Production of membrane proteins for NMR studies using the condensed single protein (cSPP) production system

Abstract

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.

Use of Amino Acids as Inducers for High-Level Protein Expression in the Single-Protein Production System

“…This system is thus termed the single-protein production (SPP) system. One of the most remarkable advantages of the SPP system is that the cell culture can be highly condensed without affecting protein yields (7,12,13). Using this cSPP system, one can achieve a cost savings of as much as 97.5% by condensing a culture 40-fold.…”

Membrane proteins, magic-angle spinning, and in-cell NMR

“…Dynamic nuclear polarization should improve sensitivity, and its application to native membrane systems is feasible (12,13). New expression systems offer even more powerful ways to suppress background signals (14,15), and membrane fractionation is a tried and true method for improving sample homogeneity and reducing background (16,17). Finally, site-specific labeling with unnatural amino acids provides residuespecific information for protein in real membranes (18).…”

Festkörper‐NMR‐Spektroskopie an komplexen Biomolekülen

“…Solche Ansätze sind besonders interessant, da durch die starke Unterdrückung der Proteinsynthese des Wirtsystems sowohl die Effizienz der Membranproteinexpression als auch das Targeting und die korrekte Insertion in die Wirtsmembran gesteigert werden. [40] Dadurch eröffnen sich neue Möglichkei-ten zur Strukturanalyse integraler Membranproteine durch FK-NMR-Spektroskopie. Des Weiteren könnten auch andere heterologe Expressionssysteme wie Kluyveromyces lactis, [41] Pichia pastoris [42] und andere Zelllinien wie Sf9-Insektenzellen [43] oder Säugetier-Zelllinien wie CHO [44] und HEK [45] in Zukunft für NMR-Studien verwendet werden.…”