Expression, isolation, and characterization of a signal sequence-appended chimeric precursor protein

This report describes the properties and the functional utility of an unprocessed precursor protein overproduced in Escherichia coli. The precursor protein is from a fusion between DNA sequences coding for the alkaline phosphatase signal sequence and the full-length of rat liver cytochrome bg. The intact precursor protein accumulated in the membranes represented to over 5% of the total bacterial protein. A procedure involving disruption of the bacterial cells by sonication, isolation of the membranes by differential centrifugation, solubilization with a polar solvent, and ion-exchange chromatography provided milligram quantities of the undegraded precursor in a homogeneous and soluble form, The chimeric precursor protein displayed a characteristic b-type hemoprotein spectrum, identical to that of the native cytochrome bs. The properties of the precursor protein have been examined by a range of biophysical and biochemical methods, Molecular modeling suggests an amphipathic structure in which a fully preserved soluble core of cytochrome be is terminally bonded by hydrophobic interactions between the amino-terminal signal sequence and the carboxy-terminal membrane anchoring hemoprotein sequence, The precursor substrate was recognized and efficiently cleaved by signal peptidase. (C) 1996 Academic Press, Inc.