The use of the condensed single protein production system for isotope-labeled outer membrane proteins, OmpA and OmpX...

Gram-negative bacteria consist of two independent membranes, the inner cytoplasmic membrane and the outer membrane. The outer membrane contains a number of β-barrel proteins such as OmpF, OmpC, OmpA, and OmpX. In this article, we explored to use the condensed Single Protein Production (cSPP) system for isotope labelling of OmpA and OmpX for NMR structural study, both of which are known to consist of eight β-strands forming a barrel in the outer membrane. Using a deletion strain lacking all major outer membrane proteins, both OmpA and OmpX were expressed well in a 20-fold cSPP system. We demonstrated that outer membrane fractions prepared from the cSPP system in M9 medium containing ¹⁵N-NH₄Cl can be directly used for NMR structural study of the outer mebrane proteins without any further purification to get excellent [¹H-¹⁵N]-TROSY spectra. This method would be quite valuable for the study of pure proteins in their native membrane environment without the need of purification and reconstitution.