Ni-NTA Affinity Chromatography to Characterize Protein-Protein Interactions During Fe-S Cluster Biogenesis

Abstract

Affinity chromatography can be repurposed to provide useful information about the specific partner protein(s) to which a protein of interest may bind as well as the relative binding affinity of that partner protein for the protein of interest. Here, we provide a protocol for an Ni-NTA affinity chromatography assay that may be utilized to uncover insightful information about the nature of protein-protein interactions during iron-sulfur (Fe-S) cluster biogenesis reactions.

Keywords: Affinity chromatography; Anaerobic; IMAC; Iron-sulfur cluster.

Fig. 1

Diagram illustrating various bait and prey protein combinations that can be used in the affinity chromatography experiment. Several control experiments are also shown (see text for more details)

Fig. 2

SDS-PAGE analysis of elution fractions from a protein–protein interaction experiment using this technique. His₆-SufA in the apo-form was used as the bait protein on a 1 mL HisTrap column as described in Methods. Apo- or holo-SufBC₂D scaffold complex (untagged) was used as the prey proteins. The SDS-PAGE analysis shows much greater retention of holo-SufBC₂D than apo-SufBC₂D, indicating a stronger interaction between holo-SufBC₂D and apo-SufA than between apo-SufBC₂D and apo-SufA. (Reprinted (adapted) with permission from Chahal HK, Dai Y, Saini A, et al. (2009). The SufBCD Fe-S Scaffold Complex Interacts with SufA for Fe-S Cluster Transfer. Biochemistry 48:10644–10,653. https:/doi.org/10.1021/bi901518y. Copyright 2009 American Chemical Society)