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Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach
Published on: October 16, 2014
Co-expression as a convenient method for the production and purification of core histones in bacteria
Megan Anderson1, Joon H Huh, Thien Ngo
1Section of Molecular Biology, Division of Biological Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0347, USA.
Abstract:
Co-expression offers an important strategy for producing multiprotein complexes for biochemical and biophysical studies. We have found that co-expression of histones H2A and H2B (from yeast, chicken or Drosophila) leads to production of soluble heterodimeric H2AH2B complexes. Drosophila histones H3 and H4 can also be produced as a soluble (H3H4)(2) heterotetrameric complex if they are co-expressed with the histone chaperone Asf1. The soluble H2AH2B and (H3H4)(2) can be purified by simple chromatographic techniques and have similar properties to endogenous histones. Our methods should facilitate histone production for studies of chromatin structure and regulatory proteins that interact with histones. We describe a simple strategy for constructing co-expression plasmids, based on the T7 RNA polymerase system, which is applicable to other systems. It offers several advantages for quickly creating plasmids to express two or more proteins and for testing different combinations of proteins for optimal complex production, solubility or activity.
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