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

I. Alloza, E. Martens, S. Hawthorne, K. Vandenbroeck

Research output: Contribution to journalArticle

16 Citations (Scopus)

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 Ni 2+-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.
LanguageEnglish
Pages137-142
Number of pages6
JournalAnalytical Biochemistry
Volume324
Issue number1
DOIs
Publication statusPublished - 2004

Fingerprint

His-His-His-His-His-His
Proteins
Calreticulin
Interleukin-12
Purification
Assays
Cells
Nitrilotriacetic Acid
Dithiothreitol
Sulfhydryl Compounds
Urea
Carrier Proteins
Resins
Peptides

Keywords

  • Affinity tag
  • Cross-linking
  • Interleukin-12
  • Protein complex
  • Proteome

Cite this

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Cross-linking approach to affinity capture of protein complexes from chaotrope-solubilized cell lysates. / Alloza, I.; Martens, E.; Hawthorne, S.; Vandenbroeck, K.

In: Analytical Biochemistry, Vol. 324, No. 1, 2004, p. 137-142.

Research output: Contribution to journalArticle

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T1 - Cross-linking approach to affinity capture of protein complexes from chaotrope-solubilized cell lysates

AU - Alloza, I.

AU - Martens, E.

AU - Hawthorne, S.

AU - Vandenbroeck, K.

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