DIRECT IN-SITU STRUCTURAL-ANALYSIS OF RECOMBINANT OUTER-MEMBRANE PORINS EXPRESSED IN AN OMPA-DEFICIENT MUTANT ESCHERICHIA-COLI STRAIN

An OmpA-deficient mutant of an OmpF/OmpC-free Escherichia coli B-e str ain was selected using phage K3. The mutant strain was characterized b y SDS-gel electrophoresis, immunoblotting, and electron microscopy. Al l major outer membrane proteins, including OmpA, were absent. This str ain was then transformed with the plasmid pMY222 encoding the K12 OmpF porin or with pBluescript-derived plasmids, encoding the porins OmpC, PhoE, and maltoporin, respectively. Following SDS extraction of outer membrane sacculi from strains expressing individual porins, crystalli ne porin arrays that allowed in situ structural analysis to be perform ed were observed. Furthermore, the absence of endogenous major outer m embrane proteins facilitated the purification of native porin-lipopoly saccharide complexes, the functionally active channels, from the saccu li of transformed strains. (C) 1993 Academic Press, Inc.