Functional proteomics protocol for the identification of interaction partners in Tetrahymena thermophila

STAR Protoc. 2021 Mar 4;2(1):100362. doi: 10.1016/j.xpro.2021.100362. eCollection 2021 Mar 19.

Abstract

We describe an optimized protocol for one-step affinity purification of FZZ-tagged proteins followed by mass spectrometry analysis for the identification of protein-protein interactions in the ciliate protozoan Tetrahymena thermophila. The FZZ epitope tag contains 2 protein A moieties (ZZ) and a 3xFLAG separated by a TEV cleavage site, which can also be employed in tandem affinity purification. This protocol is versatile and is suitable to use for other common epitope tags and can be adapted for other ciliates. For complete details on the use and execution of this protocol, please refer to Garg et al. (2019).

Keywords: Mass spectrometry; Model organisms; Molecular biology; Protein biochemistry; Protein expression and purification; Proteomics.