Production of membrane proteins for NMR studies using the condensed single protein (cSPP) production system
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In the Single Protein Production (SPP) method, all E. coli cellular mRNAs are eliminated by the induction of MazF, an ACA-specific mRNA interferase. When an mRNA for a membrane protein, engineered to have no ACA sequences without altering its amino acid sequence, is induced in the MazF-induced cells, E. coli is converted into a bioreactor producing only the targeted membrane protein. Here we demonstrate that three prokaryotic inner membrane proteins, two prokaryotic outer membrane proteins, and one human virus membrane protein can be produced at very high levels, and assembled in appropriate membrane fractions. The condensed SPP (cSPP) system was used to selectively produce isotope-enriched membrane proteins for NMR studies in up to 150-fold condensed culture without affecting protein yields, providing more than 99% cost saving for isotopes. As a novel application of the cSPP system for studies of membrane proteins prior to purification we also demonstrate, for the first time, fast detergent screening by microcoil NMR and well-resolved NMR spectra of several targeted integral membrane proteins obtained without purification.
KeywordscSPP Membrane protein NMR
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Condensed single protein production system
We thank Dr. M Suzuki for his efforts in initial phases of this work, and Dr. A McDermott for providing a clone of the gene for KcsA. This work was supported by the National Institutes of Health Grants U54 GM074958 (G.T.M. and M. I.), U54 GM75026 (G.T.M. and M. I.), 1R01 GM085449 (M.I.), and R37 CA53370 (E. W.), and partially by a research fund from Takara-Bio., Inc.
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