CLONING AND EXPRESSION OF RECOMBINANT PROTEIN OF SUMO, FUSED WITH BIOTIN ACCEPTOR PEPTIDE
Main Article Content
Authors
A.T. Kulyyassov
National Center for Biotechnology, 13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
Ye.M. Ramankulov
National Center for Biotechnology, 13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
V.V. Ogryzko
National Center for Biotechnology, 13/5, Korgalzhyn road, Astana, 010000, Kazakhstan
Institut Gustave Roussy, CNRS UMR8126, 94805, 39 Rue Camilles Desmoulin, Villejuif, France
Abstract
Post-translation modifications by SUMO are involved in a plethora of biological processes, including cell proliferation. Since SUMOylation is an essential regulatory mechanism in signal transduction, violation of the processes associated with SUMOylation can lead to various diseases, including cancer.
We adopted the proximity utilizing biotinylation method, based on co-expression of recombinant proteins within a single cell: the protein of interest fused with the biotin ligase BirA and its partner with the biotin acceptor peptide (BAP). This co-expression allows for accurate quantitative assessment of the extent of the interaction of various molecules in vivo.
Using this approach, the aim of this work was to construct a new plasmid vector containing a SUMO gene fused with a BAP and to implement its expression in HEK293T cells.
Co-expression of the BAP–SUMO and BirA-HP1g (BirA-HP1a) recombinant proteins in HEK293T cells resulted in protein biotinylation, which are SUMOylated proximal proteins (or interaction partners) of the proteins of interest BirA-HP1g and BirA-HP1a.
Keywords
protein–protein interactions, biotinylation, biotin ligase, biotin acceptor peptide, plasmids, transient transfection, western blot, SUMO
Article Details
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