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Biotechnology Letters

, Volume 40, Issue 9–10, pp 1355–1363 | Cite as

Purification of active recombinant human histone deacetylase 1 (HDAC1) overexpressed in Escherichia coli

  • Alessandra Stefan
  • Natalia Calonghi
  • Fabrizio Schipani
  • Fabrizio Dal Piaz
  • Giorgio Sartor
  • Alejandro Hochkoeppler
Original Research Paper
  • 86 Downloads

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.

Keywords

Escherichia coli Heterologous expression Human histone deacetylase 1 (HDAC1) Protein dissociation Protein purification 

Notes

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.

Supplementary material

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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Department of Pharmacy and BiotechnologyUniversity of BolognaBolognaItaly
  2. 2.Department of MedicineUniversity of SalernoFiscianoItaly
  3. 3.CSGI, University of FirenzeSesto FiorentinoItaly

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