Single protein production in living cells facilitated by a...

Details Single protein production in living cells facilitated by a... Single protein production in living cells facilitated by a...

2011-07-26

2011-07-26

Ketter, James S (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S320100

Reexamination Certificate

active

07985575

ABSTRACT:
The present invention describes a single-protein production (SPP) system in livingE. colicells that exploits the unique properties of an mRNA interferase, for example, MazF, a bacterial toxin that is a single stranded RNA- and ACA-specific endoribonuclease, which 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 yeast and human proteins, even a bacterial integral membrane protein. This novel system enables unparalleled signal to noise ratios that should dramatically simplify structural and functional studies of previously intractable but biologically important proteins. The present invention also provides an optimized condensed single protein production system.

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