Genetic and biochemical approaches for in vivo and in vitro assessment of protein oligomerization: The ryanodine receptor case study

Stanczyk, Paulina J., Lai, Francis Anthony ORCID: https://orcid.org/0000-0003-2852-8547 and Zissimopoulos, Spyros 2016. Genetic and biochemical approaches for in vivo and in vitro assessment of protein oligomerization: The ryanodine receptor case study. Journal of Visualized Experiments (113) , e54271. 10.3791/54271

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Abstract

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca2+ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by β-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Uncontrolled Keywords:	Molecular Biology, Issue 113, chemical cross-linking, co-immnoprecipitation, mammalian cell transfection, oligomerization, ryanodine receptor, self-association, yeast two-hybrid
Publisher:	Journal of Visualized Experiments
ISSN:	1940-087X
Last Modified:	01 Nov 2022 11:27
URI:	https://orca.cardiff.ac.uk/id/eprint/94998

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