the CPSase activity was allosterically regulated. Gel filtration showed that the recombinant protein had the same molecular mass as native CAD. Thus, this complex @entity1 protein is expressed and folds correctly in bacterial cells and, despite its extreme protease sensitivity, can be isolated intact. A deletion mutant that lacked the DA linker was then constructed. The kinetic parameters of the mutant protein were, for the most part, unaltered, showing that the DA linker is not essential for the proper folding or optimal functioning of the individual domains. However, a significant decrease in the thermal stability of the CPSase domain suggested that the linker helps to stabilize the complex. Moreover, the channeling of @entity12116 , determined by measuring the extent to which the exogenously added intermediate could dilute the endogenous @entity12116 pool, was appreciably reduced when the DA linker was removed. Thus, although the domains function autonomously, some of the linkers are important for interdomain interactions in CAD.
