Overproduction, purification and characterization of SecD and SecF, integral membrane components of the protein translocation machinery of Escherichia coli

Abstract

SecD and SecF proteins were overproduced by means of recombinant DNA technology. Immunoblot and amino-acid sequencing analysis revealed that the overproduced proteins are SecD and SecF. The SecD- or SecF-overproduced membrane fraction was subjected to differential solubilization. The SecD protein was then purified through ion-exchange and size-exclusion chromatographies. The SecF protein was purified through size exclusion chromatography. Proteoliposomes reconstituted from the purified SecD and SecF together with SecE and SecY were used to analyze the translocation activity. SecD and SecF did not exhibit significant effects on the translocation activity of proteoliposomes. The amounts of SecD and SecF in overproducers were determined densitometrically on a stained SDS gel and their overproduction (fold) was determined by means of immunoblot analysis. Then the number of these molecules in one normal cell were estimated. From these numbers, together with those of other Sec proteins, the number of the translocation machinery existing in one Escherichia coli cell was inferred to be around 500.