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Expression and Functional Characterization of Recombinant Human HDAC1 and HDAC3

Expression and Functional Characterization of Recombinant Human HDAC1 and HDAC3

Junling Li, Michael J Staver, Michael L Curtin, James H Holms, Robin R Frey, Rohinton Edalji, Richard Smith, Mike R Michaelides, Steven K Davidsen, Keith B Glaser

Life Sci. 2004 Apr 16;74(22):2693-705.

PMID: 15043985

Abstract:

Histone deacetylases (HDACs) are a family of enzymes involved in transcription regulation. HDACs are known to play key roles in the regulation of cell proliferation; consequently, inhibition of HDACs has become an interesting approach for anti-cancer therapy. However, expression of mammalian HDACs has proven to be difficult. All attempts to express these HDACs in E.coli, Pichia and baculovirus systems were unsuccessful. Here we present the stable expression of human recombinant His-tagged HDAC1 and HDAC3 in mammalian cells. Full-length human genes for HDAC1 and HDAC3 were cloned into the pcDNA 3.1 vector containing a N-terminal His-tag with an enterokinase cleavage site. Recombinant HDAC enzyme activity was only detected after nickel affinity purification due to high activity of endogenous HDACs; and removal of the His-tag increased activity 2-4 fold. Western blots demonstrated the nickel affinity purified rhHDAC1 preparation also contained endogenous HDAC2 and HDAC3; likewise, rhHDAC3 preparation contained endogenous HDAC1 and HDAC2. Therefore, the active HDAC preparation is actually a multi-protein and a multi-HDAC containing complex. This provides one explanation for the similar IC50 values exhibited by SAHA and MS-275 against nuclear HDACs and rhHDAC1 and 3 preparations. These results demonstrate that recombinant forms of the HDACs can be over-expressed in mammalian cells, isolated as active multi-protein complexes that contain multiple HDAC enzymes, and caution must be used when determining HDAC inhibitor in vitro selectivity.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR4248149	HDAC 2 His tag human			Price

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