Abstract
Objective
We attempted to overexpress Human Histone Deacetylase 1 (HDAC1) in Escherichia coli.
Results
A synthetic gene coding for HDAC1, and optimised for E. coli codon usage, was cloned into pBADHisB, generating pBAD-rHDAC1. This construct was used to transform E. coli TOP10, and the target protein was overexpressed and partially purified. According to its elution volume from a Superdex 200 column, the partially purified rHDAC1 was obtained in aggregated form, i.e., as an octamer. The dissociation of octameric HDAC1 was tested using several agents, among which sodium dodecyl sulfate was competent in partially dissociating rHDAC1 aggregates. When the enzyme activity was tested in vitro using 3H-acetyl-labelled histones both protein samples, aggregated and dissociated, were active. Hence, our results suggest that E. coli represents an alternative system for the production of the recombinant HDAC1.
Conclusions
We described a procedure for the overexpression in E. coli of recombinant HDAC1, the purification of which in active form can be successfully performed, although yielding an octameric aggregate.
Similar content being viewed by others
References
Amellem O, Stokke T, Sandvik JA, Pettersen EO (1996) The retinoblastoma gene product is reversibly dephosphorylated and bound in the nucleus in S and G2 phases during hypoxic stress. Exp Cell Res 227:106–115
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Conte E, Vincelli G, Schaaper RM, Bressanin D, Stefan A, Dal Piaz F, Hochkoeppler A (2012) Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core. Arch Biochem Biophys 523:135–143
Feng JH, Jing FB, Fang H, Gu LC, Xu WF (2011) Expression, purification, and S-nitrosylation of recombinant histone deacetylase 8 in Escherichia coli. Biosci Trends 5:17–22
Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130
Hassig CA, Fleischer TC, Billin AN, Schreiber SL, Ayer DE (1997) Histone deacetylase activity is required for full transcriptional repression by mSin3A. Cell 89:341–347
Hassig CA, Tong JK, Fleischer TC, Owa T, Grable PG, Ayer DE, Schreiber SL (1998) A role for histone deacetylase activity in HDAC1-mediated transcriptional repression. Proc Natl Acad Sci USA 95:3519–3524
Hu E, Chen Z, Fredrickson T, Zhu Y, Kirkpatrick R, Zhang GF, Johanson K, Sung CM, Liu R, Winkler J (2000) Cloning and characterization of a novel human class I histone deacetylase that functions as a transcription repressor. J Biol Chem 275:15254–15264
Karwowska-Desaulniers P, Ketko A, Kamath N, Pflum MKH (2007) Histone deacetylase 1 phosphorylation at S421 and S423 is constitutive in vivo, but dispensable in vitro. Biochem Biophys Res Commun 361:349–355
Li J, Staver MJ, Curtin ML, Holms JH, Frey RR, Edalji R, Smith R, Michaelides MR, Davidsen S, Glaser KB (2004) Expression and functional characterization of recombinant human HDAC1 and HDAC3. Life Sci 74:2693–2705
Rao KS, Prakash V (1993) Interaction of sodium dodecyl sulfate with multi-subunit proteins. A case study with carmin. J Biol Chem 268:14769–14775
Shevchenko A, Tomas H, Havlis J, Olsen JV, Mann M (2007) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1:2856–2860
Supporting information
Supplementary Fig. 1—Sequence of the synthetic optimised rHDAC1 gene. The NcoI and PstI restriction sites, used for the cloning into pBADHisB, are underlined. The ATG start codon in shown in bold.
Supplementary Fig. 2—(A) Purification of rHDAC1 by anion exchange chromatography (Q-Sepharose FF column, 50 mL). (B) SDS-PAGE (10% polyacrylamide) of eluted fractions. Lane M: molecular mass markers (116, 66, 45, 35, 25 kDa); lane I: sample of soluble protein extract (input); FT: flow-through fractions; W: washing fractions; numbered lanes: eluted fractions.
Supplementary Fig. 3—(A) HiTrap Heparin affinity chromatography of rHDAC1 (5 mL column). (B) SDS-PAGE (10% polyacrylamide) of eluted fractions. Lane M: molecular mass markers (116, 66, 45, 35, 25 kDa); lane I: sample of pooled fractions (input); FT: flow-through fractions; W: washing fractions; numbered lanes: eluted fractions.
Supplementary Fig. 4—(A) Poly-lysine affinity chromatography of rHDAC1 (8 mL column). (B) SDS-PAGE (10% polyacrylamide) of eluted fractions. Lane M: molecular mass markers (116, 66, 45, 35, 25 kDa); lane I: sample of pooled fractions (input); FT: flow-through fraction; W: washing fraction; numbered lanes: eluted fractions.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Stefan, A., Calonghi, N., Schipani, F. et al. Purification of active recombinant human histone deacetylase 1 (HDAC1) overexpressed in Escherichia coli. Biotechnol Lett 40, 1355–1363 (2018). https://doi.org/10.1007/s10529-018-2585-5
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-018-2585-5