Expression and functional characterization of recombinant human HDAC1 and HDAC3.
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.
Li
Junling
J
Global Pharmaceutical Research and Development, Cancer Research, R47J-AP9, Abbott Laboratories, Abbott Park, IL 60064-6121, USA.
Staver
Michael J
MJ
Curtin
Michael L
ML
Holms
James H
JH
Frey
Robin R
RR
Edalji
Rohinton
R
Smith
Richard
R
Michaelides
Mike R
MR
Davidsen
Steven K
SK
Glaser
Keith B
KB
D015153:Blotting, Western; D002455:Cell Division; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006367:HeLa Cells; D056284:Histone Deacetylase 1; D006655:Histone Deacetylases / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D013379:Substrate Specificity; D014162:Transfection; D000099186:Histone Deacetylase 3
D004791:Enzyme Inhibitors; D056284:Histone Deacetylase 1; D006655:Histone Deacetylases; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D000099186:Histone Deacetylase 3; C110550:HDAC1 protein, human