Elsevier

Analytical Biochemistry

Volume 324, Issue 1, 1 January 2004, Pages 137-142
Analytical Biochemistry

Cross-linking approach to affinity capture of protein complexes from chaotrope-solubilized cell lysates

https://doi.org/10.1016/j.ab.2003.09.017Get rights and content

Abstract

Affinity capture methods are widely used for isolation and analysis of protein complexes. Short peptide tags fused to the protein of interest normally facilitate straightforward purification and detection of interacting proteins. We investigated the suitability of applying C-terminally hexahistidine-tagged interleukin-12 (IL-12) α- and β-chains as “bait” proteins for cocapturing novel binding partners using heterologous recombinant human embryonic kidney-293 (HEK-293) cell lines. The β-chain, but not the α-chain, extracted from cell lysates was capable of binding to the Ni2+–nitrilotriacetic acid affinity resin under nondenaturing conditions. Retention of the α-chain on this matrix was dependent on treatment of cell lysates with high concentrations of chaotropes such as urea. Since under these conditions any noncovalent protein associations are destroyed, prior cross-linking of proteins interacting with the α-chain in intact cells was required. The use of the thiol-cleavable cross-linker 3,3′-dithiobis(succinimidyl proprionate) facilitated dissociation of α-chain-binding proteins by means of dithiothreitol following purification. Using this approach we were able to demonstrate a strong interaction between the endoplasmic reticulum chaperone calreticulin (CRT) and the IL-12 α-chain that was confirmed in a reciprocal anti-CRT immunoprecipitation assay. The assay presented here provides a simple approach to exposing concealed hexahistidine tags while retaining native noncovalent protein interactions and should be generally applicable in a range of pull-down or affinity capture methods aiming at analysis of protein complexes.

Section snippets

Cell culture

HEK-293 cells transfected with pVgRXR (EcR293) encoding the regulatory ecdysone receptor were obtained from Invitrogen (Breda, The Netherlands) and were routinely cultivated in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 2 mM l-glutamine, and 400 μg/ml zeocin. Cells were maintained in a 5% CO2 incubator at 37 °C.

Antibodies and immunoblot procedures

The following antibodies were used: (i) mouse anti-p35 antibody G161-566 and mouse anti-p40 antibody C8.6, both obtained from BD-Biosciences (Erembodegem,

The C-terminal hexahistidine tag of the IL-12 β-chain but not of the α-chain is exposed for binding on Ni2+–NTA

The aim of this work was to investigate whether the hexahistidine tag approach can be used for capturing proteins associating with p35 or p40 in the ER. Fig. 1A illustrates that purification of the α-chain on Ni2+–NTA from cell lysates under nondenaturing conditions was unsuccessful. Though high levels of this protein were found in 2B9 cell lysates, it did not bind to the Ni2+–NTA matrix. In contrast, both the monomeric and the homodimeric forms of the β-chain were strongly enriched following

Discussion

In this study we have developed a chaotrope/cross-linking-based strategy for extracting functional multisubunit complexes from intact cells. We have demonstrated the efficacy of this method by isolating a functional CRT–IL-12 α-chain complex from a recombinant EcR293 cell line. This strategy should be generally applicable in proteomics, pull-down, and other affinity-capture strategies aiming at identification of functional protein interactions. It should be particularly appropriate whenever

Acknowledgements

We thank Brian Walker, School of Pharmacy, Queens’ University of Belfast for advice in the synthesis and provision of calreticulin peptides.

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