Swapping for Protein Engineering

PCA can be used for quantitative pharmocological analysis of protein-protein and protein—small molecule binding in vivo. However, if the mDHFR domains are swapped for complementary fragments of another enzyme, then perhaps a PCA with ligand-dependent interacting domains such as FKBP and FRB can be used to regulate the activity of this enzyme. 

To delineate regions of the protein responsible for the observed differences and create PQQGDH enzymes with desired substrate activities and stability, Sode and co-workers have constructed a variety of domain-swapped hybrids between the E. coli and Adnetobacter calcoaceticus PQQGDHs. They identified the region between 32% and 59% of the 

The three-dimensional structure of human extracellular superoxide dismutase (EC-SOD) is unknown. Studies of structure-function relationships have been severely limited by its poor production in mammalian cell lines and failure to be expressed in prokaryotic and yeast systems. In contrast, extra- and intracellular Cu- and Zn-containing superoxide dismutases (CuZn-SOD) are expressed very well in E. coli and yeast. CuZn-SOD is homologous to a large interior fragment of EC-SOD, but lacks its extra N-terminal and C-terminal domains. Fusions of either the N-terminal domain of EC-SOD or both the N- and C-terminal domains of EC-SOD to CuZn-SOD resulted in a domain-swapped enzyme that expressed well and whose characteristics resemble EC-SOD (Stenlund and Tibell, 1999). 

Trypsin and factor Xa (fXa) are two members of the chymotrypsin family that have 38% sequence identity on the amino acid level and have distinguishable substrate specificities. Recently, the N-terminal 13-barrel of fXa and the C-terminal /3-barrel of trypsin were fused at a rationally designed site in the linker region between the two domains in order to create a hybrid fXa-trypsin protease (Hopfner et al., 1998). The fXa-trypsin hybrid was highly active and more active than either parent on three of the ten substrates assayed, as determined by k /Km. For most substrates, the activity of fXa-trypsin was an admixture of the two parents, probably because trypsin had higher activity than fXa for all the substrates tested. 

Polyketides are made by the sequential activity of domains of large, multifunctional enzymes called polyketide synthases (PKSs) (Fig. 6a and b). Polyketides are formed by the condensation and modification of acyl units derived from acyl-CoA precursors. Domains are organized in modules and each module carries out the series of steps necessary for one cycle of polyketide chain elongation. A single protein can have more than one module, and several different proteins together can make up a PKS. The number of modules determines the size of the polyketide. A growing polyketide chain is tethered to the enzyme as a thiol ester and moves sequentially from the N- to the C-terminus of a module, lengthened by two carbon units per module. The first module in a PKS 

---

Progress in utilizing domain swapping for protein engineering has been partially hindered by the lack of combinatorial methods for per-... 

---