Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions.

Autor:	Bonchuk A; Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences; bonchuk_a@genebiology.ru., Zolotarev N; Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences., Balagurov K; Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences., Arkova O; Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology, Russian Academy of Sciences., Georgiev P; Department of the Control of Genetic Processes, Institute of Gene Biology, Russian Academy of Sciences.
Jazyk:	angličtina
Zdroj:	Journal of visualized experiments : JoVE [J Vis Exp] 2022 Dec 23 (190). Date of Electronic Publication: 2022 Dec 23.
DOI:	10.3791/64541
Abstrakt:	Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble effectively in vitro. Such complexes may require co-translational assembly and the presence of molecular chaperones; either they form stable oligomers which cannot dissociate and re-associate in vitro or are unstable without a binding partner. To overcome these problems, it is possible to use a method based on the bacterial co-expression of differentially tagged proteins using a set of compatible vectors followed by the conventional pulldown techniques. The workflow is more time-efficient compared to traditional pulldown because it lacks the time-consuming steps of separate purification of interacting proteins and their following incubation. Another advantage is a higher reproducibility due to a significantly smaller number of steps and a shorter period of time in which proteins that exist within the in vitro environment are exposed to proteolysis and oxidation. The method was successfully applied for studying a number of protein-protein interactions when other in vitro techniques were found to be unsuitable. The method can be used for batch testing protein-protein interactions. Representative results are shown for studies of interactions between BTB domain and intrinsically disordered proteins, and of heterodimers of zinc-finger-associated domains.
Databáze:	MEDLINE
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