Protein-protein cross-linking in the use of the eukaryotic eGST-fusion system.
We describe here an unusual phenomenon in the isolation of protein complexes from eukaryotic cells using expressed GST-fusion proteins. Protein complexes are involved in a large number of regulatory mechanisms. Therefore, the use of tagged fusion proteins is an important tool for isolation of such protein complexes. For this purpose, we used the nuclear factor Alien, described as a corepressor for the thyroid hormone receptor, fused to the eukaryotic eGST and expressed this fusion in human cells. After affinity purification over glutathione-Sepharose using stringent washing steps, we observed several co-purifying bands migrating at molecular weights higher than the GST-Alien fusion protein. These bands appeared specifically in the GST-Alien transfected cell preparations. Surprisingly, using both Western blotting and MALDI-analyses, we revealed that these bands are composed of the GST-Alien protein itself. We hypothesize that overexpressed factors may generate unexpected cross-linking products which can confound the analyses of such affinity-purified complexes. The cross-linking products could not be eliminated by using beta-mercaptoethanol in the gel system and by boiling in SDS-sample buffer. Also, we demonstrate that Western blotting analyses using antibodies directed against both the tag-epitope and the expressed protein of interest can rapidly, reliably, and in a cost-saving manner identify such artifacts, eliminating them from the analyses of potentially interesting interaction partners. Our findings clearly show that the overexpression and purification of proteins from eukaryotic cells may generate unusual structural features that strongly influence complex formation and the migration in SDS-PAGE.
Papaioannou
Maria
M
Genetic Institute, Justus-Liebig-University, Heinrich-Buff-Ring 58-62, Giessen D-35392, Germany.
Ruppert
Thomas
T
Dotzlaw
Helmut
H
Dressel
Uwe
U
Baniahmad
Aria
A
D016477:Artifacts; D015153:Blotting, Western; D000075686:COP9 Signalosome Complex; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D046911:Macromolecular Substances; D008623:Mercaptoethanol; D008970:Molecular Weight; D010449:Peptide Mapping; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C402762:COPS2 protein, human; D046911:Macromolecular Substances; D011506:Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D008623:Mercaptoethanol; D005982:Glutathione Transferase; D000075686:COP9 Signalosome Complex