Advertisement

Purification of Histone Lysine Methyltransferase SMYD2 and Co-Crystallization with a Target Peptide from Estrogen Receptor α

  • Yuanyuan Jiang
  • Joshua Holcomb
  • Nicholas Spellmon
  • Zhe Yang
Part of the Methods in Molecular Biology book series (MIMB, volume 1366)

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.

Key words

Proteinpurification Chromatography Nickel affinity Gel filtration Crystallization Seeding X-ray crystallography 

References

  1. 1.
    Bergfors T (2003) Seeds to crystals. J Struct Biol 142:66–76CrossRefGoogle Scholar
  2. 2.
    Zhang X, Tanaka K, Yan J et al (2013) Regulation of estrogen receptor alpha by histone methyltransferase SMYD2-mediated protein methylation. Proc Natl Acad Sci U S A 110:17284–17289CrossRefGoogle Scholar
  3. 3.
    Jiang Y, Trescott L, Holcomb J et al (2014) Structural insights into estrogen receptor alpha methylation by histone methyltransferase SMYD2, a cellular event implicated in estrogen signaling regulation. J Mol Biol 426:3413–3425CrossRefGoogle Scholar
  4. 4.
    Sirinupong N, Brunzelle J, Ye J et al (2010) Crystal structure of cardiac-specific histone methyltransferase SmyD1 reveals unusual active site architecture. J Biol Chem 285:40635–40644CrossRefGoogle Scholar
  5. 5.
    Sirinupong N, Brunzelle J, Doko E et al (2011) Structural insights into the autoinhibition and posttranslational activation of histone methyltransferase SmyD3. J Mol Biol 406:149–159CrossRefGoogle Scholar
  6. 6.
    Jiang Y, Sirinupong N, Brunzelle J et al (2011) Crystal structures of histone and p53 methyltransferase SmyD2 reveal a conformational flexibility of the autoinhibitory C-terminal domain. PLoS One 6, e21640CrossRefGoogle Scholar
  7. 7.
    Papanyan Z, Markarian S (2013) Detection of oxidation of L-cysteine by dimethyl sulfoxide in aqueous solutions by IR spectroscopy. J Appl Spectrosc 80:775–778CrossRefGoogle Scholar
  8. 8.
    San-Miguel T, Perez-Bermudez P, Gavidia I (2013) Production of soluble eukaryotic recombinant proteins in E. coli is favoured in early log-phase cultures induced at low temperature. Springerplus 2:89CrossRefGoogle Scholar
  9. 9.
    Benvenuti M, Mangani S (2007) Crystallization of soluble proteins in vapor diffusion for x-ray crystallography. Nat Protoc 2:1633–1651CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Yuanyuan Jiang
    • 1
  • Joshua Holcomb
    • 1
  • Nicholas Spellmon
    • 1
  • Zhe Yang
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyWayne State University School of MedicineDetroitUSA

Personalised recommendations