POSTERS - BLAST X - University of Utah

BLAST X Poster #38
MINING THE E. COLI GFP FUSION COLLECTION
Jason Dobkowski, Aleksandra Sikora, John Brooks, Richard Zielke and Janine Maddock
Department of MCDB, University of Michigan, 830 N. University, Ann Arbor, MI 48109
Use of green fluorescent protein (GFP) has greatly increased the ability to visualize
protein localization within bacterial cells. A library of GFP fusions, expressed from an inducible
promoter, was created with each protein from every open reading frame in the Escherichia coli
genome (Kitagawa et al, 2005). The addition of the 26.9 kDa GFP, however, can lead to
protein misfolding and aggregation and the formation of polar inclusion bodies. These polar
inclusion bodies are often mistaken for bona fide polar localization of the protein fusion. Using a
purification and screening approach, we have sorted polarly localized GFP fusions into those
that form inclusion bodies and those that are likely real polar proteins. Those that form inclusion
bodies are being used to identify interacting partners that co-aggregate with the misfolded
protein. Proteins not previously not known to be polar are being verified using
immunofluorescence microscopy and the localization of known interacting partners determined.
We are particularly interested in the spatial organization of membrane bound histidine kinases
involved in sensing environmental stress. Our current line of focus is to determine whether the
activation of the signal transduction cascade has any influence of their polar localization.
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