Purification of Histone XXXX Methyltransferase SMYD2 and Co-Crystallization with a Target Peptide from @entity172 Receptor a.

@entity172 @entity3406 @entity376 @entity2975 @entity214 @entity526
Methylation of @entity172 receptor a by the histone @entity376 methyltransferase SMYD2 regulates ERa chromatin recruitment and its target gene expression. This protocol describes SMYD2 purification and crystallization of SMYD2 in complex with an ERa peptide. Recombinant SMYD2 is overexpressed in @entity526 cells. After release from the cells by French Press, SMYD2 is purified to apparent homogeneity with multiple chromatography methods. Nickel affinity column purifies SMYD2 based on specific interaction of its 6*His tag with the bead-immobilized @entity3406 ions. Desalting column is used for protein buffer exchange. Gel filtration column purifies SMYD2 based on molecular size. The entire purification process is monitored and analyzed by @entity214 - @entity2975 gel electrophoresis. Crystallization of SMYD2 is performed with the hanging drop vapor diffusion method. Crystals of the SMYD2-ERa peptide complex are obtained by microseeding using seeding bead. This method can give rise to large size of crystals which are suitable for X-ray diffraction data collection. X-ray crystallographic study of the SMYD2-ERa complex can provide structural insight into posttranslational regulation of ERa signaling.
