Technologies

The Quiescent Cell Expression System (Q-Cells)

A fundamental problem associated with the expression of recombinant proteins in growing bacteria is that energy and nutritional resources are channeled towards biomass production. The product gene must compete with host genes for the transcription-translation machinery and metabolic resources. Cambridge Microbial has developed a method of generating non-growing but metabolically active ('quiescent') E. coli cells. Growth arrest is achieved by the manipulation of endogenous cell cycle controls under culture conditions where nutrients are not limiting. Condensation of the bacterial nucleoid ensures that while most chromosomal genes are inactive, plasmid gene expression continues. Thus the entire metabolic resources of the cell are channeled towards the expression of a plasmid borne product gene (Rowe & Summers, 1999 (1.4Mb PDF) )

The quiescent state is established by over-expressing a small (70 nt) RNA (Rcd) in an hns205 mutant bacterial host. Entry into quiescence is medium-independent and has been achieved for a wide variety of E. coli strains. Quiescent cultures can be generated in shake-flasks and in fermenters at densities as high as OD600=50. Q-Cells have proved successful in producing a range of proteins including cytokines and an antibody fragment (Fab). The expression, folding and export of biologically-active Fab into the culture supernatant is more then ten times faster than in a conventional culture under the same conditions. ( Mukherjee et al 2004 (553k PDF) ).

A limitation of the 'first generation' Q-Cell system was that expression of Rcd was induced by a shift from 30°C to 42°C which means that product synthesis is at an undesirably high temperature. A recent discovery that Rcd stimulates indole production by binding to tryptophanase has led to the development of 'second generation' Q-Cells in which quiescence is induced by adding indole to the growth medium of an E.coli hns205 culture (Chant & Summers 2005). This removes any constraint on the product expression temperature and simplifies the system because no Rcd expression plasmid is required. In parallel with these improvements, the wider potential for Q-Cells is being explored for production not only of recombinant proteins, but also of hydrogen and higher alcohols as biofuels.

Included here is a PDF version of David Summers' layman's guide to the Q-Cell gene expression technology which was published by the SGM in Microbiology Today - 'A Quiet Revolution in the Bacterial Cell Factory' (358k PDF). To view this document you will need to have Adobe Acrobat Reader installed.

 

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