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Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro
Published on: August 14, 2011
Looking for a needle in a haystack: Cellular proteins that may interact with the tyrosine-based sorting signal of the
Anna Trincone1, Christel Schwegmann-Weßels1
1Institute for Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Bünteweg 17, 30559 Hannover, Germany.
Abstract:
The spike protein S of transmissible gastroenteritis virus, an Alphacoronavirus, contains a tyrosine-based sorting signal that is responsible for ERGIC retention and may be important for a correct viral assembly process. To find out whether the S protein interacts with cellular proteins via this sorting signal, a pulldown assay with GST fusion proteins was performed. Filamin A has been identified as a putative interaction candidate. Immunofluorescence assays confirmed a co-localization between the TGEV S protein and filamin A. Further experiments have to be performed to prove a significant impact of filamin A on TGEV infection. Different approaches of several researchers for the identification of cellular interaction candidates relevant for coronavirus replication are summarized. These results may help in the future to identify the role of cellular proteins during coronavirus assembly at the ER-Golgi intermediate compartment.
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