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Controlled localization of functionally active proteins to inclusion bodies using leucine zippers.


ABSTRACT: Inclusion bodies (IBs) are typically non-functional particles of aggregated proteins. However, some proteins in fusion with amyloid-like peptides, viral coat proteins, and cellulose binding domains (CBDs) generate IB particles retaining the original functions in cells. Here, we attempted to generate CBD IBs displaying functional leucine zipper proteins (LZs) as bait for localizing cytosolic proteins in E. coli. When a red fluorescent protein was tested as a target protein, microscopic observations showed that the IBs red-fluoresced strongly. When different LZ pairs with KDs of 8-1,000 µM were tested as the bait and prey, the localization of the red fluorescence appeared to change following the affinities between the LZs, as observed by fluorescence imaging and flow cytometry. This result proposed that LZ-tagged CBD IBs can be applied as an in vivo matrix to entrap cytosolic proteins in E. coli while maintaining their original activities. In addition, easy detection of localization to IBs provides a unique platform for the engineering and analyses of protein-protein interactions in E. coli.

SUBMITTER: Choi SL 

PROVIDER: S-EPMC4045587 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Controlled localization of functionally active proteins to inclusion bodies using leucine zippers.

Choi Su-Lim SL   Lee Sang Jun SJ   Yeom Soo-Jin SJ   Kim Hyun Ju HJ   Rhee Young Ha YH   Jung Heung-Chae HC   Lee Seung-Goo SG  

PloS one 20140604 6


Inclusion bodies (IBs) are typically non-functional particles of aggregated proteins. However, some proteins in fusion with amyloid-like peptides, viral coat proteins, and cellulose binding domains (CBDs) generate IB particles retaining the original functions in cells. Here, we attempted to generate CBD IBs displaying functional leucine zipper proteins (LZs) as bait for localizing cytosolic proteins in E. coli. When a red fluorescent protein was tested as a target protein, microscopic observatio  ...[more]

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