Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.