Cross-linking approach to affinity capture of protein complexes from chaotrope-solubilized cell lysates.
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) alpha- and beta-chains as "bait" proteins for cocapturing novel binding partners using heterologous recombinant human embryonic kidney-293 (HEK-293) cell lines. The beta-chain, but not the alpha-chain, extracted from cell lysates was capable of binding to the Ni(2+)-nitrilotriacetic acid affinity resin under nondenaturing conditions. Retention of the alpha-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 alpha-chain in intact cells was required. The use of the thiol-cleavable cross-linker 3,3'-dithiobis(succinimidyl proprionate) facilitated dissociation of alpha-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 alpha-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.
Alloza
Iraide
I
Biomolecular Sciences Group, School of Pharmacy, Queen's University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, UK.
Martens
Erik
E
Hawthorne
Susan
S
Vandenbroeck
Koen
K
D000345:Affinity Labels; D037282:Calreticulin / Q000737:chemistry; D002460:Cell Line; D003432:Cross-Linking Reagents; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006801:Humans; D018664:Interleukin-12 / Q000737:chemistry; D009532:Nickel / Q000737:chemistry; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010957:Plasmids; D011489:Protein Denaturation; D011506:Proteins / Q000737:chemistry*; D013388:Succinimides / Q000737:chemistry; D013438:Sulfhydryl Compounds; D014508:Urea
D000345:Affinity Labels; D037282:Calreticulin; D003432:Cross-Linking Reagents; D010455:Peptides; D011506:Proteins; D013388:Succinimides; D013438:Sulfhydryl Compounds; D018664:Interleukin-12; D006639:Histidine; D009532:Nickel; D014508:Urea; C011240:dithiobis(succinimidylpropionate); D009571:Nitrilotriacetic Acid; D004229:Dithiothreitol