ENGINEERING A DE NOVO-DESIGNED COILED-COIL HETERODIMERIZATION DOMAIN FOR THE RAPID DETECTION, PURIFICATION AND CHARACTERIZATION OF RECOMBINANTLY EXPRESSED PEPTIDES AND PROTEINS

Using the techniques of genetic engineering and the principles of prot ein de novo design, we have developed a unique affinity matrix protein tag system as a rapid, convenient and sensitive method to detect, pur ify and characterize newly expressed recombinant peptides or proteins from cell extracts. The method utilizes two de novo-designed linear pe ptide sequences that can selectively dimerize to form the stable prote in moth, the two-stranded a-helical coiled-coil. In this method, a rec ombinant bacterial expression vector pRLDE has been engineered so that one of the dimerization strands (E-coil) is expressed as a C-terminal fusion tag on newly expressed peptides or proteins, while the other ( K-coil) is either biotin-labeled for detection in a Western blot-type format or immobilized on an insoluble silica support for selective dim erization affinity chromatography. Recombinantly expressed peptides fr om Escherichia coli containing the dimerization tag have been produced , detected and purified using this method. The recombinant peptides we re easily and clearly identified using the biotin-labeled coil, while the single-step affinity purification results indicated the purity of the affinity purified expressed peptides to be >95%, as assessed by re versed-phase chromatography. The stability of the dimerization domain also allows for the purified peptide to be left attached to the matrix , thus creating a new peptide-bound column that can be used to study p eptide-protein or peptide-ligand interactions. Therefore this system o ffers a new alternative to existing peptide or protein fusion tags and demonstrates the utility of a de novo-designed system.