Single protein production in living cells facilitated by an mRNA interferase.
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.
Suzuki
Motoo
M
Department of Biochemistry, Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, New Jersey 08854, USA.
Zhang
Junjie
J
Liu
Mohan
M
Woychik
Nancy A
NA
Inouye
Masayori
M
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000378:metabolism; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005098:Exotoxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D014170:Transformation, Genetic
D001427:Bacterial Toxins; D029968:Escherichia coli Proteins; D005098:Exotoxins; D012333:RNA, Messenger; D004722:Endoribonucleases