STRUCTURAL AND FUNCTIONAL-CHARACTERIZATION OF A HIS-TAGGED PHOE PORE PROTEIN OF ESCHERICHIA-COLI

The recent elucidation of the 3-D structure of the outer membrane prot ein PhoE of Escherichia coli provides an excellent tool for a detailed analysis of the structure-function relationship of this pore-forming protein. For this purpose, a fast and efficient method fbr the purific ation of mutant porins is needed. A histidine-tag was engineered betwe en the signal sequence and the N terminus of mature PhoE. The recombin ant PhoE protein was normally assembled into the outer membrane and co uld be purified by immobilized metal affinity chromatography. Part of the total amount of the trimers dissociated into folded monomers durin g purification. The histidine-tag did not change the electrophysical c haracteristics of the protein in lipid bilayers. Hence, the method is useful for the fast purification of mutant porins for functional and s tructural characterization. (C) 1996 Academic Press.