5] In Vivo and in Vitro systems for studying bacterial membrane biogenesis

Abstract

This chapter describes the methods that are most useful for the study of the biogenesis of the major outer membrane proteins of Escherichia coli K12: lipoprotein, the matrix proteins, and ompA protein. The pro-ompA protein accumulated in the nonfluid state can be completely processed to ompA protein during a 2-min chase. During the chase, aliquots are removed from the cultures at various time intervals, and membranes are isolated. Bacterial membrane proteins are more resistant to puromycin and kasugamycin, but more sensitive to tetracycline and sparsomycin than are soluble proteins. An important approach to the study of the biosynthesis and assembly of bacterial membrane proteins is the development of RNA-dependent cell-free translation systems. Although, bacterial membrane proteins are translated in eukaryotic protein-synthesizing systems, the use of a bacterial system is not as restrictive and can reveal more details about in vivo biosynthetic mechanisms.