Ni2 chelating sepharose

DNA encoding the soluble domain of Om45p without the first 22 amino acids (predicted N‐terminal transmembrane domain) was cloned onto the expression vector pET15b (Novagen‐Addgene, Cambridge, MA, USA) and expressed in E. coli BL21 (DE3). The expressed chimeric protein containing a His6 tag on its N‐terminus, a thrombin cleavage site, and N‐terminally truncated Om45p was affinity purified with Ni²⁺ chelating Sepharose (GE Healthcare, Piscataway, NJ, USA), concentrated 10 times with a Millipore Amicon Ultra‐15 10000 NMWL concentrator (Carrigtwohill, Cork, Ireland) in a table centrifuge at 4°C and subjected to anion‐exchange chromatography in Fractogel EMD DEAE (M) (Merck KGaA, Darmstadt, Germany), in a 10 × 150 mm column (20 mM Tris/HCl pH 7.4, linear gradient elution with NaCl from 50 to 200 mM at the flow rate 1 ml/min in 25 min). The purity of the peak fractions was controlled by SDS‐PAGE and mass spectrometry. Peak fractions were concentrated to 1.69 mg of protein per ml. 500 µl of the protein were used for the generation of polyclonal antisera fin two rabbits by Davids Biotechnologie (Regensburg, Germany).

6xHis-WDR5_23–334 was produced by expression in Escherichia coli BL21Star (DE3, Invitrogen) from the pProEXHTb (Invitrogen) expression vector and affinity-purified on Ni²⁺ chelating Sepharose (GE Healthcare). After His tag cleavage with TEV protease, the protein was further purified by a second Ni²⁺ column and subsequent size exclusion chromatography on a Superdex 75 (GE Healthcare) gel filtration column pre-equilibrated with 20 mM Tris (pH 7.0), 200 mM NaCl, and 5 mM β-mercaptoethanol. 6xHis-WDS 50-361 was produced by expression in Hi5 insect cells from the pFastBacHTb (Invitrogen) vector and purified as for WDR5.

Four constructs of untagged KANSL1 (residues 1–233, 262–537, 537–773, and 777–1105) were each coexpressed with 6xHis-WDR5 (residues 23–334) in E. coli BL21Star (DE3, Invitrogen) from pRSFDuet-1 (Novagen) and pProEXHTb (Invitrogen) expression vectors, respectively. 6xHis-WDR5 was affinity-purified on Ni²⁺ chelating Sepharose (GE Healthcare) in 20 mM Tris (pH 7.0), 200 mM NaCl, and 5 mM β-mercaptoethanol. Proteins were eluted with an increasing concentration of imidazole. Samples were analyzed on SDS-PAGE. A His-tagged minimal interacting region of KANSL1 (residues 584–690; wild type or R592A mutant) was coexpressed with untagged WDR5 (residues 23–334) in E. coli BL21Star (DE3, Invitrogen) from pProEXHTb (Invitrogen) and pRSFDuet-1 (Novagen) expression vectors, respectively. Pull-down experiments were performed as above.
Constructs of KANSL2 were produced as 6xHis-GST fusion proteins in pETM30 expression vector. KANSL2_381–492 and mutated KANSL2_381–492 L411E,V413D were expressed in E. coli BL21Star (DE3, Invitrogen) and purified as for WDR5. Pure wild-type KANSL2_381–492 or its V413D mutant were mixed with pure WDR5_23–334 and loaded onto a Superdex 200 gel filtration column. Fractions containing protein were analyzed on SDS-PAGE.