The Use of the Condensed Single Protein Production System for Isotope-Labeled Outer Membrane Proteins,OmpA and OmpX in <Emphasis Type="Italic">E. coli</Emphasis> |
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Authors: | S Thangminlal Vaiphei Yuefeng Tang Gaetano T Montelione Masayori Inouye |
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Institution: | (1) Department of Biochemistry, Robert Wood Johnson Medical School and Centre for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854, USA;(2) Department of Molecular Biology and Biochemistry, Rutgers and Centre for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854, USA; |
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Abstract: | 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. |
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