title: Purification of Histone Lysine Methyltransferase SMYD2 and Co-Crystallization with a Target Peptide from Estrogen Receptor α.
abstract: Methylation of estrogen receptor α by the histone lysine methyltransferase SMYD2 regulates ERα chromatin recruitment and its target gene expression. This protocol describes SMYD2 purification and crystallization of SMYD2 in complex with an ERα peptide. Recombinant SMYD2 is overexpressed in Escherichia coli 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 nickel 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 SDS-polyacrylamide gel electrophoresis. Crystallization of SMYD2 is performed with the hanging drop vapor diffusion method. Crystals of the SMYD2-ERα 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-ERα complex can provide structural insight into posttranslational regulation of ERα signaling.
authors.affiliation: Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 540 East Canfield Street, Detroit, MI, 48201, USA.
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 540 East Canfield Street, Detroit, MI, 48201, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: Holcomb
authors.affiliation: Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 540 East Canfield Street, Detroit, MI, 48201, USA.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Spellmon
authors.affiliation: Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 540 East Canfield Street, Detroit, MI, 48201, USA. zyang@med.wayne.edu.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Yang
mesh_terms: D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003460:Crystallization; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification; D057188:Workflow
chemical_list: C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D011994:Recombinant Proteins; D011495:Histone-Lysine N-Methyltransferase; C512569:SMYD2 protein, human
keywords: Chromatography; Crystallization; Gel filtration; Nickel affinity; Protein purification; Seeding; X-ray crystallography