Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies

Romier, C.; Ben Jelloul, M.; Albeck, S.; Buchwald, G.; Busso, D.; Celie, P.H.N.; Christodoulou, E.; De Marco, V.; van Gerwen, S.; Knipscheer, P.; Lebbink, J.H.; Notenboom, V.; Poterszman, A.; Rochel, N.; Cohen, S.X.; Unger, T.; Sussman, J.L.; Moras, D.; Sixma, T.K.; Perrakis, A.

doi:10.1107/S0907444906031003

Figure 3
Coomassie blue-stained polyacrylamide gels of various stages of protein production referring to the case studies. In all gels MW is molecular-weight markers and the size of relevant bands is indicated with a number at the side or above each band. A label indicating each protein of interest is at the side of each gel, with an arrow pointing to the corresponding height of each protein band. (a) Expression and co-expression of yeast (y) and human (h) TAF_II6 (T6) and TAF_II9 (T9). All samples represent proteins retained on cobalt beads. Lanes 1, 2, 5 and 6, single expression of His-tagged yTAF_II6, yTAF_II9, hTAF_II6 and hTAF_II9, respectively. Lanes 3, 4, 7 and 8, co-expression of His-yTAF_II6/yTAF_II9, His-yTAF_II9/yTAF_II6, His-hTAF_II6/hTAF_II9 and His-hTAF_II9/hTAF_II6, respectively. (b) Co-expression of the complex formed between hTAF_II4 and hTAF_II12. Lane 1 shows the purified complex. (c) Expression of His-ER-LBD and co-expression of His-ER-LBD with SRC-1 fragment. Lane 1, soluble fraction of His-ER-LBD expression; lane 2, His-ER-LBD after affinity purification on cobalt beads; lane 3, soluble fraction of His-ER-LBD and SRC-1 co-expression; lane 4, His-ER-LBD–SRC-1 complex after affinity purification on cobalt beads through the His-tag on ER-LBD. (d) Co-expression of the complex formed between human VDR, RXR and a fragment of Drip205. Lane 1 shows the purified complex. Note that MW and lane 1 are both from the same gel, but at opposing sides and are depicted together. (e) Co-expression of the complex formed between VirE1 and VirE2. Lane 1 shows the purified complex. (f) Co-expression trials of human Cdt1 and geminin. Lane 1, cells before induction. Lane 2, co-expression from two plasmids. Lane 3, co-expression from one plasmid, producing one transcript. Lane 4, co-expression from pETDuet. All lanes present total cell extract. Lane 5 (pasted from a different gel), the complex after affinity purification through the His tag on Cdt1. (g) Co-expression trials of mouse Ring1b and Bmi1 Ring domain fragments. Lane 1, proteins retained in Ni²⁺ beads after co-expression of His-tagged Ring domains. Lane 2, proteins retained in glutathione beads after co-expression of GST-tagged Ring1b and untagged Bmi1. (h) Co-expression trials of human Rad18 and HR6B. Lanes 1 and 2, proteins retained in Ni⁺ beads after co-expression of His-tagged contructs, using one plasmid (lane 1) or two plasmids (lane 2) for the co-expression experiment. (i) Co-expression of human MSH2 and MSH6 in baculovirus-infected Sf9 insect cells. Lane 1, uninfected cells; lane 2, cells infected with virus for MSH2 72 h post-infection; lane 3, cells infected with virus for His-tagged MSH2; lane 4, cells infected with virus for MSH6; lane 5, co-expression of MSH2 and MSH6 from separate viruses; lane 6, co-expression from separate viruses coding for His6-MSH2 and MSH6; lane 7, co-expression of MSH2 and MSH6 from a single baculovirus; lane 8 (pasted from a different gel), purified His-tagged MutSα. (j) Co-expression of the SCF components in insect cells followed by Ni²⁺-bead affinity purification. Lanes 1–3, using separate viruses for Rbx1 (1), Cul1 (2) and Skp1-F-box (3); lane 4, after co-transfection and optimization using all three viruses. (k) Co-expression of CTD-activating kinase (CAK) subunits co-expressed in insect cells. Lane 1, purified Flag-Cdk7; lane 2, Flag-Cdk7/cyclin H; lane 3, Flag-Cdk7/cyclin H/MAT1.

BIOLOGICAL
CRYSTALLOGRAPHY

ISSN: 1399-0047

Volume 62| Part 10| September 2006| Pages 1232-1242

doi:10.1107/S0907444906031003

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