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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.

MazF from Escherichia coli is an endoribonuclease that specifically cleaves mRNAs at ACA sequences. Its induction in mammalian cells has been shown to cause programmed cell death. Here we explored if a bacterial MazF-MazE toxin-antitoxin system can be used for gene therapy. For this, we first constructed a tetracycline-inducible MazF expression system in human embryonic kidney cells (T-Rex 293-mazF). Solid tumors were formed by injecting T-Rex 293-mazF cells into nude mice. All 8 mice injected with the cells developed solid tumors, which regressed upon induction of MazF. In 4 mice, tumors completely regressed, while in the remaining 4 mice, tumors reappeared after apparent significant regression, which was found to be due to the lack of presence of functional MazF. Notably, the MazF-mediated regression of the tumors was counteracted by the expression of its cognate antitoxin MazE. These results indicate that a bacterial MazF-MazE toxin-antitoxin system may have potential to be used as a therapeutic tool.

By taking advantage of MazF, an ACA codon-specific mRNA interferase, Escherichia coli cells can be converted into a bioreactor producing only a single protein of interest by using an ACA-less mRNA for the protein. In this single-protein production (SPP) system, we engineered MazF by replacing two tryptophan residues in positions 14 and 83 with Phe (W14F) and Leu (W83L), respectively. Upon the addition of an inducer (IPTG [isopropyl-␤-D-thiogalactopyranoside]), the mutated MazF [MazF(⌬W)] can still be produced even in the absence of tryptophan in the medium by using a Trp auxotroph, while a target protein having Trp residues cannot be produced. However, at 3 h after the addition of IPTG, the addition of tryptophan to the medium exclusively induces production of the target protein at a high level. A similar SPP system was also constructed with the use of a His-less protein [MazF(⌬H)] and a His auxotroph. Using these dual-induction systems, isotopic enrichments of 13 C, 15 N, and 2 H were highly improved by almost complete suppression of the production of the unlabeled target protein. In both systems, isotopic incorporation reached more than 98% labeling efficiency, significantly reducing the background attributable to the unlabeled target protein.

Background: Hepatitis B virus (HBV) infection is closely related to the development of not only acute or chronic hepatitis, but also hepatocellular carcinoma. Among the HBV genes, the X gene has been implicated in the carcinogenicity of this virus as a major causative factor by its ability to activate viral and cellular genes in trans via protein-protein interaction with cellular factors without binding to DNA.

At present, only 0.9% of PDB-deposited structures are of membrane proteins in spite of the fact that membrane proteins constitute approximately 30% of total proteins in most genomes from bacteria to humans. Here we address some of the major bottlenecks in the structural studies of membrane proteins and discuss the ability of the new technology, the Single-Protein Production (SPP) system, to help solve these bottlenecks.

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