The next best thing after cryoprobe and TROSY?


Single Protein Production in Living Cells Facilitated by an mRNA Interferase

Motoo Suzuki,1 Junjie Zhang,1 Mohan Liu,2 Nancy A. Woychik,2 and Masayori Inouye1,*

1 Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854
2 Department of Molecular Genetics, Microbiology and Immunology, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854


We designed a single-protein production (SPP) system in living E. coli cells that exploits the unique properties of MazF, a bacterial toxin that is an ssRNA- and ACA-specific endoribonuclease. In effect, MazF functions as an “mRNA interferase,” because it efficiently and selectively degrades all cellular mRNAs in vivo, resulting in a precipitous drop in total protein synthesis. Concomitant expression of MazF and a target gene engineered to encode an ACA-less mRNA results in sustained and high-level (up to 90%) target expression in the virtual absence of background cellular protein synthesis. Remarkably, target synthesis continues for at least 4 days, indicating that cells retain transcriptional and translational competence despite their growth arrest. SPP technology works well for E. coli (soluble and membrane), yeast, and human proteins. This expression system enables unparalleled signal to noise ratios that should dramatically simplify structural and functional studies of previously intractable but biologically important proteins.