Purification of metal-dependent lysine deacetylases with consistently high activity

Tasha B Toro; Richard G Painter; Rashad A Haynes; Elena Y Glotser; Melyssa R Bratton; Jenae R Bryant; Kyara A Nichols; Asia N Matthew-Onabanjo; Ashley N Matthew; Derek R Bratcher; Chanel D Perry; Terry J Watt

doi:10.1016/j.pep.2017.08.009

Purification of metal-dependent lysine deacetylases with consistently high activity

Protein Expr Purif. 2018 Jan:141:1-6. doi: 10.1016/j.pep.2017.08.009. Epub 2017 Aug 24.

Authors

Affiliations

¹ Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125-1098, USA. Electronic address: ttoro@xula.edu.
² Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125-1098, USA. Electronic address: rgpainter@gmail.com.
³ Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125-1098, USA. Electronic address: rhaynes1@xula.edu.
⁴ Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125-1098, USA. Electronic address: eglotser@xula.edu.
⁵ Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125-1098, USA. Electronic address: mbratton@xula.edu.
⁶ Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125-1098, USA. Electronic address: jbryant001@hotmail.com.
⁷ Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125-1098, USA. Electronic address: knichols@xula.edu.
⁸ Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125-1098, USA. Electronic address: asia.matthew2@umassmed.edu.
⁹ Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125-1098, USA. Electronic address: Ashley.matthew2@umassmed.edu.
¹⁰ Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125-1098, USA. Electronic address: dbratch89@yahoo.com.
¹¹ Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125-1098, USA. Electronic address: chanelperry07@gmail.com.
¹² Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125-1098, USA. Electronic address: tjwatt@xula.edu.

Abstract

Metal-dependent lysine deacetylases (KDACs) are involved in regulation of numerous biological and disease processes through control of post-translational acetylation. Characterization of KDAC activity and substrate identification is complicated by inconsistent activity of prepared enzyme and a range of multi-step purifications. We describe a simplified protocol based on two-step affinity chromatography. The purification method is appropriate for use regardless of expression host, and we demonstrate purification of several representative members of the KDAC family as well as a selection of mutated variants. The purified proteins are highly active and consistent across preparations.

Keywords: Histone deacetylase; Lysine deacetylase; hdac.

MeSH terms

Animals
Circular Dichroism
Cobalt / chemistry
Cobalt / metabolism*
Electrophoresis, Polyacrylamide Gel
Escherichia coli / genetics
Histone Deacetylases / chemistry
Histone Deacetylases / genetics
Histone Deacetylases / isolation & purification*
Histone Deacetylases / metabolism*
Humans
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / isolation & purification
Recombinant Proteins / metabolism
Repressor Proteins / chemistry
Repressor Proteins / genetics
Repressor Proteins / isolation & purification*
Repressor Proteins / metabolism*
Sf9 Cells / metabolism

Substances

Recombinant Proteins
Repressor Proteins
Cobalt
HDAC8 protein, human
Histone Deacetylases

Abstract

MeSH terms

Substances

Grants and funding